Kinetic characterization of VopT, a mono-ADP-ribosyltransferase toxin from Vibrio
parahaemolyticus
by
Amanda Poole
A Thesis
presented to
the University of Guelph
In partial fulfillment of requirements
for the degree of
Master of Science
in Biochemistry
Guelph, Ontario, Canada
© Amanda Poole, September 2016
ABSTRACT
KINETIC CHARACTERIZATION OF VOPT, A MONO-ADP-RIBOSYLTRANSFERASE
TOXIN FROM VIBRIO PARAHAEMOLYTICUS
Amanda N. Poole
University of Guelph, 2016
Advisor:
Prof. Rod Merrill
Pathogenic bacteria cause many human infections, and most employ virulence factors
that cause cell or tissue damage in the host. One important virulence factor group is the monoADP-ribosyltransferase (mART) family. Vibrio parahaemolyticus is a pathogenic bacterium that
encodes a virulence factor, VopT, a mART toxin. VopT modifies Ras, a small GTPase involved
in cell signaling, leading to intestinal epithelial tissue damage. VopT is classified as an ExoS-like
toxin as it requires the binding of a 14-3-3 protein for activation and shares high sequence
identity to other ExoS-like members. Glycohydrolase activities were assessed for wild-type and
catalytic variants, the first inhibitors of VopT were identified with IC50 values between 4 and 45
µM, and an ExoT homology model was produced that provides insight into the FAS-dependent
activity of VopT. Characterization of VopT has increased our understanding of mART enzymes
and may lead to the development of effective therapeutic compounds.
Acknowledgements
I’d first like to thank my advisor, Prof. Rod Merrill, for giving me the opportunity to
work and study in his lab as well as his support throughout my Masters degree. I also would like
to thank Profs. David Josephy and Fred Brauer for being active participants on my advisory
committee and for valuable discussions. Thank you to Profs. John Dawson and Andrew Bendall
as well for being a part of my examination committee.
I would like to thank past and present lab members for their help with my project: Kayla
Heney for investigating the interaction between VopT and VocC, and Madison Wright for
growing various cultures, preparing some VopT variants, and assisting me in the lab whenever
possible. I am also grateful to Miguel Lugo for preparing the ExoT homology model and
providing valuable insights to VopT and other ExoS-like mART members. I am also grateful to
Ravi Ravulapalli and Debajoti Dutta for assistance in the initiation of the VopT project.
I’d also like to thank members of my lab, Bronwyn Lyons, Dan Krska, Tom Keeling,
Stephanie Carlin, Madison Turner, Kayla Heney, Madison Wright, Jesse Rentz, Chantal Wood,
Jordan Willis, and other students of our second floor hallway, Patrick, Evan Mallette, Charles
Wroblewski, Brianna Guild, Dave Martynowicz, Megan Brasher, Olivia Grafinger, Connor
Randall, and Haidun Liu, for their support, assistance, and friendship over the past few years.
I thank my friends Kristen, Steph, and Christina for motivating me to pursue and finish
graduate school and for our adventures together that always gives me a story to laugh about.
I am extremely grateful to my whole family and, in particular, Brandes, for always
believing in me and for their never-ending support.
iii
Declaration of work performed
In some cases, it has been necessary to include work done by other students. All work
presented in this thesis is my own work with the following exceptions: all cloning and site-directed
mutagenesis were performed by Tom Keeling, Dawn White, and Madison Wright. In silico
identification of VopT was done by Robert Fieldhouse, and the yeast cytotoxicity assay was
performed by Zachari Turgeon. The ExoT homology model was made by Miguel Lugo.
iv
Table of Contents
ABSTRACT ..........................................................................................................................................ii
Acknowledgements ..............................................................................................................................iii
Declaration of work performed ............................................................................................................ iv
List of Figures .....................................................................................................................................viii
List of Tables ........................................................................................................................................ ix
List of Abbreviations ............................................................................................................................. x
Chapter 1: Literature Review ................................................................................................................ 1
1.0
Bacterial infections ................................................................................................................. 2
1.1
Bacterial virulence factors...................................................................................................... 3
1.2
Antivirulence strategy ............................................................................................................ 6
1.3
mART toxins .......................................................................................................................... 8
1.3.1
1.3.1.2
Classification of mART toxins ....................................................................................... 9
CT-like group toxins ................................................................................................. 11
1.4
Structure of mART toxins .................................................................................................... 14
1.5
Mechanism of mART toxins................................................................................................ 18
1.6
Inhibitors of mART toxins ................................................................................................... 20
1.7
Discovery of novel mARTs ................................................................................................. 23
1.8
Vibrio parahaemolyticus ...................................................................................................... 24
1.9
VopT and associated proteins .............................................................................................. 25
1.9.1
Ras protein .................................................................................................................... 25
1.9.2
14-3-3 (FAS) protein .................................................................................................... 28
1.9.3
VopT ............................................................................................................................. 30
1.9.4
Yeast cytotoxicity assay for VopT ............................................................................... 30
Chapter 2: Materials and methods ....................................................................................................... 35
2.0
Materials ............................................................................................................................... 36
2.1
Yeast cytotoxicity assay ....................................................................................................... 36
2.2
Heat-shock transformation of E. coli BL21 cells ................................................................ 36
2.3
Growth of cells and gene expression ................................................................................... 37
2.4
Sonication ............................................................................................................................. 37
2.5
Purification of His6-tagged proteins ..................................................................................... 38
2.5.1
Purification of VopT and VopT variants...................................................................... 38
2.5.2
Purification of VocC..................................................................................................... 40
v
2.5.3
Purification of hRas ...................................................................................................... 41
2.5.4
Purification of FAS ....................................................................................................... 42
2.5.5
Purification of ExoS ..................................................................................................... 42
2.6
Thermal shift analysis .......................................................................................................... 43
2.7
Activity assays ...................................................................................................................... 44
2.7.1
ɛAMP standard curves .................................................................................................. 44
2.7.2
Optimizing protein quantities ....................................................................................... 45
2.7.3
Incubation time ............................................................................................................. 45
2.7.4
Effect of VocC on activity ............................................................................................ 47
2.7.5
Glycohydrolase and transferase activity – Michaelis-Menten analysis ....................... 47
2.7.6
Metal dependence screen.............................................................................................. 47
2.7.7
Inhibitor screen ............................................................................................................. 48
2.7.8
Effect of boiling on VopT activity ............................................................................... 48
2.8
Binding experiments ............................................................................................................ 49
2.8.1
NAD+-binding............................................................................................................... 49
2.8.2
FAS binding .................................................................................................................. 49
2.8.3
VocC binding ................................................................................................................ 49
2.9
Origin fitting ......................................................................................................................... 50
Chapter 3: Results and discussion ....................................................................................................... 51
3.1
Purification of His6-tagged proteins ..................................................................................... 52
3.1.1
Purification of wild-type VopT and VopT variants ..................................................... 52
3.1.2
Purification of VocC..................................................................................................... 52
3.1.3
Purification of hRas ...................................................................................................... 56
3.1.4
Purification of FAS ....................................................................................................... 56
3.1.5
Purification of ExoS ..................................................................................................... 56
3.2
Thermal shift analysis using RT-PCR ................................................................................. 56
3.3
Activity Assays..................................................................................................................... 60
3.3.1
Optimizing protein concentrations ............................................................................... 64
3.4.2
Incubation time ............................................................................................................. 68
3.4.3
Effect of VocC on VopT glycohydrolase activity........................................................ 68
3.3.4
Glycohydrolase activity ................................................................................................ 71
3.3.5
Metal dependence ......................................................................................................... 76
3.3.6
Inhibitor search ............................................................................................................. 78
3.3.7
Effect of boiling on VopT activity ............................................................................... 86
vi
3.4
Tryptophan-fluorescence binding assays ............................................................................. 88
Chapter 4: Conclusion ......................................................................................................................... 91
4.1
Conclusions .......................................................................................................................... 92
4.2
Future directions ................................................................................................................... 94
References............................................................................................................................................ 97
vii
List of Figures
Figure 1. Classification of T3SS2 among bacterial secretion systems ............................................... 4
Figure 2. Domain organization of mART enzymes .......................................................................... 10
Figure 3. Multiple sequence alignment and phylogenetic tree of CT and DT mART toxins .......... 15
Figure 4. Structure of mART toxins.................................................................................................. 17
Figure 5. Proposed mechanism for mART toxins ............................................................................ 19
Figure 6. Examples of inhibitors of mART toxins............................................................................ 22
Figure 7. MAPK and PKB pathways ................................................................................................ 26
Figure 8. Crystal structure of Ras (PDB:4EFL) ................................................................................ 27
Figure 9. Monomeric crystal structure of 14-3-3 (FAS) protein (PDB: 2O02) ................................ 29
Figure 10. Growth of S. cerevisiae expressing wild-type or variant VopT proteins .......................... 32
Figure 11. Homology model of ExoT, an ExoS-like mART.............................................................. 34
Figure 12. Examples of ɛAMP curves used to calculate enzymatic activity ...................................... 46
Figure 13. Recombinant wild-type and variant VopT purification .................................................... 53
Figure 14. Recombinant ΔN64 purification ........................................................................................ 54
Figure 15. Recombinant VocC purification ........................................................................................ 55
Figure 16. Recombinant hRas purification ......................................................................................... 57
Figure 17. Recombinant FAS purification .......................................................................................... 58
Figure 18. Recombinant ExoS purification......................................................................................... 59
Figure 19. Thermal profiles for VopT and associated proteins .......................................................... 63
Figure 20. Optimal FAS concentration for VopT and ExoS activity ................................................. 65
Figure 21. ExoT homology model ...................................................................................................... 66
Figure 22. Optimal hRas concentration............................................................................................... 67
Figure 23. Effect of pre-incubation time on VopT glycohydrolase activity....................................... 69
Figure 24. Effect of VocC on VopT GH activity................................................................................ 70
Figure 25. Glycohydrolase MM curves for VopT and VopT variants ............................................... 74
Figure 26. The effect of metal salts on VopT and ExoS glycohydrolase activity .............................. 77
Figure 27. Effect of DMSO on VopT glycohydrolase activity........................................................... 80
Figure 28. Inhibitor screen for VopT glycohydrolase activity ........................................................... 81
Figure 29. IC50 curves for VopT glycohydrolase activity................................................................... 83
Figure 30. Glycohydrolase activity of VopT after boiling.................................................................. 87
Figure 31. Binding of NAD+, VocC, and FAS to VopT..................................................................... 89
viii
List of Tables
Table 1.
Table 2.
Table 3.
Table 4.
Expression conditions of VopT and associated proteins .................................................... 39
Melting temperatures of VopT and associated proteins ..................................................... 61
Kinetic values for VopT and VopT variants GH activity ................................................... 72
Structures of inhibitors of VopT glycohydrolase activity .................................................. 84
ix
List of Abbreviations
ABC – ATP-binding cassette
ADP – Adenine diphosphate
ADPRT – Adenine diphosphate-ribosyltransferase
ARTT – ADP-ribosyl-turn-turn
CT – Cholera toxin
DALDL – Aspartate-alanine-leucine-aspartate-leucine
DMSO – Dimethyl sulfoxide
DT – Diphtheria toxin
DTT – Dithiothreitol
ɛAMP – Etheno adenine monophosphate
EDTA – Ethylenediaminetetraacetic acid
eEF2 – Eukaryotic elongation factor two
ɛNAD+ – Etheno nicotinamide adenine dinucleotide
EXE – Glutamate-X-glutamate
ExoA – Exotoxin A
ExoS – Exoenzyme S
ExoT – Exoenzyme T
FAS – Factor activating ExoS
GAP – GTPase activation proteins
GDP – Guanosine diphosphate
GH – Glycohydrolase
GNP – Phosphoaminophosphonic acid-guanylate ester
GTP – Guanosine triphosphate
x
GOLD – Genomic Online Database
IC50 – Median inhibitory concentration
IMAC – Immobilized metal affinity chromatography
IPTG – Isopropyl β-D-1-thiogalactopyranoside
LB – Luria broth
M6 – 2-(4-oxo-3,4-dihydrophthalazin-1-yl)acetate
M15 – (2S)-3-(4-hydroxyphenyl)-2-[2-(4-oxo-3,4-dihydrophthalazin-1-yl)acetamido]propanoate
M19 – methyl-(2S)-2-[2-(2,4-dioxo-1,2,3,4-tetrahydroquinazolin-1-yl)acetamido]-3-(1H-indol-3-yl)
propanoate
MAPK – Mitogen-activated protein kinase
mART – Mono-ADP-ribosyltransferase
MM – Michaelis-Menten
NAD+ – Nicotinamide adenine dinucleotide
1,8-NAP – 1,8-naphthalamide
NATA – N-acetyl-tryptophanamide
P6C – 8-fluoro-2,3,4,5-tetrahydro-1H-pyrido[4,3-c]isoquinolin-6-one
PARP – Poly-ADP-ribosyltransferase
PKB – Protein kinase B
PMSF – Phenylmethanesulfonyl fluoride
PN – Phosphate-nicotinamide
PT – Pertussis toxin
SBTI – Soybean trypsin inhibitor
SDS-PAGE – Sodium dodecyl sulfate polyacrylamide gel electrophoresis
Sec – General secretory pathway
SN1 – Unimolecular nucleophilic substitution
xi
STS – Serine-threonine-serine motif
T1SS – Type I secretion system
T2SS – Type II secretion system
T3SS – Type III secretion system
T4SS – Type IV secretion system
T5SS – Type V secretion system
T6SS – Type VI secretion system
TDH – Thermostable direct hemolysin
Tm – Denaturation temperature
TSA – Thermal shift assay
WHO – World Health Organization
xii
Chapter 1: Literature Review
1
1.0
Bacterial infections
Bacterial infections are one of the most prevalent ailments that humans experience. These
can lead to potentially lethal diseases. Bacteria responsible for these infections are pathogenic
and usually encode various virulence factors (often proteins) that elicit the infection phenotype.
A group of virulence factors is the mono-ADP-ribosyltransferase (mART) family which transfers
an ADP-ribose group to various macromolecules in host cells from NAD+. This modification
causes altered macromolecular function and aids the progression of bacterial infection.
Ingestion of pathogenic bacteria is a main route of infection as food or water may become
contaminated1. Bacterial diseases are most common in areas of the world where access to and
use of hygienic methods is limited, but it is also routinely seen in the developed world1. For
example, acute diarrhea is observed globally. A study surveying bacterial pathogens in diarrheal
patients determined that pathogenic Vibrio parahaemolyticus strains were the most frequent
causative agents, followed by Shigella and Salmonella strains2. Bacterial infections such as these
are usually treated with antibiotics.
Antibiotics kill bacteria by inhibiting crucial processes within the cell. Antibiotics have
been produced for therapeutic use since the time of Florey (1941); however, development of new
classes of antibiotics has slowed considerably over the last three decades3,4. New antibiotics are
continuously required because bacteria develop resistance to these drugs over time due to the
large selection pressure on bacteria to survive3,5. Bacteria can also pass resistance genes to other
bacteria via horizontal gene transfer, subsequently increasing the number of bacterial species that
are drug resistant5. In 2014, the World Health Organization (WHO) reported that we may no
longer be able to treat even simple infections in the near future, since antibiotic resistance in
pathogenic bacteria is developing faster than the introduction of new antibiotics. Consequently,
2
fewer antibiotics on the market are effective; therefore, new strategies for the treatment of
bacterial infections are required.
I will discuss the need for a new bacterial treatment approach; mART toxin classification,
structure, and function; and finally the novel mART toxin, VopT, produced by V.
parahaemolyticus. The purpose of this thesis work was to characterize the enzyme kinetics,
binding parameters, and inhibitory compounds of VopT, a mART toxin from Vibrio
parahaemolyticus.
1.1
Bacterial virulence factors
Pathogenic bacteria release virulence factors into, or in the vicinity of, host cells6,7.
Virulence factors are often protein agents that facilitate infection. Most of these proteins have
specific targets involved with protein synthesis, cytoskeletal structure, DNA replication,
intracellular signalling, and other crucial cellular processes8,9. Upon modification of the
macromolecule targets by the bacterial virulence factors, host/target cells will eventually undergo
apoptosis, causing tissue damage. Some examples of virulence factors include adhesion and
attachment proteins, secreted proteases, and exotoxins and some have the ability to enter host
cells independently while others require specialized transport into the host10.
Bacterial secretion systems are common among bacteria, using type 1 through 6 to
translocate effector proteins (Fig. 1). Type III secretion systems (T3SS) are encoded among
many pathogenic, Gram-negative bacteria, including Yersinia spp., Pseudomonas aeruginosa,
Salmonella spp., and Shigella spp.11,12 and are responsible for directly translocating virulence
factors from the producing bacterium into the host cell cytoplasm11,13–16. Similar to T3SS, the
T1SS translocates effector proteins across both bacterial membranes in a single step using
different machinery, while the T2SS translocates effector in two steps17. First, effector proteins
3
Figure 1. Classification of T3SS2 among bacterial secretion systems
Figure
1. Classification
of T3SS2
amongused
bacterial
secretion
systems.infection
There areor6 types
There are
six types of secretion
systems
by bacterial
to promote
death of nearby
cells. Each type uses different machinery to secrete effector proteins out of the bacterial cell.
4
are transported to the periplasm by the general secretory pathway (Sec translocon) and then an
ABC (ATP-binding cassette) transporter allows the secretion of the effector across the outer
membrane18. The T3SS and bacterial flagellum are evolutionarily related and are comprised of
similar structures19,20. The T3SS apparatus is composed of many proteins and traverses both
bacterial membranes and the membrane of host cells. A basal body component spans both the
inner and outer bacterial membranes and connects with a cytoplasmic export apparatus19,20. A
needle-like structure is anchored into the export apparatus while other proteins act as
translocation pores in the host membrane to connect the bacterial and host cytoplasms16,21,22. The
length of the needle varies across species, but may be based on the ruler protein’s length20. The
ruler protein is hypothesized to be anchored in the bacterial cytoplasm with its globular Cterminal domain extended outside of the bacterium. Once the needle length is long enough to
interact with the globular C-terminal domain, construction is terminated and ―substrate
switching‖ occurs. Substrate switching is the event at which the T3SS needle stops being
constructed and virulence factors start to be translocated across the bacterial membrane through
the needle apparatus.
Some secreted virulence factors require chaperone proteins for delivery to the T3SS
apparatus. Once at the apparatus, chaperone proteins dissociate and virulence factors initiate
unfolding to pass through the narrow needle-like structure14. Once in the host cell, virulence
factors refold into an active conformation. Chaperones bind specific virulence factors and
chaperone genes are encoded proximate to their binding partners; however, not all virulence
factors require a chaperone to be translocated22,23.
A notable virulence factor is thermostable direct hemolysin (TDH), present in pathogenic
V. parahaemolyticus strains. TDH lyses blood cells causing hemolysis, cytotoxicity, and
5
enterotoxicity of the host cell24. TDH acts as a porin, causing secretion of chloride and increasing
intracellular levels of free calcium25,26. The passage of these ions and water molecules through
the epithelial cell membrane promotes the onset of diarrhea15 and morphological changes to the
epithelial cell, including chromatin condensation, and nuclei and cellular shrinkage27. When the
TDH gene was deleted from the bacterial genome, the host cell no longer experienced hemolysis;
however, it still succumbs to infection. This suggests that TDH is not the only virulence factor
encoded by V. parahaemolyticus13,24. Other virulence factors include VopP, VopC, VopV, and
VopT, whose post-translational modifications alter normal host cell function13,26,28.
1.2
Antivirulence strategy
Bacterial infections among humans are common and are generally treated with
antibiotics; however, many bacterial strains are now resistant to antibiotics5,29. These bacteria are
resistant because antibiotics target crucial processes in bacteria, causing cell death, which places
strong selective pressure upon the bacteria. Therefore, novel antibiotics must be constantly
developed to treat resistant bacteria; however, the number of new antibiotics has plummeted over
the last 20 years3. Because it is a long and expensive process to develop, test, and approve new
drugs, large pharmaceutical companies have lost interest in antibiotic development, as it is not a
profitable venture30. Antibiotic development is a poor investment and the drug has a short market
life since bacteria quickly develop resistance, rendering the antibiotic useless.
Furthermore, antibiotics are often under- or overused which ―select[s] for the emergence of
resistant pathogens‖31. For example, when antibiotic treatment ends prematurely, there is a small
population of bacteria that survive antibiotic therapy. These drug-resistant bacteria may cause
another, more serious infection, since this population of bacteria is already resistant to the treatment.
Another antibiotic must then be administered to treat this infection and the cycle continues. An
6
example of antibiotic overuse would be physicians prescribing drugs to treat an unknown ailment,
whether or not it is a bacterial infection31.
An alternative strategy to combat bacterial infection is to inactivate the virulence factors that
cause infection, rendering the offending bacterium less or even avirulent3,29. This strategy involves
the development of small molecules to disable or inhibit virulence factors; these small drug
molecules are called antivirulence compounds. Antivirulence compounds have two significant
advantages over antibiotic therapy. First, they have specific targets that are not essential for bacterial
survival. By disarming pathogenic bacteria instead of killing them, the selective pressure on bacteria
to evolve drug resistance is reduced8. Second, specificity towards virulence factors means that host
microbiota is unaffected, thereby eliminating undesired side effects that are caused by antibiotic
treatment in the gut3,29. Researchers screen for these compounds to develop potent neutralizing
agents against bacterial virulence and success is now beginning to be reported32,33. However, the
discovery of antivirulence compounds only offers proof-of-principle that this approach is potentially
viable to treat bacterial infections, since it is unlikely that the first generation small molecules will
pass through clinical trials and be put on the market. A more sophisticated drug discovery platform
must be developed and fostered, requiring huge resources typical of the pharmaceutical industry.
ADP-ribosyltransferase toxins are examples of virulence compounds that participate in infection
and serve as antivirulence targets. Our research group characterizes the structure and function of
bacterial ADP-ribosyltransferase toxins, develops inhibitors for these toxins, and analyzes the effect
of these toxins on eukaryotic cell culture. Overall, the use of antibiotics is not a sustainable
treatment for bacterial infections because bacteria gain resistance over time. The antivirulence
strategy focuses on disarming harmful bacteria of their ―weapons‖ and not threatening their
survival, which reduces pressure for bacteria to evolve drug-resistance genes.
7
1.3
mART toxins
Enzymes that transfer ADP-ribose moieties from NAD+ to a target macromolecule are
referred to as ADP-ribosyltransferases (ADPRTs) and those that transfer a single ADP-ribose in
particular are called mono-ADP-ribosyltransferases (mARTs); enzymes that transfer more than
one ADP-ribose unit to a target are called poly-ADP-ribose polymerases (pARPs). The action of
transferring the ADP-ribose to a target macromolecule is called a transferase reaction, while the
hydrolysis of NAD+ is called a glycohydrolase reaction. Glycohydrolase reactions occur in the
absence of the target macromolecule and are usually much slower than transferase reactions.
MART enzymes are present in all branches of life. Many organisms use mARTs to
regulate pathways within a cell. For example, humans have cytoplasmic mARTs that are
involved in DNA repair, regulating cell cycle, and apoptosis34–36. Cleverly, some opportunistic
bacteria use these enzymes to mimic host mART activity and modify alternate targets, impairing
cell processes37. These pathogenic bacteria are responsible for a number of human infections,
including diarrhea, whooping cough, cholera, diphtheria38,39, and lung infections in immunecompromised patients, such as those who suffer from cystic fibrosis40.
Cholera is a gastrointestinal disease caused by the consumption of 01 and 0139 strains of
Vibrio cholerae that have contaminated drinking water or food sources41. Vibrio cholera
colonizes intestinal epithelia and secretes a mART toxin called cholera toxin (CT) that modifies
G-proteins42. The colonization of the epithelia both damages the cell lining and reduces surface
area for the uptake of water and nutrients, causing large fluid loss and diarrhea symptoms1.
Prior to mass vaccination programs, diphtheria was one of the leading causes of
childhood death. This was due to diphtheria toxin (DT), a mART toxin from Corynebacterium
diphtheriae43. Upon contamination, this bacterium takes up residence in the respiratory tract and
8
releases DT, causing the neck to swell, among other symptoms. In extreme cases, the neck can
swell so excessively that the patient eventually suffocates44. Notably, diphtheria is still prevalent
in developing countries with limited vaccination programs.
P. aeruginosa is well known as an opportunistic pathogen that infects wounds and afflicts
immune-compromised individuals, such as those suffering from AIDS, cancer, and cystic
fibrosis45. P. aeruginosa colonizes the lungs in these immune-deficient hosts and secretes a
number of toxins, including exotoxin A (ExoA) and S (ExoS), both mART toxins40,46. Infection
of P. aeruginosa is very challenging to treat, due to the multiplicity of virulence factors and
adaptive responses of this pathogen. MART toxins are found in many strains of bacteria and a
large number of these disrupt intracellular processes and are detrimental to eukaryotic cells.
1.3.1
Classification of mART toxins
There are two main classes of mARTs: cholera-like toxins (CT) and diphtheria-like
toxins (DT)37,47. The CT-like group is vast and contains numerous members and it is divided into
PT-like, C2-like, C3-like, and ExoS-like groups. These groups have a few highly conserved
motifs and conserved core structure, but differ in their domain organization (Fig. 2). DT and CT
group toxins can also be categorized by their physiological targets; many of the known mARTs
modify eukaryotic elongation factor 2 (eEF2), actin, small GTPases, or signalling G-proteins48.
1.3.1.1 DT-like group toxins
DT-like toxins are grouped together because they share the same macromolecule target.
They also contain conserved catalytic residues distinct from CT-like members, even though they
both catalyze the transfer of ADP-ribose from NAD+. The conserved features are aromatic
residues followed by histidine, to position the NAD+ molecule, two tyrosines separated by 10
residues to stabilize the NAD+-binding pocket, and a primary glutamate that catalyzes the
9
B
A
C
D
Figure 2. Domain organization of mART enzymes
Figure A shows a DT-like group example that is a single-chain polypeptide containing all three
domains. The catalytic and translocation domains are linked by one disulphide bond. B, CT/PTlike mARTs contain two polypeptide domains which associate together in a 1:5 ratio of catalytic:
binding. C, C2-like binary mARTs contain A and B domains which associate in a 1:1 ratio. D,
C3-like mARTs are single-chain polypeptides with only one domain. Because they lack
translocation and binding domains, they require assistance into target cells. The figure was taken
from Simon and Barbieri37.
10
reaction. Diphtheria toxin (DT) is produced and secreted by C. diphtheria and causes diphtheria
disease. DT was the first DT-like group toxin member to be characterized. ExoA from P.
aeruginosa and cholix from V. cholerae are the only other known mART toxins that belong to
this DT-like group39,49. All members transfer an ADP-ribose moiety from NAD+ to the conserved
diphthamide residue, a post-translationally modified histidine, in eukaryotic elongation factor 2.
Normally, during protein synthesis, eEF2 undergoes a conformational change to allow tRNA
movement and growth of the peptide50. ADP-ribosylated eEF2 becomes rigid and cannot adopt
different conformations, thus preventing polypeptide growth50,51. This modification induces a
conformation of eEF2 that impairs its function which results in wide-spread apoptosis followed
by cell death52–54. DT-like group proteins are single polypeptides with three domains attributed to
receptor binding, membrane translocation, and catalysis37 (Fig. 2a). The receptor domain of DT
binds the heparin-specific epidermal growth factor on the host cell membrane, and then
undergoes endocytosis. DT is subjected to proteolysis, which initiates the insertion of two helices
from the translocation domain into the endosomal membrane. The catalytic domain is then
translocated into the cytoplasm, where it finds the ribosomes and catalyzes its reaction37,55. DTlike group members are distinct from CT-like toxins in their domain organization (one
polypeptide, three domains), catalytic residues (His, 2 Tyr, and Glu), and their macromolecule
target (eEF2).
1.3.1.2 CT-like group toxins
The CT-like group is named after cholera toxin from V. cholera and has numerous
members. It is divided into CT/PT-like, C2-like, C3-like, and ExoS-like groups, based on domain
organization and macromolecule targets. The CT-like group contains three conserved regions
within the catalytic domain: an arginine (R) residue to position the NAD+ molecule, a S-T-S
11
motif to stabilize the NAD+-binding pocket, and an E(Q)-X-E motif that participates in
catalysis37,56.
The CT/PT-like group contains mARTs that have an AB5 subunit structure and modify
small GTP-binding proteins57 (Fig. 2b). The CT/PT-like group is named after cholera-like toxins
and is further classified as pertussis-like toxins (PT). These subunits are expressed separately and
interact non-covalently37 (Fig. 2b). The A subunit contains a catalytic domain where the transfer
of ADP-ribose occurs, while the B subunits contain a membrane-binding domain responsible for
interacting with host cell membranes and the translocation of A subunits across the
membrane37,58. Five B subunits are required for translocation of the A subunit. The CT/PT-like
members modify arginine residues in the GTP-binding proteins. Members include PT, cholera,
and heat-labile Escherichia coli toxins37,59. ADP-ribosylation of GTP-binding proteins retains
Gα-proteins in their GTP-bound states, preventing dissociation56,57. Gα-proteins in their GTPbound states stimulate adenylate cyclase producing an increase in cAMP levels within the cell.
High cAMP levels cause fluid loss from intestinal cells and initiate diarrhea57.
C2-like group members include Iota toxin from Clostridium perfringens, VIP from
Bacillus cereus, and C2 toxin from Clostridium botulinum37,60. C2-like toxins contain A- and Bsubunits, but have a simple binary AB subunit organization (Fig. 2c). Functions of each subunit
are the same as the CT/PT-like subunits; however, the B-subunits require protease activation
prior to interaction with the host cell membrane and introduction of the A-subunit into the cell5.
C2-like members ADP-ribosylate R177 in G-actin, and when G-actin is modified,
depolymerization of F-actin occurs and the actin complex structure becomes compromised,
leading to morphological changes and apoptosis59,61.
12
C3-like toxins target Rho GTPases or low molecular weight G-proteins involved in cell
morphology, adhesion, and microtubule dynamics37,59,62. After these macromolecules are
covalently modified with ADP-ribose, they can still bind with their physiological partner(s);
however, modified Rho has an increased affinity for a dissociation inhibitor, resulting in a
reduction of Rho signalling37,62. ADP-ribosylated Rho prevents actin polymerization and leads to
impairment of the cytoskeleton and apoptosis63. C3-like toxins do not have B-subunits to aid in
translocation into host cells, so C3-like toxins must use other methods of host cell entry37 (Fig.
2d). Some N-terminal helices confer selective internalization of macrophages using non-specific
pinocytosis64 while other pathogenic bacteria express secretion systems to translocate virulence
factors directly into the host cell cytoplasm and elicit infection12,65,66. Examples of C3-like toxins
include C3bot1 and 2 from C. botulinum, C3cer from Bacillus cereus, and HopU1 from
Pseudomonas syringae67–69.
Another subgroup of CT-like toxins is the ExoS-like mARTs. These mARTs resemble
exoenzyme S, ExoS, from P. aeruginosa, in their amino acid sequence and depend on binding a
eukaryotic co-factor, 14-3-3, for activation. The 14-3-3 proteins are involved with signalling
pathways and have many binding partners70. ExoS was identified in 1978 by Iglewski and
colleagues and classified as a mART enzyme46. ExoS was distinct from ExoA, a DT-like toxin
from P. aeruginosa, since it did not modify eEF2. It was then discovered that ExoS contained
two domains: the GTPase-activating protein (GAP) and ADP-ribosylation domains71, and that
there is another enzyme similar to ExoS expressed by P. aeruginosa, denoted ExoT72. Both rely
on the binding of 14-3-3 proteins for activity. Aeromonas salmonicida AexT is another enzyme
closely related to ExoS and ExoT73; all three enzymes were termed the ExoS-like mART family.
These ExoS-like members require the binding of 14-3-3 proteins (Factor Activating exoenzyme
13
S, FAS) to be enzymatically active, have a conserved motif for FAS binding, have two distinct
activities in two separate domains, and share relatively high sequence identity26,71,74–77. All ExoSlike members are single polypeptides that are secreted into target cells using the type III
secretion system26,78. Unfortunately, the structure of this group of toxins is unknown since no
crystal structures have been published. These mARTs belong to the larger CT-like toxin group
since they contain the conserved R, S-T-S, and E/Q-X-E motifs and do not target eEF2.
ExoS and ExoT share about 76% sequence identity79 and about 45% sequence identity to
VopT within the ADP-ribosylation domain26. CT-like mARTs usually have low sequence
identity, so 45% identity is unusual and significant. Even though VopT doesn’t have the GAP
domain, it is still classified as an ExoS-like toxin, because it requires the FAS protein to be
enzymatically active and shares high sequence identity with ExoS-like members compared to
other mART enzymes (Fig. 3). Novel mARTs are continuously being discovered and
characterized to further the understanding of all mART enzymes56 and this is one of the
mandates of the Merrill research program39,80–84.
1.4
Structure of mART toxins
Although mART toxins catalyze the transfer of ADP-ribose from NAD+, their overall
structures, target proteins, and their primary sequences are diverse81. However, their NAD+binding and catalytic sites are relatively conserved, especially within the CT class81 (Fig. 3).
There are three regions within the catalytic domain where both sequence and structure are
conserved37 (Fig. 3, 4). Region 1 contains an aromatic residue immediately followed by an
arginine (R) residue in CT group members; histidine replaces the arginine in DT group
members37,56. Region 1 aids in positioning of the NAD+ molecule by hydrogen bonding one of
the phosphate groups85. When the crystal structures of Iota and DT are superimposed, the
14
Figure 3. Multiple sequence alignment and phylogenetic tree of CT and DT mART toxins
A) CT-like and B) DT-like phylogenetic trees to show relatedness among mART members. C)
Multiple sequence alignments for CT-like and D) DT-like mARTs are represented in Logos format
to show the conservation of sequence in the three catalytic regions. VopT is shown to be most
related to ExoS, ExoT, and AexT from the ExoS-like group of CT-like toxins. HopU1 is classified
as C3-like toxin but not ExoS-like. The three conserved regions of DT-like and CT-like toxins are
labeled 1 – 3 to show high conservation. This figure was taken from Fieldhouse et al.56
15
conserved arginine and a histidine are arranged almost identically. Region 2 contains a serinethreonine-serine (S-T-S) motif that positions the nicotinamide ring of NAD+ 56,86. The DT group
contains two tyrosine residues separated by 10 residues, Y-(X10)-Y, which stabilize the
nicotinamide ring by aromatic ring stacking38. This region contains a β-strand and a tilted α-helix
responsible for providing the scaffold structure of the active site cavity and the nicotinamide
pocket87. Region 3 is responsible for catalysis and contains essential glutamate residue(s). DT
group members possess only one glutamate in this site whereas CT group members have a
glutamate/glutamine-X-glutamate (E/Q-X-E) motif37,56. The activity of DT was decreased 300fold when the conserved glutamate was substituted for serine88. C3cer activity was decreased by
over 1000-fold when both catalytic residues were replaced89. The variation of glutamate and
glutamine in the first conserved position determines the modified amino acid residue in the target
macromolecule. mART toxins with the E-X-E motif target arginine residues, whereas Q-X-Econtaining mARTs target either asparagine or cysteine90. The catalytic glutamate is positioned
opposite the arginine in region 1 (in CT group members) or histidine (in DT group members) on
an anti-parallel β-strand39,91. These regions position NAD+ (region 1), stabilize the NAD+ pocket
(region 2), and promote catalysis (region 3)37.
MARTs also have phosphate nicotinamide (PN) and ADP-ribosylating toxin turn-turn
(ARTT) loops that are responsible for NAD+ binding and substrate recognition, respectively. The
PN loop is about 8 residues long and includes an aromatic residue that rearranges to interact with
the nicotinamide moiety of NAD+
92
. This loop is flexible and shows bound and unbound
conformations. The ARTT loop is also about 8 residues long and contains the catalytic motif
E/Q-X-E; three positions N-terminal from this motif is a conserved aromatic residue. The
aromatic and E/Q-position residues are required for transferase activity, as the aromatic residue
16
A
Helix 1
B
Q178
S140
R95
E180
Figure 4. Structure of mART toxins
C3larvin is a new mART toxin and shows the characteristic structure of this group of enzymes93.
A) The overall structure of mARTs includes a β-sheet core surrounded by six α-helices. Helix 1
is involved in target recognition. C3larvin is coloured by secondary structure; α-helices: green, βsheets: orange, loops: cyan. B) Three regions are conserved in the active site and are labeled in B
as follows: R95 (region 1), S140 designating the STS motif (region 2), and Q178 and E180
designating the E/QxE motif (region 3) in mARTs.
17
interacts with the target macromolecule while the E/Q residue catalyzes the ADP-ribosyl
transfer94. This loop is also flexible and has two conformations upon NAD+ binding (an ―in‖ and
―out‖ conformation). Once NAD+ is bound, the aromatic and E/Q-position residues are in the
―out‖ conformation so that they can interact with their target molecule. This is the case for C3
toxin, where the tyrosine and glutamine residues interact with RhoA94. The PN and ARTT loops
are responsible for NAD+ binding and substrate recognition.
1.5
Mechanism of mART toxins
The mechanism of the transferase reaction is still unclear despite many attempts to
elucidate it. Multiple research groups have proposed a number of mechanisms whereby mART
enzymes catalyze the reaction9,47,48,51. Three groups have co-crystallized a mART enzyme with
its target protein51,59,94; however, static crystal structures are not sufficient to reveal the
mechanism. Instead, molecular dynamics calculations and molecular mechanics studies are
required to decipher this mechanism95,96.
The active site is located in domain 3 of DT group members (or in the A subunit for CT
group members) and it catalyzes the transfer of ADP-ribose from NAD+ to its target protein. The
reaction mechanism is classified as an SN1 nucleophilic substitution, where a residue from the
target macromolecule attacks the C1 anomeric carbon on the N-ribose ring of NAD+, and the
nicotinamide ring acts as the leaving group47,97 (Fig. 5).
First, NAD+ and the target protein bind to the mART toxin. The primary catalytic
glutamate hydrogen bonds with the 2’ hydroxyl group of the N-ribose ring and arginine (or
histidine for DT members) hydrogen bonds with the adenine ribose ring57,98. A subtle shift of the
loop around NAD+ is thought to promote the release of the nicotinamide moiety and the
formation of an oxocarbenium ion47. This was proposed by Tsuge et al. upon the comparison of
18
+
+
Figure 5. Proposed mechanism for mART toxins
This scheme features an SN1 nucleophilic attack of a protein residue. First, the primary catalytic
Glu hydrogen bonds the 2’ hydroxyl and promotes the cleavage of the C-N glycosidic bond. This
creates an oxocarbenium ion containing an electrophilic anomeric carbon on the N-ribose ring.
Then, rotation of the P-O bond releases enzyme-induced conformational strain and positions the
ribose and the attacking residue (shown as Arg) in proximity. Finally, the target protein residue
is activated by the secondary catalytic Glu to become a stronger nucleophile, and attacks the
electrophilic C1 in the N-ribose ring. The Arg residue is now labeled with ADP-ribose in the
target protein. This figure was modified from Fieldhouse et al.47
19
Iota-actin and ExoA-eEF2 crystal structures59. The oxocarbenium ion is electrophilic at the
anomeric carbon and is, therefore, susceptible to nucleophilic attack. A rotation of the O-P bond
by the N-ribose is believed to position the ribose and moves the attacking residue into closer
proximity for the transfer of the ADP-ribose moiety, which may relieve strain within the
molecule59,99. There is a large distance between the anomeric carbon of the oxocarbenium ion
and the target residue, based upon crystal structures of the enzyme-substrate complex; a rotation
of the O-P bond was suggested as this may also reduce the strain in the oxocarbenium ion and
reduce the distance between these molecules59. Finally, the secondary Glu/Gln acts as a base to
activate the attacking residue on the target protein to create a stronger nucleophile. The target
residue attacks the oxocarbenium ion forming a new glycosidic bond and then dissociates from
the mART toxin. Site-directed mutagenesis was used to replace the catalytic Glu with neutral
residues to prove this residue was required for cleavage of NAD+ and hence the activity of the
mART enzyme39,85,100,101. However, replacing the upstream Glu/Gln prevented only the
transferase reaction; the hydrolysis of NAD+ was still observed, since this residue is not required
for cleavage of the glycosidic bond. In the case of C3cer, variants of the catalytic motif were
generated, Q183E and E185Q, and 45% of glycohydrolase activity was still observed for
Q183E89. The mechanism for general mART catalysis was based on crystal structures of ExoA
and iota toxin51,102. A crystal structure of a mART toxin and a transition-state analogue would
provide considerable insight to the mechanism, but none exists at this time.
1.6
Inhibitors of mART toxins
Before antibiotics were available, antibodies or anti-toxins were used to treat bacterial
infections. This was the treatment used for diphtheria in the early 1900’s. When the animal was
given a non-lethal dose of the toxin, an antibody against the toxin was produced, and then serum
20
containing antibodies were collected from the animal. The antibodies bound and neutralized DT,
eliminating symptoms in patients103. In the present day, many bacterial infections for which a
vaccine is not available are treated with antibiotics which kill not only the infectious bacteria, but
also avirulent species. Recently, the focus is shifting again in the search for another treatment
strategy to combat bacterial infections.
The antivirulence strategy uses small molecule compounds that target virulence factors
produced by a pathogenic bacterium. mART toxins are good drug targets since they are often
required and important in helping to establish infection, damage host cells or disrupt immune
responses, and participate in several bacterial disease states33. The Merrill group has employed
several drug discovery and screening approaches to identify lead inhibitors for Scabin, Certhrax,
C3larvin, Plx2A, Vis, VahC, Photox, Cholix, and ExoA, which have recently been
characterized39,80–84,93,104.
Crystal structures of mART toxins in complex with substrates/ligands can be used to
generate virtual screens to model inhibitor candidates within the active site and can yield a list of
potential inhibitors for in vitro testing against the target toxin/enzyme (Fig. 6). It was
demonstrated that after a library of compounds was tested for the inhibition of ExoA, multiple
inhibitors contained moieties resembling the nicotinamide group of NAD+. The best inhibitor
was crystallized with ExoA and the mART-inhibitor crystal structure showed that the inhibitor
was indeed bound within the NAD+-binding pocket105. Inhibitors that resemble NAD+ bind
mARTs using ring-stacking interactions of aromatic residues in the PN loop and near the
catalytic site with the nicotinamide moiety33. Successful inhibitors are then tested in vivo in
mammalian cells to determine the efficacy of the inhibitor in a target cell system to protect
21
PJ34
M19
P6
V30
P1
1,8-NAP
+
Nicotinamide
NAD+
Figure 6. Examples of inhibitors of mART toxins
These structures are small molecule compounds effective against mART toxins. P1, P6, V30,
PJ34 and 1,8-NAP show inhibitory properties against mART toxins33. M19 was one of the
inhibitors from the M-series that was tested against VopT glycohydrolase activity. Most of these
inhibitors resemble the structure of nicotinamide moiety in NAD+ and include a benzamido
group fused with a hetero ring structure previously observed for ExoA inhibitors33.
22
against the mART toxin. This research is a proof-of-principle approach to select lead compounds
with potential for virulence inhibition and further development.
1.7
Discovery of novel mARTs
To discover novel mART toxins, Fieldhouse et al. conducted an in silico search based on
fold-recognition and multiple sequence alignments using the Genomic Online Database (GOLD,
www.genomesonline.org)47. This database now contains over 9000 fully sequenced bacterial
genomes and these can be searched for specific sequences or genes. Within the CT or DT group,
there is only about 15% homology of shared sequence in the catalytic region56, so reliance on
primary sequence had to be reduced to account for the lack of sequence homology. Foldrecognition was used since the tertiary structure of mARTs in the catalytic domain has a higher
degree of homology56. This is logical, as the NAD+-binding pocket is a highly conserved motif
throughout all mARTs in order to bind this common substrate in a conformation conducive to CN bond scission and nucleophilic substitution.
Characteristics, including structure, secretion, cell entry, activation, substrate binding,
and reaction mechanism, were proposed using bioinformatics approaches, and then in vivo toxin
activity was tested using a yeast-growth defective assay47. This test ensured that the putative
mART enzyme caused cytotoxicity of eukaryotic cells by observing a defective growth
phenotype in yeast (model) and also ensured that the toxin was a mART since conserved
catalytic mART residues were substituted by site-directed mutagenesis, allowing the growth
phenotype to be restored. Using this technique, several new cytotoxic mARTs were identified,
including VopT from V. parahaemolyticus (unpublished data, Robert Fieldhouse). This is an
important area of research as many mARTs participate in the infection process caused by
bacterial pathogens.
23
1.8
Vibrio parahaemolyticus
Vibrio parahaemolyticus is a halophilic gram-negative bacterium found in coastal waters.
It can cause gastroenteritis and diarrhea in humans after ingestion of undercooked, contaminated
seafood11,13–15,22–27,106,107 4 to 96 hours after consumption, lasting for up to three days14. V.
parahaemolyticus has a short life cycle of 8 to 12 minutes107 which may account for the rapid
development of symptoms. This bacterium is responsible for gastrointestinal disease outbreaks
around the world13,15 and is the leading cause of gastroenteritis16, but it is most prevalent in
Japan, as this country is a large consumer of raw seafood14. However, not all strains of V.
parahaemolyticus cause illness; some are avirulent and hence do not have pathogenicity islands
in their DNA, which often encode for the virulence factor genes associated with infection15,107.
One pathogenic V. parahaemolyticus strain has a 3.29 Mbp genome in the form of two
circular chromosomes107. When compared to the related V. cholerae genome, the second
chromosome of the V. parahaemolyticus strain is much larger and the size difference is likely
due to gene acquisition by horizontal transfer107. A pathogenicity island is located on
chromosome 2 in the V. parahaemolyticus strain, unlike V. cholerae, and because the island has
a different G+C content, it is suggested that the island was acquired recently14. The pathogenicity
island encodes a few virulence factors and they are similar to other bacterial virulence factors.
These include VopP, which is homologous to Yersinia spp. YopP and YopJ, VopC, which is
homologous to necrotizing factor from E. coli, and VopT, which is homologous to ExoT from P.
aeruginosa. TDH is also encoded by pathogenic V. parahaemolyticus and is a thermostable
direct hemolysin that causes hemolysis of blood cells as mentioned earlier24. A collection of
genes for a secretion system similar to Yersinia spp. are also encoded within the pathogenicity
islands26,107. These secretion systems are termed type III secretion systems (T3SS).
24
T3SS can be divided into two systems, T3SS1 and T3SS2, which are found on
chromosome 1 and 2, respectively11,22,107. T3SS1 is found in all strains of V. parahaemolyticus as
it is required for survival13, but T3SS2 is only present in pathogenic strains11,26,107 to ―mediate
bacterial invasion of the host cell‖13. T3SS1 causes cytotoxicity of the host cell while T3SS2
causes enterotoxicity11,13,14,16,23,106. The detailed mechanism of T3SS2 is still elusive, mainly
because in vitro assay systems have not been successful in analyzing T3SS2 activity26. The most
common infections and diseases caused by V. parahaemolyticus are gastroenteritis, septicemia,
and wound infections, which are partially caused by the secretion of VopT into host
cells13,14,106,107.
1.9
VopT and associated proteins
1.9.1
Ras protein
Ras is a small GTPase protein, involved in protein kinase B (PKB) and mitogen-activated
protein kinases (MAPK) cascades, that must be localized to the cell membrane for activity108
(Fig. 7). The Ras protein is ADP-ribosylated by VopT, which leads to cell apoptosis26 (Fig. 8).
This signal transduction protein is partially responsible for regulating cell growth and
differentiation. GDP-bound Ras is inactive while GTP-bound RAS is active and has increased
signalling properties109. GTPase activation proteins (GAPs) inactivate Ras proteins hydrolyzing
the bound GTP to GDP thereby converting Ras into its inactive form. Human Ras (hRas) is
regulated by ADP-ribosylation by PARP-1 in human cells and altered ADP-ribosylation will
change hRas expression110. This change in expression may cause heightened pathway signalling
and lead to cell proliferation. VopT ADP-ribosylates hRas as well but the exact mechanism by
which this ADP-ribosylated Ras causes cell apoptosis is unknown26. Even though Ras is
25
Figure 7. MAPK and PKB pathways
Ras is a signalling protein that is involved in both of these pathways. RAF, RAL-GDS and P13K
are three of the best characterized effectors stimulated by Ras that each stimulates a different
downstream effector involved in different signalling pathways. Ras is ADP-ribosylated by VopT
causing altered signalling properties and eventually cell death. The exact mechanism of how Ras
causes apoptosis is still unknown. This figure was adapted from Coleman et al.109
26
Figure 8. Crystal structure of Ras (PDB:4EFL)
This crystal structure shows human Ras protein bound to phosphoaminophosphonic acidguanylate ester (GNP), a non-hydrolyzable analogue of GTP, and coordinated with Mg2+. HRas
is coloured by secondary structure; α-helices: green, β-sheets: orange, loops: cyan, and GNP
coloured by element.
27
regulated by ADP-ribosylation in human cells, VopT may ADP-ribosylate Ras in a different
location thus causing different signalling capabilities.
Additionally, various Ras variants have been shown to be constitutively activated and
cause cell proliferation, inhibition of apoptosis, and tumorigenesis111. For example, in mammary
epithelial cells, these Ras variants cause incomplete G1 cell cycle arrest and more rapid cell
cycle progression resulting in breast cancer112. These oncogenic mutations of Ras are present in
about 30% of all cancers111. Some clinical trials are being conducted using viruses for repression
of tumors caused by the activated Ras variant. A virion, Reovirus, has been discovered that
causes apoptosis in cells with activated Ras variant and may also induce apoptosis in several
other types of cancer cells113. Disruption of Ras signalling will cause either cell proliferation or
apoptosis. When Ras is modified by VopT, signalling pathways are disrupted and the cell
undergoes apoptosis.
1.9.2
14-3-3 (FAS) protein
Factor Activating exoenzyme S (FAS), a protein from the 14-3-3 family, is ubiquitous in
eukaryotic cells114 (Fig. 9). The 14-3-3 proteins do not have enzymatic activity, but are thought
to activate multiple proteins involved in vesicle transport and antimicrobial function in
macrophages70,114. They can also regulate Akt phosphorylation in intestinal epithelial cells as
part of the Akt/PKB signalling pathway115. The 14-3-3 proteins are found in many human tissues
in multiple forms (β, γ, ɛ, ε, σ, τ, δ) and are widely distributed among eukaryotes, but not
bacteria. This partially explains why ExoS, a FAS-dependent mART enzyme, is not toxic to
bacterial strains114. Because members of the 14-3-3 protein family have so many binding partners
(brain tissue has significant amounts of 14-3-3 proteins), this family could be used as a
therapeutic target for cancer and neurogenerative disorders116. The 14-3-3 proteins are highly
28
A
B
Figure 9. Monomeric crystal structure of 14-3-3 (FAS) protein (PDB: 2O02)
This crystal structure was determined by Ottman et al. who were able to crystallize zeta 14-3-3
protein (FAS) with a small peptide from ExoS that contained the FAS-binding sequence70. A)
The ExoS peptide is coloured blue with the conserved FAS-binding sequence coloured purple.
FAS is coloured by secondary structure; α-helices: green, loops: cyan. B) rotated view of A by
90°.
29
conserved regulatory proteins involved with signalling pathways via protein-protein
interactions117. The 14-3-3 proteins usually interact with their binding partner through
phosphoserine motifs (RSXpSXP), but some binding partners, including human telomerase,
amyloid β–protein precursor, and ExoS, do not require a phosphate group70,118,119. ExoS encodes
a FAS-binding motif which primarily uses hydrophobic contacts to bind the conserved
amphipathic groove of FAS70,119. There are several isoforms of 14-3-3 that activate ExoS to a
similar degree120,121.
1.9.3
VopT
Selectively secreted by the T3SS2, VopT is one of the virulence factors expressed by V.
parahaemolyticus strain RIM221063326. VopT is a 26.8 kDa mART enzyme that requires the
interaction of FAS, a member of the 14-3-3 protein family, to be enzymatically active. VopT
targets Ras, a small GTPase protein involved in cell signalling, which causes cytotoxicity
towards Caco-2 and HCT-8 eukaryotic cells16,26,56. The ADP-ribosylation of Ras is proposed to
prevent proper protein-protein interactions between Ras and its binding partners, disruption of
signalling pathways, and cell apoptosis. However, the mechanism of how this modification alters
downstream signalling of Ras is unknown16,26. VopT is believed to require a chaperone protein,
VocC, to be delivered to the T3SS apparatus23. The VocC gene is close to the VopT gene and is
located within a pathogenicity island that contains T3SS2 genes as well. VopT has high sequence
homology to ExoS and ExoT from P. aeruginosa and requires the binding of FAS to be active;
therefore, it is classified as an ExoS-like mART toxin.
1.9.4
Yeast cytotoxicity assay for VopT
Wild-type VopT was cytotoxic to the yeast cells without induction of its gene. This was
observed since a threshold level of Cu2+ must be present in the media in order to support
30
metabolic processes (eg., Cu2+-containing redox enzymes); therefore, even in the relative
absence of Cu2+, some VopT was expressed (Fig. 10). Addition of a Cu2+ chelator would reduce
the induction of the gene but would harm the yeast cell because some redox enzymes require
Cu2+. The large decrease in cell growth upon expression of VopT was significant since the CUP1
promoter is weak and hence very low levels of the VopT protein were able to disrupt cellular
processes in yeast cells. Furthermore, there is no known repressor for the CUP1 promoter. In the
presence of higher Cu2+ doses, more wild-type VopT was produced and almost completely
inhibited yeast cell growth. Expression levels of catalytic variants of VopT were also assayed to
account for cytotoxicity caused by reasons other than mART enzyme activity (i.e., membrane
interactions, interference of signal pathways, etc.). As expected, the growth-defect phenotype
was restored with the single catalytic variants (E186A and E188A) at 0.25 mM Cu2+; however,
both of these variants showed cytotoxicity at 0.5 mM Cu2+. These variants were able to catalyze
the VopT reaction, albeit at a much slower rate, but their cytotoxic effect was still significant at
0.75 mM Cu2+. The double catalytic variant (E186A/E188A) was much less toxic to yeast at
higher Cu2+ doses. It is noteworthy that at higher Cu2+ doses, there is the appearance of Cu2+
toxicity to overall yeast growth (0.75 mM and higher). Importantly, even with reduced activity,
VopT was still cytotoxic and had a detrimental effect on yeast. These results show that VopT is a
mART toxin that is cytotoxic to yeast due to the presence of its mART enzyme activity;
therefore, VopT has shown to be a eukaryotic cell toxin.
Previous work on VopT has been conducted in our lab, including preliminary MichaelisMenten plots of VopT transferase activity and an initial search for inhibitors of VopT transferase
activity. However, activity assays have not given consistent results, largely due to protein
folding/stability issues. A hand-crafted homology model of ExoT, prepared in our lab, was based
31
CuSO4 Concentration
Figure 10. Growth of S. cerevisiae expressing wild-type or variant VopT proteins
Wild-type VopT shows cytotoxicity without the addition of exogenous CuSO4 and the catalytic
variants also show cytotoxicity at higher levels of copper induction. VopT wild-type, dark grey;
E186A variant, light grey; E188A variant, black; E186A/E188A double variant, white. Error bars
show the SD of eight replicates. The 100% horizontal line represents growth of yeast expressing a
non-toxic aminotransferase protein. Experiment performed and figure prepared by Zachari Turgeon
(unpublished data).
32
on several mART templates and optimized with data from the literature and provides important
insights into VopT structure and function such as why ExoS-like mARTs require the binding of
FAS for activation (Fig. 11).
VopT is an ExoS-like mART enzyme from V. parahaemolyticus and contributes to
infection and cytotoxicity caused by this bacterium. Inhibitors effective against VopT activity
may provide a useful approach to prevent and or treat infections caused by V. parahaemolyticus.
The research objective of this thesis work is to characterize VopT as a putative virulence factor of V.
parahaemolyticus. VopT was expressed and purified, the assay conditions were optimized, a
thorough study of its kinetics and substrate binding was completed, and inhibitors of its enzymatic
activity were identified. This was done using pre-existing protocols already implemented in our
lab.
33
Figure 11. Homology model of ExoT, an ExoS-like mART
This structure was based on several mART templates and fine-tuned to experimental ExoS data
obtained from the literature. Unlike VopT, ExoT contains a GAP domain shown in blue. The
mART domain is shown in red and the short green segment is the FAS-binding motif located at
the C-terminus. The homology model was prepared by Dr. Miguel Lugo (unpublished data)
using the MOE software suite (2015).
34
Chapter 2: Materials and methods
35
2.0
Materials
Materials were purchased either from Thermo Fisher (Waltham, MA) or Sigma-Aldrich
(St. Louis, MO). FPLC purifications used an Amersham Biosciences FPLC ÄKTA system
placed in a 4 °C refrigerated cabinet. The FPLC system used the ÄKTA UNICORN 4.1 system
control software.
2.1
Yeast cytotoxicity assay
Saccharomyces cerevisiae is quite versatile as a eukaryotic expression model and can be
used in cell-based assays to identify toxic proteins by observing a growth-defect phenotype upon
intracellular expression of the putative toxin genes122. Briefly, S. cerevisiae was grown to midlog phase and was co-transformed with a linearized pRS415 Cup1 plasmid and an insert
containing the putative toxin gene flanked with homologous regions to the plasmid. The insert
and plasmid were recombined in vivo by the yeast cell to produce a plasmid which contained the
toxin gene under the control of a CUP1 promoter. Successful transformants were grown in liquid
culture in 96-well plates with various amounts of CuSO4 to induce expression. If the protein was
toxic to the host yeast cells, a growth-defect phenotype was observed. This work was previously
performed by Zachari Turgeon, a former lab member.
2.2
Heat-shock transformation of E. coli BL21 cells
BL21 cells were obtained from Structural Genomics Consortium (SGC) and stocks of
cells were previously made by a student in the lab using the RbCl2 method123. Plasmids were
transformed into Escherichia coli BL21 cells unless otherwise stated. E. coli BL21 cells and
plasmid DNA were thawed on ice in microfuge tubes. Plasmid DNA, about 100 ng, was added to
an 100 µL aliquot of BL21 cells (3.0 × 109 cells/mL) with gentle mixing. The cells were
incubated on ice for 40 min before heat shocking at 42 °C for 45 s. Cells were immediately
36
incubated on ice for 2 min and then Luria broth (LB), 800 µL, was added to the microfuge tube.
Cells were incubated at 37 °C for 1 h with shaking and then a 120 µL aliquot of cell suspension
was pipetted and spread onto LB plates containing 30 µg/mL kanamycin. Plates were left for 20
min and were then inverted and incubated at 37 °C overnight.
2.3
Growth of cells and gene expression
Transformed BL21 cells were scraped from LB plates and transferred to 50 mL LB media
flasks containing the appropriate antibiotic. The flasks were incubated for 90 min at 37 °C with
shaking and then 25 mL cell suspension was transferred to 1 L yeast extract tryptone (2xYT)
medium flasks containing 30 µg/mL kanamycin. The 2xYT medium flasks were incubated at 37
°C with shaking until OD600 = 0.6 – 1.0 was reached and gene expression was induced with the
addition of isopropyl β-D-1-thiogalactopyranoside (IPTG). The amount of IPTG added depended
on the protein being overexpressed (Table 1). Cells were collected by centrifuging at 3750 × g
for 12 min, resuspended in lysis buffer (25 mM TRIS pH 7.5, 500 mM NaCl, and 10% glycerol),
and stored at –20 °C until purification.
2.4
Sonication
The Heat Systems Misonex Sonicator XL 2020 instrument was used to lyse the cells. Cell
suspensions in lysis buffer were kept on ice during the entire process. The sonicator was used at
power level 6 with 20% power and the cell suspension was sonicated for 45 s six times. A 45 s
pause after each sonication prevented sample overheating and provided gentle mixing of the
sample.
37
2.5
Purification of His6-tagged proteins
VopT, VocC, hRas, FAS, and ExoS were purified using immobilized metal affinity
chromatography. Columns, gradients, and buffers are described below.
2.5.1
Purification of VopT and VopT variants
The gene encoding VopT was previously cloned (Dawn White) into a pET-H6 plasmid
which contains an N-terminal hexahistidine tag (His6-tag). VopT transformed cells were grown
at 37 °C for 3 – 4 h after induction with 1 mM IPTG while VopT tryptophan variants were
grown at 16 °C for 16 h with shaking after induction. The cells were resuspended in 50 mM
TRIS pH 7.6, 500 mM NaCl, 10% glycerol. It was found to be essential to use Fisher Brand urea
for the following purification as the BioBasic brand was not able to resolubilize VopT for
purification. Most VopT variants were previously made by Dawn White while Madison Wright
generated Y144W and Y191W variants using site-directed mutagenesis.
Before sonication, 1 mg DNAse, 1 mg CHAPS, and 0.2 mM PMSF were added to the
cell suspension. After sonication, the sample was centrifuged at 27,000 × g for 50 min. The
supernatant (supernatant # 1) was discarded while the pellet was resuspended with mixing in 50
mM TRIS pH 8.0, 100 mM NaCl, 2 mM EDTA, 0.5% deoxycholate, 0.5 mg/mL lysozyme.
Lysozyme was dissolved with mixing 10 min before use. The sample was centrifuged at 13,000
× g for 20 min. The supernatant (supernatant # 2) was discarded and the pellet was resuspended
with mixing in 50 mM TRIS pH 8.0, 1 M urea. The sample was incubated on ice for 20 min and
then centrifuged at 13,000 × g for 20 min. The supernatant (supernatant # 3) was discarded and
the pellet was resuspended in 50 mM TRIS pH 8.0, 2 M urea. The sample was incubated for 20
min on ice and then centrifuged at 13,000 × g for 20 min. The supernatant (supernatant # 4) was
discarded and the pellet was resuspended in 25 mM TRIS pH 8.0, 500 mM NaCl, 6 M urea
38
Table 1. Expression conditions of VopT and associated proteins
Protein
Growth
temperature (°C)
37
Growth time (h)
Induction OD600
IPTG (mM)
3–4
1.0
1
VopT
VopT Trp16
16
1.0
1
variants
37
3
0.6
1
FAS
16
16
1.0
0.4
hRas
16
16
1.0
1
VocC
37
3
1.0
1
ExoS
To maximize protein expression, proteins were grown under different conditions. VopT-Trp
variants grew slower than wild-type VopT so they were grown for 16 h.
39
(buffer 1). The sample was incubated on ice for 30 min and then centrifuged at 27,000 × g for 30
min. The pellet was discarded and the supernatant was loaded onto a 5 mL HiTrap IMAC HP
column (GE Healthcare, Uppsala, Sweden) equilibrated with buffer 1. First, a refolding protocol
was initiated, which reduced the urea concentration from 6 M to 0 M using a linear gradient over
300 mL of buffer at a flow rate of 0.5 mL/min. Then, a FPLC elution protocol was initiated that
increased the imidazole concentration using a linear gradient from 0 to 250 mM over 180 mL
while eluting VopT. Purity was assessed on a SDS-PAGE gel including the flow-through, all
supernatants, and VopT-containing fractions. VopT-containing fractions were pooled and
dialyzed into 25 mM TRIS pH 8.0, 500 mM NaCl and then were flash-frozen in liquid N2 and
stored at –80 °C.
2.5.2
Purification of VocC
The gene encoding VocC was previously cloned into a pET-TEV plasmid which contains
an N-terminal His6-tag (Tom Keeling). VocC transformed cells were grown at 16 °C for 16 h
with shaking after induction with 1 mM IPTG at an OD600 = 1.0. Cells were pelleted and
resuspended in 50 mM TRIS pH 7.6, 500 mM NaCl, 10% glycerol. Before sonication, 1 mg
DNAse, 1 mg CHAPS, and 0.2 mM PMSF were added to the cell suspension. The sonicated
sample was centrifuged for 50 min at 27,000 × g and then loaded onto a 5 mL HiTrap IMAC HP
column (GE healthcare, Uppsala, Sweden). His-tagged VocC protein was eluted with an
increasing, linear imidazole gradient from 0 to 250 mM in 50 mM TRIS pH 7.6, 500 mM NaCl,
5% glycerol, 2 mM DTT buffer. Purity was assessed using SDS-PAGE. Fractions containing
pure VocC were collected and dialyzed into 25 mM TRIS pH 7.6, 500 mM NaCl, 5% glycerol, 2
mM DTT and stored at –80 °C.
40
2.5.3
Purification of hRas
The gene encoding hRas was previously cloned into a pET-TEV plasmid containing an
N-terminal His6-tag as a gift from I. Stagljar (Biochemistry Dept., Univ. of Toronto). HRas
transformed cells were grown to an OD600 = 1.0 and then induced with 400 µM IPTG. Cultures
were grown for 16 h with shaking at 16 °C. Cells were then spun down and resuspended in lysis
buffer, 25 mM TRIS pH 7.6, 500 mM NaCl, 10% glycerol. Cell suspensions were flash-frozen
with liquid N2 and stored at –20 °C.
Before sonication, 1 mg DNAse, 1 mg CHAPS, and 0.2 mM PMSF were added to the
cell suspension. Samples were centrifuged at 27,000 × g for 50 min and the supernatant was
collected. MgCl2 was added to the lysate for a final concentration of 5 mM and then mixed at 4
°C for 15 – 30 min before loading onto a bench-top chelating immobilized nickel Sepharose fast
flow resin (GE Healthcare, Uppsala, Sweden). The column was handmade with 2 mL of resin
and purification was performed at 4 °C. Lysate was passed over the column once and washed
with 10 column volumes of buffer 2 (25 mM TRIS pH 7.6, 500 mM NaCl, 2% glycerol). Then,
the column was washed with 5 column volumes of 10%, 25%, and 40% buffer 3 (buffer 2
supplemented with 250 mM imidazole). HRas was eluted with 15 mL of 100% buffer 3. SDSPAGE was performed to assess the purity. HRas-containing fractions were pooled and dialyzed
into 25 mM TRIS pH 7.6, 50 mM NaCl, 2 mM DTT.
Dialyzed hRas was then loaded onto a 5 mL HiTrap Q HP column (GE Healthcare,
Uppsala, Sweden) equilibrated with dialysis buffer. The column was washed with 25 mM TRIS
pH 7.6, 50 mM NaCl, 2 mM DTT and hRas was eluted with an increasing linear gradient of 25
mM TRIS pH 7.6, 500 mM NaCl, 2 mM DTT over 200 mL. Purity was assessed using SDSPAGE. Fractions containing pure hRas were dialyzed again into 25 mM TRIS pH 7.6, 150 mM
41
NaCl, 2 mM DTT, 5% glycerol. Aliquots of purified protein were then flash-frozen and stored at
–80 °C.
2.5.4
Purification of FAS
The gene encoding FAS was previously cloned into pET-15b vector containing an N-
terminal His6-tag as a gift from I. Stagljar (Biochemistry Dept., Univ. of Toronto). FAS
transformed cells were grown to an OD600 = 0.6, induced with 1 mM IPTG, and grown at 37 °C
for 3 h. Cells were then spun down and resuspended in lysis buffer, 25 mM TRIS pH 7.6, 500
mM NaCl, 10% glycerol. Cell suspensions were flash-frozen with liquid N2 and stored at –20 °C.
FAS was purified using a similar protocol as hRas purification above except that the
chelating Sepharose resin contained immobilized Zn2+ instead of Ni2+ and MgCl2 was not mixed
with the lysate prior to purification. The column was also handmade 2 mL of resin and the
purification was performed at 4 °C. These differences were because FAS does not coordinate
Mg2+, unlike hRas, and immobilized Zn2+ columns gave a higher protein yield. Also, anionexchange chromatography was not performed with FAS and the protein was dialyzed into 25
mM TRIS pH 7.5, 150 mM NaCl, 0.5 mM DTT before flash-frozen and stored at –80 °C.
2.5.5
Purification of ExoS
The gene encoding ExoS was previously cloned into a pET-H6 plasmid which contains
an N-terminal His6-tag (Tom Keeling). ExoS transformed cells were grown at 37 °C for 3 h with
shaking after being induced with 1 mM IPTG at an OD600 = 1.0. Cells were pelleted and
resuspended in 50 mM TRIS pH 7.6, 500 mM NaCl, 10% glycerol. Before sonication, 1 mg
DNAse, 1 mg CHAPS, and 0.2 mM PMSF were added to the cell suspension. Samples were
centrifuged at 27,000 × g for 50 min and the supernatant was collected. Purification was
performed using a bench top chelating immobilized nickel Sepharose fast flow resin (GE
42
Healthcare, Uppsala, Sweden). The column was handmade with 2 mL of resin and purification
was performed at 4 °C. Lysate was passed over the column twice before washing the column
with buffer 4 (25 mM TRIS pH 8.0, 500 mM NaCl, 5 mM imidazole) with 10 column volumes.
Then, the column was washed with 5 column volumes of 10%, 20%, 40% buffer 5 (buffer 4 with
250 mM imidazole). ExoS was eluted with 15 mL 100% buffer 5 and ExoS was dialyzed into 25
mM TRIS pH 7.9, 50 mM NaCl. Dialyzed ExoS was then loaded onto a 5 mL HiTrap Q HP
column (GE Healthcare, Uppsala, Sweden) equilibrated with dialysis buffer. The column was
washed with 25 mM TRIS pH 7.9, 50 mM NaCl and then ExoS was eluted using an increasing
linear gradient of 25 mM TRIS pH 7.9, 1 M NaCl over 200 mL. Purity was assessed using SDSPAGE. Fractions containing pure ExoS were dialyzed again into 25 mM TRIS pH 7.9, 50 mM
NaCl, concentrated to 1 mg/mL, and stored at –80 °C.
2.6
Thermal shift analysis
The Applied Biosystems StepOnePlus™ RT-PCR instrument was used to test samples in
a 96-well plate. Protein samples were tested at concentrations between 0.25 and 0.5 mg/mL with
1X SyproOrange® dye. SyproOrange® is a hydrophobic fluorophore which is quenched in
aqueous solution. Upon denaturation or unfolding, the dye will bind the exposed hydrophobic
regions of proteins and dye fluorescence will increase. Protein samples were tested in triplicate.
Samples were heated from 4 – 85 °C using a linear gradient over 55 min while the fluorescence
of the SyproOrange® dye was monitored in the Cybergreen channel with excitation and emission
wavelengths of 470 and 570 nm, respectively. Applied Biosystems StepOne Software v2.3 was
used to calculate the first derivative of the change in fluorescence and a peak indicated the
melting point (Tm) of the proteins. The Tm is defined as the midpoint temperature of the
transition between folded and unfolded protein.
43
2.7
Activity assays
All activity assays were performed on a Cary Eclipse fluorescence spectrometer (Varian,
Palo Alto, California) at room temperature with plastic Eppendorf cuvettes (VWR, Radnor, PA)
with a 10 mm path length and a voltage of 600 volts. The Cary Eclipse monitored the change in
fluorescence over time for activity assays and allowed the quenching of fluorescence to be
observed for binding assays (section 2.8). Only one sample at a time was tested. All activity
assays were monitored using etheno-NAD+ (ɛNAD), a fluorescent NAD+ analog. Upon cleavage
of the nicotinamide N-glycosidic bond, fluorescence of ɛADP-ribose increases 10-fold due to
reduced intramolecular quenching of the etheno group124. Excitation and emission wavelengths
of 305 and 405 nm, respectively, were used with a 5 nm band pass filter.
2.7.1
ɛAMP standard curves
Ethenoadenosine-monophosphate (ɛAMP) standard curves were used to convert the
enzyme activity from fluorescence intensity (F.I.)/min to µM/min. From a stock of ɛAMP,
samples ranging between 0 and 10 µM ɛAMP were made in triplicate with assay buffer (25 mM
TRIS pH 8.0, 500 mM NaCl) and monitored for 5 min on the Cary Eclipse. The average
fluorescence intensity for each sample was plotted against ɛAMP concentration. The inverse
slope was multiplied by the slope generated from the Cary Eclipse to calculate the formation of
ɛADP-ribose in molar concentrations (µM/min).
Eq. 1
Eq. 2
44
ɛAMP curves were generated to convert the observed slopes of activity for VopT samples
into enzymatic reaction velocities. Figure 12 shows two curves used to calibrate change in
fluorescence to change in ɛNAD+ concentration in units of µM/min. These curves were
generated in separate experiments to show that the calculated slopes were reproducible over
time.
2.7.2
Optimizing protein quantities
The amounts of FAS and hRas incubated with VopT were varied to observe the effect on
glycohydrolase (GH) and ADP-ribosyltransferase (ADPRT) activity, respectively. All samples
were tested at 100 µM ɛNAD, since this is the saturating ɛNAD concentration for VopT
determined in Results 3.4.4, and protein mixtures were incubated for 20 min at room temperature
to allow protein-protein interactions to form. The concentrations of FAS and hRas at which
VopT enzymatic activity reached a maximum were then used for all further GH and ADPRT
reactions unless otherwise stated.
2.7.3
Incubation time
Since VopT requires the presence of FAS to show GH activity, determined in Results
3.4.1, incubation time was investigated to ensure that VopT and FAS were allowed to reach
equilibrium for binding. In a volume of 15 µL, 6 µM VopT and 12 µM FAS were incubated
together for a specified amount of time at room temperature in 25 mM TRIS pH 8.0, 500 mM
NaCl. Then, a 10 µL sample of the incubation mix was added to 50 µL ɛNAD solution in a
cuvette. Final concentrations were 100 µM ɛNAD, 1 µM VopT, and 2 µM FAS.
45
Figure 12. Examples of ɛAMP curves used to calculate enzymatic activity
These ɛAMP curves had slopes of 19.280 and 19.222, and R-squared values of 0.997 and 0.995,
respectively. Error bars are the SD of 4 measurements.
46
2.7.4
Effect of VocC on activity
VocC is hypothesized to be a chaperone to some effector proteins secreted through the
T3SS2 system, including VopT, VopC, and VopL23. It was investigated whether VocC had an
effect on VopT GH activity. In a volume of 15 µL, 6 µM VopT, 12 µM FAS, and a variable
amount of VocC were incubated together for 20 min at room temperature in 25 mM TRIS pH
8.0, 500 mM NaCl. Then, 10 µL of the incubation mix was added to 50 µL ɛNAD solution in a
cuvette and then activity was measured. Final concentrations were 100 µM NAD, 1 µM VopT, 2
µM FAS, and 1 – 8 µM VocC.
2.7.5
Glycohydrolase and transferase activity – Michaelis-Menten analysis
For GH tests, VopT and FAS were incubated together for 20 min at room temperature in
VopT assay buffer (25 mM TRIS pH 8.0, 500 mM NaCl). Then, a 10 µL aliquot of this
incubation mixture was added to the cuvette which contained between 0 and 100 µM ɛNAD for
final concentrations of 1 and 2 µM for VopT and FAS, respectively. The concentration of ɛNAD
was varied to generate Michaelis-Menten curves and provide values for KM and kcat. ADPRT
assays were performed with a saturating concentration of ɛNAD (100 µM) and in the presence of
hRas in the protein incubation mixture. VopT and FAS sample stocks were diluted 10X to allow
hRas to reach higher concentrations in comparison to VopT and FAS. Michaelis-Menten curves
for ADPRT activity were produced by varying the hRas between 0 and 40 µM while holding the
concentration of ɛNAD constant at a saturating level.
2.7.6
Metal dependence screen
To investigate whether VopT is activated by metal ions, GH and ADPRT activities were
tested in the presence of divalent metal ions. A final concentration of 5 mM was used for all
metal salts dissolved in assay buffer.
47
2.7.7
Inhibitor screen
Similar to the metal dependence screen described previously, GH activity was tested in
the presence of potential inhibitors. Inhibitors were dissolved in 100% DMSO so the effect of
DMSO on VopT glycohydrolase activity had to be investigated first. DMSO was added to VopT
activity samples up to a final content of 5%. Then inhibitors were tested at a concentration of 100
µM and DMSO content of 5%. The P6- and M-series of inhibitors were previously developed by
our lab based on NAD+-bound mART crystal structures and designed to be competitive
inhibitors. These series were tested for VopT inhibition. V30 and PJ34 are effective against
many mART toxins so they were tested against VopT activity as well33,83. Then, IC50 values
were determined for some inhibitors. Concentrations of VopT, FAS, and ɛNAD were held
constant while inhibitor concentration was varied. IC50 values were calculated using Origin Pro
8.0 (Northampton, MA).
2.7.8
Effect of boiling on VopT activity
Previously in our lab, VopT was boiled and then a sample was run on a SDS-PAGE gel.
This was conducted to test whether VopT would appear as one conformation, instead of
monomers and dimers, on the gel after boiling. However, activity of this boiled sample was not
tested. About 600 µL of VopT (1 mg/mL) was incubated in boiling water (100 °C) for 1 h.
Aliquots of 50 µL were taken at the specified time intervals, between 1 and 60 min, and kept on
ice until activity could be tested. VopT (6 µM) and FAS (12 µM) were incubated in a total
volume of 15 µL for 20 min at room temperature. An aliquot, 10 µL, of the incubation was
added to 50 µL ɛNAD in a cuvette to initiate the reaction. Final concentrations were 1 µM VopT,
2 µM FAS, and 100 µM ɛNAD. Even though protein precipitate was observed upon boiling, the
volume of VopT incubated with FAS was kept the same.
48
2.8
Binding experiments
Binding assays were performed on the Cary Eclipse with excitation and emission
wavelengths 295 and 340 nm, respectively, and band pass = 5 nm. Tyrosine fluorescence binding
assays used excitation and emission wavelengths of 290 and 305 nm, respectively. N-Acetyl
tryptophanamide (NATA) correction titrations were performed to account for inner-filter effects.
2.8.1
NAD+-binding
Tryptophan-variants of VopT were generated using site-directed mutagenesis to
introduce a tryptophan residue in place of a Tyr to determine binding constants (Kd) using
tryptophan fluorescence. VopT Y191W variant, 5 µM, samples were prepared in 25 mM TRIS
pH 8.0, 500 mM NaCl and titrated with NAD+ for final concentrations 0.5 µM – 1.5 mM.
2.8.2
FAS binding
The Kd for FAS binding VopT was investigated. FAS contains tryptophan residues while
VopT does not, making this procedure possible. FAS was diluted in assay buffer (25 mM TRIS
pH 8.0, 500 mM NaCl) to a concentration of 5 µM in quartz cuvettes. Samples were then titrated
with VopT protein for final concentrations 0.06 – 5.2 µM.
2.8.3
VocC binding
The Kd for VocC binding VopT was determined using tryptophan fluorescence. The
Y191W variant was diluted in assay buffer (25 mM TRIS pH 8.0, 500 mM NaCl) to a
concentration of 5 µM in quartz cuvettes. Samples were then titrated with VocC protein for final
concentrations 1.0 nM – 1.5 µM.
49
2.9
Origin fitting
Activity assays were fit to the Michaelis-Menten equation using OriginPro v8.0.
Michaelis-Menten curves were conducted three times and then the three data slopes for each
concentration of ɛNAD were averaged. This average was converted to activity using equations 1
and 2 (Page 42) and the standard deviation was calculated for the average. Then, the average and
the standard deviation was put into OriginPro and fitted to the Michaelis-Menten equation.
Binding constants were calculated using a similar technique. Three fluorescence values
were obtained from each concentration of NAD+, VocC, or wild-type VopT, depending on the
binding assay. The 1 – relative fluorescence value was calculated and then, all three values were
averaged and the standard deviation was calculated. The average and standard deviation was put
in OriginPro and fitted to one site-binding equation.
50
Chapter 3: Results and discussion
51
3.1
Purification of His6-tagged proteins
Recombinant forms of VopT, VocC, hRas, FAS, and ExoS were all purified using
immobilized-metal-affinity chromatography (IMAC) after being overexpressed in E. coli BL21
cells. Protein samples after each step of purification were run on a SDS-PAGE gel to assess
purity.
3.1.1
Purification of wild-type VopT and VopT variants
Wild-type and variants of VopT were isolated from cell pellets by resuspension in buffers
containing 1 – 6 M urea. No VopT-associated protein purifications involved buffers containing
urea. The final VopT supernatant was subjected to IMAC. Interestingly, VopT was present in a
monomeric and dimeric form when run on a SDS-PAGE gel (Fig. 13). It is not known why this
occurred. VopT-containing fractions were dialyzed, concentrated, and stored at –80 °C.
Purification yielded about 8 mg protein/L culture of wild-type VopT whereas VopT variants only
yielded about 5 mg protein/L culture.
ΔN64 was the only VopT variant with a slightly different purification procedure; a
significant amount of ΔN64 was present in one of the supernatants, so this fraction was
extensively dialyzed to remove urea from the buffer and was not subjected to IMAC purification.
Figure 14 shows the purity of this VopT variant.
3.1.2
Purification of VocC
VocC was purified from cell lysate using IMAC and then subjected to size-exclusion
chromatography (Fig. 15). Purified VocC was dialyzed, concentrated, and stored at –80 °C.
Purification yielded about 10 mg protein/L culture.
52
1
2
3
4
5
6
75 kDa
Dimer, 53.6 kDa
50 kDa
Monomer, 26.8 kDa
25 kDa
Figure 13. Recombinant wild-type and variant VopT purification
Lanes 1 – 4 contain the supernatants of resuspended pellets, lane 5 is the final supernatant that
was loaded onto the IMAC column, and lane 6 is eluted VopT. Some VopT was present in the
last unused supernatant yet a significant amount was present in the ―lysate‖.
53
1
2
3
4
5
75 kDa
50 kDa
25 kDa
ΔN64 Monomer, 20 kDa
Figure 14. Recombinant ΔN64 purification
Lanes 1 – 4 contain supernatants of resuspended pellets. Lane 5 is the final supernatant that is
normally loaded onto the IMAC column; however, the fraction shown in Lane 4 was collected as
it contained the largest amount of ΔN64 and was the purest sample.
54
1
2
3
75 kDa
50 kDa
25 kDa
VocC, 16.9 kDa
Figure 15. Recombinant VocC purification
VocC was purified using IMAC. Lane 1 contains the flow-through sample, lane 2 is concentrated
VocC after IMAC, and lane 3 is purified VocC after size-exclusion chromatography.
55
3.1.3
Purification of hRas
HRas was isolated from cell lysate using bench-top IMAC at 4 °C and further purified
using anion-exchange chromatography. Some proteolysis was observed even though protease
inhibitors were present in the cell lysate as the hRas appears as two closely migrating bands after
anion-exchange purification (Fig. 16). Eluted hRas was dialyzed, concentrated, and stored at –80
°C. Purification yielded about 4 mg protein/L culture.
3.1.4
Purification of FAS
FAS was isolated from cell lysate using bench-top IMAC at 4 °C. No further purification
was required (Fig. 17). Eluted FAS was dialyzed, concentrated, and stored at –80 °C.
Purification yielded about 30 mg protein/L culture.
3.1.5
Purification of ExoS
ExoS was isolated from cell lysate using bench-top IMAC and further purified using
anion-exchange chromatography (Fig. 18). Eluted ExoS was dialyzed, concentrated, and stored
at –80 °C. Purification yielded about 3 mg protein/L culture.
3.2
Thermal shift analysis using RT-PCR
Protein samples were subjected to thermal shift analysis (TSA) to observe if proteins
were properly folded. HRas, FAS, and VocC gave reproducible and sharp peaks resulting in
confident Tm estimates at which the protein likely cooperatively unfolds. This suggests that these
proteins are in one conformation and are hypothesized to be properly folded. However, not all
proteins, whether they are properly folded or not, will give reproducible, sharp Tm values. This
may be due, in part, to the presence of multiple conformations, uncooperative unfolding, the
presence of hydrophobic patches on protein surfaces, or the presence of stabilizing agents 125. In
56
1
2
3
75 kDa
50 kDa
25 kDa
hRas, 22.2 kDa
Figure 16. Recombinant hRas purification
HRas was purified using bench-top IMAC and then anion-exchange chromatography. Lane 1
contains the cell lysate, lane 2 is hRas after IMAC, and lane 3 is purified hRas after anionexchange chromatography. Some proteolysis was observed.
57
1
2
75 kDa
50 kDa
FAS, 27.7 kDa
25 kDa
Figure 17. Recombinant FAS purification
FAS was purified using bench-top IMAC. Lane 1 contains the flow through cell lysate, and lane
2 is FAS after IMAC. No proteolysis was observed.
58
1
2
3
75 kDa
50 kDa
ExoS, 49 kDa
25 kDa
Figure 18. Recombinant ExoS purification
Lane 1 contains the cell lysate, lane 2 is ExoS after IMAC, and lane 3 is ExoS after anionexchange chromatography.
59
each of these cases, an increase in background fluorescence of the hydrophobic dye (Sypro®
Orange) may occur, preventing the accurate determination of the melting point. For these
reasons, the instrument and the results were used in a qualitative approach to test whether the
proteins unfolded cooperatively and showed a stable Tm value (above the temperature used in
experiments).
VopT is one such protein that gave high background fluorescence and rather broad peaks
with poor resolution (Table 2, Fig. 19a). All VopT variants had similar thermal profile as wildtype VopT, except ΔN64. ΔN64 had a similar trend as the rest of the VopT variants but the trend
appeared to be shifted to higher temperatures suggesting that it has a more stable overall
conformation. The broad and multi-component melting curve for VopT protein could be due to
the presence of multiple conformations; however, ΔN64 appears as a monomeric protein in
solution unlike wild-type VopT and still possesses multiple peaks. This suggests that the core
structure of VopT also plays a role in uncooperative unfolding and that VopT may have multiple
solution conformations unrelated to its oligomeric state(s).
HRas, FAS, and VocC gave reproducible thermal profiles suggesting they cooperatively
unfold and are properly folded at room temperature (Fig. 19c-e). There is no published report on
TSA data for native hRas, FAS, or VocC proteins so Tm values cannot be compared; however,
the relatively high Tm values and cooperative folding transitions for these proteins suggest that
they are properly folded (Table 2).
3.3
Activity Assays
One main goal of this thesis was to characterize in vitro glycohydrolase and transferase
activity of VopT. However, due to the requirement of an activator protein, FAS, assay conditions
first had to be optimized for protein concentration and incubation time with FAS.
60
Table 2. Melting temperatures of VopT and associated proteins
Sample
Melting Point (°C) Comments
WT VopT
Poor resolution
E186A
Poor resolution
E188A
Poor resolution
E186A/E188A
Poor resolution
R122A
Poor resolution
S146/S148
Poor resolution
Y141W
Poor resolution
Y191W
Poor resolution
ΔN64
Poor resolution
hRas
53.9 ± 0.2
Distinct and reproducible peak
FAS
61.4 ± 0.2
Distinct and reproducible peak
VocC
71.8 ± 0.2
Distinct and reproducible peak
All VopT variants were tested on the ThermoCycler as well as the target, activator, and
chaperone proteins of VopT in triplicate. No VopT samples gave a sharp melting point but the
associated proteins did produce sharp, well-defined peaks.
61
A
B
C
D
54 °C
61 °C
62
E
72 °C
Figure 19. Thermal profiles for VopT and associated proteins
Thermal shift profiles of VopT and associated proteins: A) Wild type VopT, B) ΔN64, C) FAS,
D) hRas, E) VocC. Each protein was run in triplicate in the StepOnePlus RT-PCR instrument in
96-well plates and all three runs are shown for each sample. All VopT variants had similar
thermal profiles to the wild type VopT shown above so they were not included.
63
3.3.1
Optimizing protein concentrations
Optimal FAS and hRas concentrations were determined by measuring VopT activity at
various protein concentrations. VopT glycohydrolase activity showed a dose-dependent response
for increasing FAS (Fig. 20). VopT absolutely requires FAS to be active and the current
homology model of ExoT built on using seven mART templates and based on the ExoS
literature, suggests that the FAS-binding sequence is folded into the NAD+-binding pocket (Fig.
21). Then, upon FAS binding, the sequence leaves the binding pocket and facilitates the binding
and cleavage of NAD+. Activity dependence of FAS was also seen with ExoS transferase activity
reported by Fu et al. and activity appeared to be maximal at a ratio of 1:1 (enzyme:FAS) (see
Fig. 4 in Fu et al.)114. This result suggests that members of the ExoS-like family, which rely on
the binding of FAS, may reach maximal activity when FAS is in a stoichiometric ratio of 1:1
with the enzyme. The ExoS literature is silent on this matter as ExoS is the only other member of
this family to show the dose-dependent relationship with FAS114.
As for transferase function, VopT showed maximal activity around 40 µM hRas (Fig.
22). Multiple groups have used 400-fold excess of Ras to ExoS to ensure maximal activity126.
These results show that VopT and ExoS have a different affinity for Ras. Perhaps this is because
ExoS has a wide range of ADP-ribosylation targets, whereas VopT has only been shown to
modify Ras. VopT may have a specialized binding area that is specific for Ras as compared to
ExoS, since ExoS binds multiple proteins. However, VopT transferase activity was only 2-fold
higher than its glycohydrolase activity, ExoS transferase activity was almost 50-fold higher, and
other mART enzymes transferase activities can be over 1000-fold greater93,127. MichaelisMenten (MM) curves for transferase activity were not produced for VopT variants.
64
Figure 20. Optimal FAS concentration for VopT and ExoS activity
VopT has maximal glycohydrolase activity when FAS is at the same concentration as VopT (1
µM). To ensure sufficient protein interaction, subsequent glycohydrolase assays used FAS in
excess of VopT for a final concentration of 2 µM. Error bars shown in A are the SD for triplicate
measurements.
65
Figure 21. ExoT homology model
This homology model was based on seven mART templates with 92% of residues modeled with
greater than 90% confidence (Miguel Lugo, unpublished data). ExoT has 46% sequence identity
with VopT and is proposed to be structurally comparable to VopT since they are both ExoS-like
members. ExoTΔC34 is shown in blue and Vis toxin is shown in red for comparison. NAD+ is
also modeled into this structure and is represented as green sticks. The conserved FAS-binding
motif in ExoT (shown in yellow) is folded into the NAD+-binding site. This prevents the binding
of NAD+ and prevents the reaction from proceeding. Therefore, it is hypothesized that ExoS-like
mART members require the binding of FAS to cause the FAS-binding motif to leave the active
site and allow NAD+ to bind.
66
Figure 22. Optimal hRas concentration
When hRas was incubated with VopT and FAS at room temperature, VopT transferase activity
reached an activity maximum with hRas being 40-fold excess of VopT. VopT was at a final
concentration of 100 nM instead of 1 µM to allow increased hRas concentration in relation to
VopT. KM and kcat values were determined to be 1.545 ± 0.4 µM and 0.63 ± 0.005 min-1,
respectively. Error bars are the SD of triplicate measurements.
67
Glycohydrolase activity of VopT was maximal at 1 µM FAS, while transferase activity
was highest around 40 µM hRas. All glycohydrolase and transferase reactions as reported later in
this thesis used FAS in 2-fold excess and hRas in 40-fold excess, in relation to VopT
concentration, in order to obtain consistent and maximal enzymatic activity.
3.4.2
Incubation time
Since VopT requires FAS as an accessory factor to be enzymatically active, incubation
time of these proteins was investigated to determine the time required for stable binding and
maximal activation. VopT was active without incubation; however, activity slightly increased
with incubation time (Fig. 23). It was decided that VopT and FAS (and hRas for transferase
reactions) would be incubated at room temperature for 20 min before testing activity to allow
sufficient time for the complex to reach equilibrium. ExoS was commonly incubated with FAS
but no data describing the effect of incubation time on activity has been published. ExoS was
routinely incubated for 15 to 30 min at room temperature46,72,126. Therefore, all members of the
ExoS-like family should be incubated with FAS at room temperature for 20 min prior to
measuring activity.
3.4.3
Effect of VocC on VopT glycohydrolase activity
VocC was proposed as a chaperone to deliver VopT to the T3SS2 apparatus in V.
parahaemolyticus23 but the effect, if any, of VocC on VopT activity has not been investigated.
No significant increase in VopT activity was observed in the presence of VocC, so VocC was not
used in any future activity assays involving VopT (Fig. 24). This result was not surprising, since
VocC is expected to bind VopT in the N-terminal region and deliver VopT to the T3SS2 needle
apparatus, which would thread VopT from N- to C-terminus through the T3SS2 needle and into
the host cell cytoplasm23,128. VocC is not delivered into the eukaryotic cell with VopT; it remains
68
Figure 23. Effect of pre-incubation time on VopT glycohydrolase activity
Samples were tested for GH activity with 100 nM VopT and 200 nM FAS. Activity shown above
does not have a strong correlation and does not illustrate that activity is maximized after
incubation. Incubation of proteins for 20 minutes was chosen to use in all future assays since
ExoS is routinely pre-incubated with FAS between 15 and 30 minutes. Error bars are the SD of
triplicate measurements.
69
Figure 24. Effect of VocC on VopT GH activity
Variable amounts of VocC was incubated with 1 µM VopT and 2 µM FAS for 20 minutes before
testing. Assays were conducted in triplicate with VocC ranging between 1 and 7.8 µM. Error
bars are the SD of triplicate measurements.
70
in the bacterial cell along with the chaperone’s other protein effectors, including VopC23. This
means VocC does not have the opportunity to bind or affect VopT function in the eukaryotic
cell. Therefore, it was surmised that it should not have any effect on VopT enzymatic activity.
3.3.4
Glycohydrolase activity
Glycohydrolase Michaelis-Menten curves for VopT and several variants were generated
by varying the amount of ɛNAD (Fig. 25). KM and kcat values are shown in Table 3. Graphs
showed good reproducibility and good fits to the Michaelis-Menten equation when fitted in
OriginPro 8.1 with R-squared values greater than 0.993.
Among the catalytic variants (E186A, E188A, E186A/E188A), the KM did not increase
and enzymatic activity was not significantly reduced as observed for catalytic ExoS variants
(Fig. 25a-d, Table 3)127. E188 in VopT hydrogen bonds the 2’ hydroxyl in the ribose ring of
NAD+, which promotes the cleavage of the nicotinamide bond. E186 in VopT is responsible for
activating the nucleophilic molecule that will attack the oxocarbenium intermediate and accept
the ADP-ribose moiety. It was anticipated that the E188A variant may show increased KM and
lower kcat values, as this glutamate residue is extremely important in enzyme catalysis and
substrate binding; the E186A variant would only have slightly decreased glycohydrolase activity
since it is mostly involved with activating the receptor molecule for transferase activity127. The
KM value increased 5-fold for the ExoS variant E381D and 3-fold for E379D in transferase
assays, so it was expected that the KM for catalytic VopT variants would increase as well129.
However, these results were not observed for any of the catalytic VopT variants. Also, the
activity was not decreased substantially for any of the catalytic variants and theoretically, the
double catalytic variant should have experienced even lower activity than the single E186A or
E188A variants, but this was not observed. Altogether, the kcat for VopT catalytic variants were
71
Table 3. Kinetic values for VopT and VopT variants GH activity
Prep
KM (µM)
kcat (min-1)
% Activity
23.3 ± 1.2
0.74 ± 0.01
Wild-type
100
22.5 ± 1.2
0.31 ± 0.01
E186A
43
27.9 ± 1.7
0.27 ± 0.01
E188A
37
0.44 ± 0.02
E186A/E188A 20.0 ± 1.5
60
17.5 ± 2.3
0.12 ± 0.005
R122A
16
21.8 ± 1.5
0.10 ± 0.001
S146A/S148A
14
26.7 ± 2.2
0.30 ± 0.01
Y144W
42
19.6 ± 1.7
0.36 ± 0.01
Y191W
50
25.2 ± 1.2
0.57 ± 0.02
ΔN64
79
Michaelis-Menten parameters are summarized with percent activity compared to wild-type
VopT. Errors are the SD of triplicate measurements.
72
A
B
C
D
E
F
73
G
I
H
J
Figure 25. Glycohydrolase MM curves for VopT and VopT variants
Michaelis-Menten curves were prepared for a number of VopT variants: A) wild-type, B)
E186A, C) E188A, D) E186A/E188A, E) R122A, F) S146A/S148A, G) Y144W, H) Y191W, I)
ΔN64-VopT. Panel J is an example of residuals after data were fit. These residuals were from the
Y191W GH curve. Curves were fit with OriginPro 8.1 using the general hyperbolic MM model.
R-squared values were above 0.993 in all cases. Error bars are the SD of triplicate measurements.
74
much higher than expected compared to other catalytic mART variants. For example, Iota toxin
experienced a complete loss of activity when the catalytic residues were replaced with alanine100
and C2 toxin had no activity when the catalytic glutamates were substituted with glutamine101.
ExoS E379D variant had 25% wild-type glycohydrolase activity while the E381D variant had
only 0.05%127. Other groups have also shown this trend in activity loss by catalytic
variants85,89,101.
Conserved structural motifs in VopT were modified as well. It was proposed that R122 of
VopT provides structural integrity to the NAD+ binding pocket while the STS motif positions the
NAD+ molecule in the NAD+-binding pocket. These VopT variants showed reduced activity but
similar KM values compared to wild-type VopT activity (Fig. 25ef; Table 3). This was
unexpected, since both of these motifs aid in binding NAD+, so it was expected that variants of
these motifs would have had reduced binding affinity and activity caused by the poorly formed
binding pocket. Kinetic studies of these variants have not been described for ExoS; however,
other mART toxins have had their activity completely abolished upon substitution of the
conserved arginine and the first serine in the S-T-S motif100,101.
Enzymatic activities of the catalytic and structural VopT variants were reduced, but not
as severely as other reported mART variants. In comparison to previously characterized mART
catalytic variants, VopT variants were not as sensitive to single-site mutagenesis. Therefore,
further investigation is required to understand the underlying cause for this difference.
Tryptophan variants were also produced to investigate binding using tryptophan
fluorescence; however, enzymatic activity of these variants was first assessed to determine if the
enzyme retained wild-type activity. Unfortunately, these variants were only 50% active so the
substitution influenced native enzymatic activity (Fig. 25gh; Table 3). The tyrosine residues
replaced with tryptophan were not involved with enzymatic activity, but the substitution may
75
have affected folding of the protein as the tryptophan side chain is bulkier than tyrosine and does
not hydrogen bond with neighbouring residues as tyrosine may. This is highly probable since
Y144 is two residues away from the S-T-S motif and Y191 is two residues from the E-X-E
motif. The tryptophan residues may have disrupted hydrogen bonding with NAD+ or the
orientation of the S-T-S or E-X-E motifs.
As mentioned earlier, VopT possesses a long N-terminal region that extends from the
core of the enzyme. The ΔN64 variant had this region truncated and was tested for activity. As
expected, this variant had a similar KM and retained most of the native enzymatic activity (Fig.
25i; Table 3). Thus, the N-terminal region is not involved in enzymatic activity and is
hypothesized to provide the binding region for VocC, the chaperone protein of VopT.
Overall, the enzyme activity of VopT was much lower than ExoS. This could arise from
purification of VopT from inclusion bodies. The protein was recovered from cell lysates as an
unfolded protein and it had to be refolded during the purification procedure. However, another
explanation is that VopT may possess intrinsically lower enzyme activity than ExoS.
Unfortunately, there is no structure of any member of the ExoS family as they have so far all
proved recalcitrant to protein crystallization.
3.3.5
Metal dependence
Various divalent metal salts were added to wild-type VopT at a final concentration of 5
mM and the samples were tested for glycohydrolase activity. Most of the metals showed
significant inhibition of glycohydrolase activity, even though some of these metal ions are
commonly coordinated in enzyme active sites (Fig. 26a).
76
A
120
100
% Activity
80
60
40
20
0
None
ZnCl2
CuCl2
ZnSO4
CuSO4
CoSO4
NiSO4
CaCl2
MgCl2
MnCl2
EDTA
-20
B
120
100
% Activity
80
60
40
20
0
-20
None
EDTA
MgCl2
NiSO4
ZnCl2
Figure 26. The effect of metal salts on VopT and ExoS glycohydrolase activity
Relative activity is represented as % activity compared to the wild-type enzyme activity.
Samples were conducted in triplicate with a final concentration of 5 mM divalent metal. Both
VopT (A) and ExoS (B) were inhibited by zinc and nickel salts. Error bars are the SD of
triplicate measurements. The 100% horizontal line represents activity of VopT and ExoS without
the addition of metal ions.
77
MART enzymes are not metalloenzymes, but there is one mART that contains an iron
cofactor130. This iron-sulfur center was stabilized by two cysteines and two histidines in the
active site and was required for transferase activity, but not glycohydrolase activity. Based on the
homology model of VopT produced in our lab, VopT does not have two cysteines or histidines
orientated in such a way as to coordinate iron as seen for NarE, so it is unlikely that VopT binds
iron in this manner.
Metal chlorides and sulfates were tested to ensure that the observed inhibition was due to
the presence of the metal and not the counter ion. However, regardless of the counter ion, Zn 2+
and Cu2+ still showed complete inhibition of VopT at 5 mM (Figure 26a). VopT was slightly
more active in the presence of Mg2+, but the increase in activity was not significant. Also, VopT
had higher activity with EDTA. This result suggests that VopT had little or no metal ion
requirement for activity, although it is well known that EDTA can bind to proteins and directly
affect their conformation and/or function131. Transferase activity was not tested with these
metals.
ExoS was also tested with some metals and showed similar results to VopT; both ZnCl 2
and NiSO4 showed almost complete inhibition, while Mg2+ appeared to slightly enhance ExoS
glycohydrolase activity (Figure 26b). However, ExoS showed partial inhibition when assayed
with EDTA. Since Zn2+ and Ni2+ gave a similar effect on activity for both VopT and ExoS, it is
possible that all ExoS-like mARTs are sensitive to these metals.
3.3.6
Inhibitor search
No inhibitors have been previously identified for VopT and only a few potent inhibitors
have been found for ExoS121,126. Two previously developed series of inhibitors from our lab, the
P6- and M-series, were tested against VopT glycohydrolase activity and multiple compounds
78
were found to be effective inhibitors. Many members of the P6-series inhibited Scabin toxin
from S. scabies with IC50 values between 40 and 120 µM80 and C3larvin, another mART toxin
recently characterized in our lab, was inhibited by M3, a member of the M-series93.
Since inhibitors were dissolved in 100% DMSO, the effect of DMSO on glycohydrolase
activity was also investigated. Figure 27 shows that VopT had slightly increased activity as
DMSO content increased (up to 5% by volume), which was previously observed for ExoS 121.
Therefore, if a decrease in activity was observed with VopT, it can be attributed solely to the
inhibitor.
For the inhibitor screens, the final DMSO content was 5%, so a control with 5% DMSO
was included in the samples tested (Fig. 28). 1,8-Naphthalimide (1,8-NAP) was tested at 10 µM
instead of 100 µM, as it was previously identified as an inhibitor of VopT that gave an IC50 value
of 5.6 µM (unpublished data, Danielle Visschedyk). P6C was tested at 10 µM as well because it
completely inhibited VopT activity at 100 µM. IC50 curves were produced from inhibition data
and IC50 values were determined for P6C, M15, and NiSO4 as these were some of the best
inhibitors (Fig. 29). 1,8-NAP was not used to construct an IC50 curve since a previous lab
member (Danielle Visschedyk) had already determined the IC50 value (unpublished).
All of the inhibitors were designed to be NAD+ competitive, so most contain a ring
structure similar to the nicotinamide moiety of NAD+ (Table 4). Most of these compounds have
been shown to exhibit a competitive inhibition mechanism80,83,93,132. For example, 1,8-NAP, P6C,
M6, M15, and M19 have a benzamide ring mimicking the nicotinamide ring in NAD+.
Furthermore, M6 and M15 have an acid group to resemble the phosphates linking the two ribose
79
Figure 27. Effect of DMSO on VopT glycohydrolase activity
As DMSO concentration increased in assay samples, activity also increased. Error bars are the
SD of 4 measurements.
80
120
100
% Activity
80
60
40
*
*
20
0
Figure 28. Inhibitor screen for VopT glycohydrolase activity
Relative activities are represented as % activity compared to the VopT glycohydrolase activity sample without the addition of
inhibitors or DMSO. Bars with an asterisk denote inhibitors used to calculate an IC50 value and the 100% horizontal line is a reference
to VopT activity in absence of inhibitors. Error bars are the SD of triplicate measurements.
81
A
B
82
C
D
Figure 29. IC50 curves for VopT glycohydrolase activity
Some of the most potent inhibitors were chosen to conduct IC50 curves with A) P6C, B) M15,
and C) NiSO4. NiSO4 exhibits biphasic inhibition and was fitted with a two-site inhibition model.
D) shows the residuals for the NiSO4 biphasic curve. Error bars are the SD of triplicate
measurements.
83
Table 4. Structures of inhibitors of VopT glycohydrolase activity
Inhibitor
Structure
IC50 (µM)
1,8-NAP
5.59 ± 1.05
P6C
4.04 ± 0.13
M6
ND
M15
44.7 ± 1.3
84
M19
ND
17 ± 5.5
NiSO4 · 6H20
862 ± 110
+
+
NAD
-
All of the compounds resemble NAD+ but inhibit VopT. Not all of the compounds had an IC50
value determined and are denoted ND.
85
rings in NAD+ and M19 contains an adenine ring. All of these features allow the inhibitory
compound to masquerade as a substrate and function as competitive inhibitors for the enzyme.
NiSO4 also inhibited VopT glycohydrolase activity, but exhibited a biphasic-inhibition
profile. This suggests that VopT has activity modes for this divalent metal. It might suggest that
there are two binding sites with one site having higher affinity for Ni2+ than the other. However,
this would need to be tested in a metal-binding experiment. ExoS also appeared to be inhibited
by Ni2+, so this whole family of enzymes may be susceptible to Ni2+ inhibition. On the other
hand, it is possible that all mART enzymes, or a fraction of them, are sensitive to Ni2+ since
preliminary tests of Scabin glycohydrolase activity in the presence of NiSO4 showed complete
inhibition at 5 mM as well (Stephanie Carlin, unpublished data).
3.3.7
Effect of boiling on VopT activity
Surprisingly, upon boiling VopT, a significant amount of enzymatic activity was still
observed. The graph in Figure 30 shows that almost 20% of its enzyme activity was retained
after 60 min. Protein precipitates formed within the first 2 min of boiling so precipitatecontaining aliquots were spun down to clarify the sample. Not all protein in the boiled samples
precipitated, since activity was still observed, so some protein remained folded and had
enzymatic activity.
When ExoS was first characterized, ExoS was boiled up to 30 min and transferase
activity was tested at various time intervals. ExoS displayed tolerance to boiling by retaining
more than 60% activity after 10 min, but then quickly lost stability at approximately 20 min (see
Table 3 in Iglewski et al.)46. Boiling caused VopT activity to decrease immediately but a
fraction of activity persisted beyond 60 min. Based on the homology model of ExoT, the FASbinding sequence is folded into part of the NAD+-binding pocket (Fig. 21). If this is also the case
86
Figure 30. Glycohydrolase activity of VopT after boiling
A) Data obtained for VopT glycohydrolase activity after rapid boiling incubation (100 °C). B)
Activity data published by Iglewski et al. when this group first characterized ExoS46. ExoS (open
circles) is able to tolerate boiling better than ExoA (solid circles), another mART toxin from P.
aeruginosa. Error bars for VopT are the SD of triplicate measurements; SD was not published by
Iglewski et al. for ExoS or ExoA so there are no error bars.
87
for VopT, the folded-in sequence would produce a more compact protein that has had an empty
pocket filled; thus, it may be more thermostable133. Better packing would also contribute to
higher thermostability133. However, at this point in time, the increased stability of VopT caused
by the placement of the FAS-binding sequence is only hypothetical and further modeling is to be
conducted. However, it is not clear how activity was still observed after incubation for 60 min in
boiling water.
3.4
Tryptophan-fluorescence binding assays
As previously described in the Methods section, the dissociation constant (Kd) can be
determined by monitoring the quenching of tryptophan fluorescence since it is well known that
indoles form ground-state complexes with DNA bases such as adenine in NAD+
134
. Below are
the results of multiple experiments determining the Kd for NAD+, FAS, and VocC with VopT.
NAD+ and VocC were titrated into Y191W separately to determine binding affinity, and
the dissociation constants were estimated to be 68.3 ± 23.1 µM and 287 ± 103 nM, respectively
(Fig. 31a-d). These values may not be accurate, since the quenching of tryptophan fluorescence
could not be saturated for either condition and the curve fits were not completely satisfactory as
the residuals do not show a random trend. Also, the Kd determined for NAD+ may be different
for the wild-type VopT enzyme since Y191W was used in its place; Y191W only showed about
50% of wild-type VopT activity so the active site and binding affinity of NAD+ may be slightly
different. However, the Kd for NAD+ was within the range of 9 to 100 µM for experimentally
determined Kd values as reported for other mART enzymes80,81,93,132. Even though Y191W was
used as a proxy for wild-type VopT, VocC binding should not be affected as VocC is proposed
to bind the N-terminal region of VopT. The binding affinity of VocC with its effector proteins
has not been discussed in the literature, so the binding data provided the first insight into the
88
A
B
C
D
E
F
Figure 31. Binding of NAD+, VocC, and FAS to VopT
A) Tryptophan fluorescence quenching observed upon NAD+ binding to Y191W, C) VocC
binding to Y191W, E) FAS binding to wild-type VopT. Fluorescence values were divided by the
initial NAD+-free fluorescence value to obtain relative fluorescence and then was subtracted
from 1. Graphs B, D, and F are the residuals from NAD+, VocC, and FAS binding, respectively.
All of the residual plots have the same trend of decreasing and then increasing as the titration
continues suggesting that the residuals do not appear to be random and that the data collected
may be inaccurate. Error bars are the SD of triplicate measurements.
89
binding affinity of VocC with one of its effector proteins. These results were comparable with
other chaperone-effector binding affinities because dissociation constants for other T3SS
structural protein chaperones have been reported to be between 60 and 500 nM135,136 and can be
as low as 1 nM137.
Using the same principle as the previous experiment, wild-type VopT was titrated into a
solution of FAS, the activator protein that contains two tryptophan residues. The binding
constant was determined to be 20.13 ± 1.28 nM (Fig. 31ef). This result was close to previously
determined binding constants for FAS as dissociation constants between 80 and 150 nM have
been reported for most binding partners138–140. Tir, an effector protein from E. coli, bound the τisoform of FAS with a Kd of only 10 nM141. FAS is a high-affinity binding protein so the Kd of
20 nM for FAS binding VopT was similar to previously determined results.
In summary, VopT was identified as a potential mART enzyme from in silico analysis
and it was concluded that it is indeed a mART enzyme. VopT was shown to be cytotoxic to both
yeast and mammalian cell lines upon expression and is an ExoS-like mART toxin that modifies
Ras. Catalytic variants of VopT were generated but activity was not compromised as much as
other characterized mARTs, so further investigation is required to understand this difference.
Binding affinities were determined for VopT with FAS, VocC, and NAD+, and multiple activesite inhibitors, competitive against the NAD+ substrate, were identified. The characterization of
VopT has increased the understanding of ExoS-like toxins and should provide the basis for the
development of potent inhibitors against this family of virulence factors.
90
Chapter 4: Conclusion
91
4.1
Conclusions
Antibiotics have been used to treat bacterial infections since the time of Florey (1941),
but a new strategy for treating and managing these infections is now desperately needed, because
many infectious bacteria are now resistant to multiple antibiotic drugs due to the evolution and
spread of resistance genes. Ideally, new antibiotic compounds would be developed to treat these
infections; however, bacteria are gaining resistance to antibiotics faster than we can discover
them.
One emerging approach is the antivirulence strategy, targeting virulence factors of
infectious bacteria and disabling them by the application of antivirulence compounds. By doing
so, bacteria are not killed but have had their virulence factors (weaponry) inactivated or
neutralized. MART toxins are an example of virulence factors that help elicit the infection
phenotype. Since this strategy does not target vital cellular processes in the pathogen, bacteria
will likely not gain resistance to these compounds as quickly as they do for antibiotics, since the
selective pressure to survive has been alleviated.
In this thesis, VopT, a mART toxin from V. parahaemolyticus, was further characterized.
Previous research included an in silico search for mART toxins which (Rob Fieldhouse)
discovered VopT based on its conserved catalytic residues and characteristic mART protein fold.
Subsequently, VopT was tested in vivo in yeast and it was shown to be cytotoxic, since it caused
a growth-defect phenotype. This in vivo assay also showed that VopT required its catalytic
residues for yeast-induced cytotoxicity and it was therefore classified as a mART toxin against a
eukaryotic cell host.
Independently, VopT was also identified by Kodama et al. and shown to be a mART
toxin, based on in vivo and preliminary in vitro assays26. Kodama et al. classified VopT as being
92
ExoS-like since it required the binding of FAS for activity and shared 45% sequence identity
with ExoS and ExoT, mART toxins from P. aeruginosa.
In this thesis, VopT was expressed in E. coli and purified from inclusion bodies for
kinetic characterization. VopT had both transferase activity towards hRas and glycohydrolase
activity, and VopT was FAS-dependent, congruent with the findings from Kodama et al.
Glycohydrolase activity followed Michaelis-Menten kinetics and the KM and kcat values were
comparable to those of other mART toxins. The KM for wild-type VopT (23.3 ± 1.2 µM) is
comparable to previously determined mART KM values as seen by C3larvin (34 ± 12 µM), ExoS
(37 ± 0.7 µM), and Iota toxin (23 µM)72,93,100. The glycohydrolase kcat value (0.74 ± 0.01 min-1)
was within the range of most mART toxins: Cholix (600 ± 180 min-1), Scabin (94 ± 2 min-1),
ExoS (2.68 ± 0.35 min-1), Iota toxin (0.2 min-1), C3larvin (1.3 ± 0.05 x 10-3 min-1)39,80,93,100,127.
Multiple VopT catalytic variants were produced and tested for activity. Surprisingly, the
variants retained a significant amount of activity compared to other mART toxins, so further
investigation is required. Inhibitor screens were conducted using a compound library previously
developed to mimic NAD+ and competitively inhibit mART toxins. Multiple compounds from
the P6- and M-series have previously been shown to inhibit a number of mART toxins; however,
this is the first report of VopT inhibitors. Inhibitors of VopT glycohydrolase activity included
P6C, M15, and NiSO4, with IC50 values of 4.04 ± 0.13, 44.7 ± 1.3, and 17 ± 5.5 µM,
respectively. NAD+, VocC, and FAS bind VopT and binding experiments were performed to
determine KD values for these binding partners. The KD values determined for NAD+ and VocC,
8 µM and 12 nM, respectively, are only preliminary estimates since binding curves did not
completely saturate and the curve fits were not ideal. The KD value for FAS was determined to
be 20 nM and this agreed with previously determined values reported for other FAS-binding
partners138–141. A homology model of another ExoS-like mART, ExoT, was developed in our lab
93
(unpublished results) and provided valuable insights for VopT structure and FAS-binding. This
homology model led to the proposal that the helix containing the FAS-binding motif is normally
folded into the active site, preventing NAD+ binding. Upon FAS binding, the motif interacts
strongly with FAS, causing it to vacate the active site.
4.2
Future directions
It is puzzling that the VopT catalytic variants (E186A, E188A, and E186A/E188A),
involving substitution of the classic mART catalytic signature motifs, resulted in relatively small
reductions in enzymatic activity compared with ExoS. These catalytic variants should be reevaluated due to the discrepancy between the variant activities of VopT with other catalytic
variant mART toxins, especially within the ExoS family. One concern from this thesis work is
the folded integrity of VopT. It is always a concern when a protein requires folding from
inclusion bodies (misfolded/aggregated protein), as was needed for VopT purification from E.
coli cells. Some strategies for studying the folding of a protein include circular dichroism (CD)
spectroscopy, thermal shift analysis (TSA), and protein nuclear magnetic resonance (NMR)
spectroscopy. Further investigation of protein stability of VopT could be completed using some
of the above methods. The diminished effect of replacement of conserved catalytic signature
residues on the VopT enzyme activity may be due to an already compromised wild-type protein.
Future studies could involve the preparation of more constructs of VopT involving N- and Cterminal truncations, fusion protein partners, etc., in an effort to produce a more stable protein
with improved aqueous solubility and higher enzymatic activity. However, it is possible that
VopT has unusual and intrinsically different mART enzymatic activity from other ExoS family
members.
94
Several inhibitors were identified as effective against VopT glycohydrolase activity;
however, the IC50 values were only determined for a few of them. More IC50 values should be
generated to expand the information initially collected. Also, inhibitors with different scaffold
structures from other libraries should be tested against VopT glycohydrolase activity including
the K-series, the most recently developed series of inhibitors in our lab, and also previously
reported ExoS inhibitors126. Additionally, VopT inhibitors should be tested against ExoS to
evaluate their general efficacy against the ExoS-like mART toxins. Effective inhibitors should
also be evaluated, to determine the inhibition mechanism.
VopT modifies hRas, but it may also target other macromolecules, as reported for
ExoS37,142. A wide search of macromolecule targets, including many ExoS substrates, should be
used to investigate the target specificity of VopT; examples would include other Ras family
proteins, Rab proteins, Rho proteins, vimentin, and soybean trypsin inhibitor (STBI).
A homology model of ExoT was prepared, since there are no crystal structures of any
ExoS-like members. It is anticipated that future work involving a VopT homology model built
similarly to the ExoT model may shed light on the structure and function of this perplexing
member of the ExoS family, as well as the design of catalytic variants and biochemical
experiments. Ultimately, a crystal structure would take precedence, but significant efforts have
been invested to get crystal structures for both VopT and ExoS without success (unpublished
data). Notably, there is a crystal structure of an ExoS peptide containing the FAS-binding motif
that shows the ExoS peptide bound to the FAS protein and it revealed its orientation and possible
binding mechanism. This should be attempted with VopT because VopT has residues
surrounding the FAS-binding motif that are different from either ExoS or ExoT; therefore, there
may be a difference in the FAS-binding mode for VopT. Also, the length of VopT peptide should
be varied to determine the optimal length that may be crystallized in complex with the FAS
95
protein. These crystals would provide valuable insights pertaining to the interaction of FAS
protein with VopT and may also shed light on the apparent insensitivity of VopT to variation of
its catalytic signature.
Another future direction would be to infect eukaryotic target cells like Caco-2 or HCT-8
cells with V. parahaemolyticus. Then, the efficacy of inhibitors in vivo could be assessed by
monitoring whether the inhibitors show a protective or therapeutic effect against VopT in the
eukaryotic cells. Then, the addition of inhibitors would provide an initial assessment of their
efficacy against VopT cell-induced damage and possibly the treatment of V. parahaemolyticus
infections. This would be an important step towards characterizing inhibitors and their
therapeutic properties.
Bacteria cause numerous infections and these infections are usually treated with
antibiotics. However, the rate at which bacteria are gaining resistance to these compounds is
outpacing our antibiotic development programs. Another strategy to treat these infections is the
disarmament of harmful bacteria using small antivirulence compounds. This strategy has not
been well tested and it requires considerably more research and validation; however, this
approach certainly provides a new avenue that may prove complementary to antibiotic therapy.
MART toxins are examples of virulence factors that help elicit bacterial infection and represent
an attractive target for antivirulence therapy. The study of the structure and function of mART
toxins and their molecular mechanisms provides the basis for future success involving
antivirulence therapy for the treatment of bacterial infections.
96
References
(1) Clements, A., Young, J. C., Constantinou, N., and Frankel, G. (2012) Infection strategies of
enteric pathogenic Escherichia coli. Gut Microbes 3, 71–87.
(2) Zhang, Y., Zhao, Y., Ding, K., Wang, X., Chen, X., Liu, Y., and Chen, Y. (2014) Analysis of
bacterial pathogens causing acute diarrhea on the basis of sentinel surveillance in Shanghai,
China, 2006-2011. Jpn. J. Infect. Dis. 67, 264–8.
(3) Cegelski, L., Marshall, G. R., Eldridge, G. R., and Hultgren, S. J. (2008) The biology and future
prospects of antivirulence therapies. Nat. Rev. Microbiol. 6, 17–27.
(4) Lee Ligon, B. (2004) Sir Howard Walter Florey—the force behind the development of
penicillin. Semin. Pediatr. Infect. Dis. 15, 109–114.
(5) Baron, C. (2010) Antivirulence drugs to target bacterial secretion systems. Curr. Opin.
Microbiol. 13, 100–5.
(6) Schmitt, C. K., Meysick, K. C., and O’Brien, A. D. (1999) Bacterial toxins: friends or foes?
Emerg. Infect. Dis. 5, 224–34.
(7) Mainil, J. (2013) Escherichia coli virulence factors. Vet. Immunol. Immunopathol. 152, 2–12.
(8) Clatworthy, A. E., Pierson, E., and Hung, D. T. (2007) Targeting virulence: a new paradigm for
antimicrobial therapy. Nat. Chem. Biol. 3, 541–8.
(9) Wirthmueller, L., and Banfield, M. J. (2012) mADP-RTs: versatile virulence factors from
bacterial pathogens of plants and mammals. Front. Plant Sci. 3, 142.
(10) Barth, H., Aktories, K., Popoff, M. R., and Stiles, B. G. (2004) Binary bacterial toxins:
biochemistry, biology, and applications of common Clostridium and Bacillus proteins.
Microbiol. Mol. Biol. Rev. 68, 373–402, table of contents.
(11) Ono, T., Park, K.-S., Ueta, M., Iida, T., and Honda, T. (2006) Identification of proteins secreted
via Vibrio parahaemolyticus type III secretion system 1. Infect. Immun. 74, 1032–42.
(12) Barison, N., Gupta, R., and Kolbe, M. (2013) A sophisticated multi-step secretion mechanism:
how the type 3 secretion system is regulated. Cell. Microbiol. 15, 1809–17.
(13) Zhang, L. (2013) Virulence determinants for Vibrio parahaemolyticus infection 16, 70 – 77.
(14) Broberg, C. A. (2011) Vibrio parahaemolyticus cell biology and pathogenicity determinants.
Microbes Infect. 13, 992 – 1001.
(15) Ceccarelli, D., Hasan, N. A., Huq, A., and Colwell, R. R. (2013) Distribution and dynamics of
epidemic and pandemic Vibrio parahaemolyticus virulence factors. Front. Cell. Infect.
Microbiol. 3, 97.
(16) O’Boyle, N., and Boyd, A. (2014) Manipulation of intestinal epithelial cell function by the cell
contact-dependent type III secretion systems of Vibrio parahaemolyticus. Front. Cell. Infect.
Microbiol. 3, 114.
(17) Masi, M., and Wandersman, C. (2010) Multiple signals direct the assembly and function of a
type 1 secretion system. J. Bacteriol. 192, 3861–9.
(18) Korotkov, K. V, and Hol, W. G. J. (2008) Structure of the GspK–GspI–GspJ complex from the
enterotoxigenic Escherichia coli type 2 secretion system. Nat. Struct. Mol. Biol. 15, 462–
468.
(19) Galkin, V. E., Schmied, W. H., Schraidt, O., Marlovits, T. C., and Egelman, E. H. (2010) The
structure of the Salmonella typhimurium type III secretion system needle shows divergence
from the flagellar system. J. Mol. Biol. 396, 1392–1397.
(20) Bergeron, J. R. C., Fernández, L., Wasney, G. A., Vuckovic, M., Reffuveille, F., Hancock, R.
E. W., and Strynadka, N. C. J. (2016) The Structure of a type 3 secretion system (T3SS)
ruler protein suggests a molecular mechanism for needle length sensing. J. Biol. Chem. 291,
97
1676–1691.
(21) Kodama, T., Hiyoshi, H., Gotoh, K., Akeda, Y., Matsuda, S., Park, K.-S., Cantarelli, V. V, Iida,
T., and Honda, T. (2008) Identification of two translocon proteins of Vibrio
parahaemolyticus type III secretion system 2. Infect. Immun. 76, 4282–4289.
(22) Waddell, B., Southward, C. M., McKenna, N., and Devinney, R. (2014) Identification of
VPA0451 as the specific chaperone for the Vibrio parahaemolyticus chromosome 1 type
III-secreted effector VPA0450. FEMS Microbiol. Lett. 353, 141–50.
(23) Akeda, Y., Kodama, T., Saito, K., Iida, T., Oishi, K., and Honda, T. (2011) Identification of the
Vibrio parahaemolyticus type III secretion system 2-associated chaperone VocC for the
T3SS2-specific effector VopC. FEMS Microbiol. Lett. 324, 156–64.
(24) Park, K.-S., Ono, T., Rokuda, M., Jang, M.-H., Iida, T., and Honda, T. (2004) Cytotoxicity and
enterotoxicity of the thermostable direct hemolysin-deletion mutants of Vibrio
parahaemolyticus. Microbiol. Immunol. 48, 313–8.
(25) Raimondi, F., Kao, J. P., Fiorentini, C., Fabbri, A., Donelli, G., Gasparini, N., Rubino, A., and
Fasano, A. (2000) Enterotoxicity and cytotoxicity of Vibrio parahaemolyticus thermostable
direct hemolysin in in vitro systems. Infect. Immun. 68, 3180–5.
(26) Kodama, T., Rokuda, M., Park, K. S., Cantarelli, V. V., Matsuda, S., Iida, T., and Honda, T.
(2007) Identification and characterization of VopT, a novel ADP-ribosyltransferase effector
protein secreted via the Vibrio parahaemolyticus type III secretion system 2. Cell.
Microbiol. 9, 2598–2609.
(27) Naim, R., Yanagihara, I., Iida, T., and Honda, T. (2001) Vibrio parahaemolyticus thermostable
direct hemolysin can induce an apoptotic cell death in Rat-1 cells from inside and outside of
the cells. FEMS Microbiol. Lett. 195, 237–44.
(28) Hiyoshi, H., Kodama, T., Saito, K., Gotoh, K., Matsuda, S., Akeda, Y., Honda, T., and Iida, T.
(2011) VopV, an F-Actin-binding type III secretion effector, is required for Vibrio
parahaemolyticus-induced enterotoxicity. Cell Host Microbe.
(29) Escaich, S. (2008) Antivirulence as a new antibacterial approach for chemotherapy. Curr.
Opin. Chem. Biol. 12, 400–8.
(30) Talbot, G. H., Bradley, J., Edwards, J. E., Gilbert, D., Scheld, M., and Bartlett, J. G. (2006) Bad
bugs need drugs: an update on the development pipeline from the Antimicrobial Availability
Task Force of the Infectious Diseases Society of America. Clin. Infect. Dis. 42, 657–68.
(31) Yoshikawa, T. T. (2002) Antimicrobial resistance and aging: beginning of the end of the
antibiotic era? J. Am. Geriatr. Soc. 50, S226–9.
(32) Moreau, F., Desroy, N., Genevard, J. M., Vongsouthi, V., Gerusz, V., Le Fralliec, G., Oliveira,
C., Floquet, S., Denis, A., Escaich, S., Wolf, K., Busemann, M., and Aschenbrenner, A.
(2008) Discovery of new Gram-negative antivirulence drugs: structure and properties of
novel E. coli WaaC inhibitors. Bioorg. Med. Chem. Lett. 18, 4022–6.
(33) Turgeon, Z., Jørgensen, R., Visschedyk, D., Edwards, P. R., Legree, S., McGregor, C.,
Fieldhouse, R. J., Mangroo, D., Schapira, M., and Merrill, A. R. (2011) Newly discovered
and characterized antivirulence compounds inhibit bacterial mono-ADP-ribosyltransferase
toxins. Antimicrob. Agents Chemother. 55, 983–991.
(34) Lindgren, A. E. G., Karlberg, T., Ekblad, T., Spjut, S., Thorsell, A.-G., Andersson, C. D.,
Nhan, T. T., Hellsten, V., Weigelt, J., Linusson, A., Schüler, H., and Elofsson, M. (2013)
Chemical probes to study ADP-ribosylation: synthesis and biochemical evaluation of
inhibitors of the human ADP-ribosyltransferase ARTD3/PARP3. J. Med. Chem. 56, 9556–
68.
(35) Bürkle, A. (2005) Poly(ADP-ribose). The most elaborate metabolite of NAD+. FEBS J. 272,
98
4576–89.
(36) Di Paola, S., Micaroni, M., Di Tullio, G., Buccione, R., and Di Girolamo, M. (2012)
PARP16/ARTD15 is a novel endoplasmic-reticulum-associated mono-ADPribosyltransferase that interacts with, and modifies karyopherin-1. PLoS One 7.
(37) Simon, N. C., Aktories, K., and Barbieri, J. T. (2014) Novel bacterial ADP-ribosylating toxins:
structure and function. Nat. Rev. Microbiol. 12, 599–611.
(38) Fieldhouse, R. J., Jørgensen, R., Lugo, M. R., and Merrill, A. R. (2012) The 1.8 Å cholix toxin
crystal structure in complex with NAD+ and evidence for a new kinetic model. J. Biol.
Chem. 287, 21176–88.
(39) Jørgensen, R., Purdy, A. E., Fieldhouse, R. J., Kimber, M. S., Bartlett, D. H., and Merrill, A. R.
(2008) Cholix toxin, a novel ADP-ribosylating factor from Vibrio cholerae. J. Biol. Chem.
283, 10671–8.
(40) Stover, C. K., Pham, X. Q., Erwin, A. L., Mizoguchi, S. D., Warrener, P., Hickey, M. J.,
Brinkman, F. S., Hufnagle, W. O., Kowalik, D. J., Lagrou, M., Garber, R. L., Goltry, L.,
Tolentino, E., Westbrock-Wadman, S., Yuan, Y., Brody, L. L., Coulter, S. N., Folger, K. R.,
Kas, A., Larbig, K., Lim, R., Smith, K., Spencer, D., Wong, G. K., Wu, Z., Paulsen, I. T.,
Reizer, J., Saier, M. H., Hancock, R. E., Lory, S., and Olson, M. V. (2000) Complete
genome sequence of Pseudomonas aeruginosa PAO1, an opportunistic pathogen. Nature
406, 959–64.
(41) Wilson, R., Lieb, S., Roberts, A., Stryker, S., Janowski, H., Gunn, R., Davis, B., Riddle, C. F.,
Barrett, T., Morris, J. G., and Blake, P. A. (1981) Non-O group 1 Vibrio cholerae
gastroenteritis associated with eating raw oysters. Am. J. Epidemiol. 114, 293–8.
(42) Gill, D. M., and Meren, R. (1978) ADP-ribosylation of membrane proteins catalyzed by
cholera toxin: basis of the activation of adenylate cyclase. Proc. Natl. Acad. Sci. U. S. A. 75,
3050–4.
(43) Vitek, C., and Wharton, M. (1998) Diphtheria in the former Soviet Union: reemergence of a
pandemic disease. Emerg. Infect. Dis. 4, 539–550.
(44) Kleinman, L. C. (1992) To end an epidemic. Lessons from the history of diphtheria. N. Engl. J.
Med. 326, 773–7.
(45) Armstrong, S., Yates, S. P., and Merrill, A. R. (2002) Insight into the catalytic mechanism of
Pseudomonas aeruginosa exotoxin A. Studies of toxin interaction with eukaryotic
elongation factor-2. J. Biol. Chem. 277, 46669–75.
(46) Iglewski, B. H., Sadoff, J., Bjorn, M. J., and Maxwell, E. S. (1978) Pseudomonas aeruginosa
exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A. Proc. Natl.
Acad. Sci. U. S. A. 75, 3211–5.
(47) Fieldhouse, R. J., Turgeon, Z., White, D., and Rod Merrill, A. (2010) Cholera- and anthrax-like
toxins are among several new ADP-ribosyltransferases. PLoS Comput. Biol. 6, 1–19.
(48) Tsuge, H., and Tsurumura, T. (2014) Reaction mechanism of mono-ADP-ribosyltransferase
based on structures of the complex of enzyme and substrate protein. Curr. Top. Microbiol.
Immunol.
(49) Iglewski, B. H., and Sadoff, J. C. (1979) Toxin inhibitors of protein synthesis: production,
purification, and assay of Pseudomonas aeruginosa toxin A. Methods Enzymol. 60, 780–93.
(50) Gomez-Lorenzo, M. G., Spahn, C. M., Agrawal, R. K., Grassucci, R. A., Penczek, P.,
Chakraburtty, K., Ballesta, J. P., Lavandera, J. L., Garcia-Bustos, J. F., and Frank, J. (2000)
Three-dimensional cryo-electron microscopy localization of EF2 in the Saccharomyces
cerevisiae 80S ribosome at 17.5 A resolution. EMBO J. 19, 2710–8.
(51) Jørgensen, R., Merrill, A. R., Yates, S. P., Marquez, V. E., Schwan, A. L., Boesen, T., and
99
Andersen, G. R. (2005) Exotoxin A-eEF2 complex structure indicates ADP ribosylation by
ribosome mimicry. Nature 436, 979–84.
(52) Ogura, K., Yahiro, K., Tsutsuki, H., Nagasawa, S., Yamasaki, S., Moss, J., and Noda, M.
(2011) Characterization of Cholix toxin-induced apoptosis in HeLa cells. J. Biol. Chem.
286, 37207–15.
(53) Sharma, A. K., and FitzGerald, D. (2010) Pseudomonas exotoxin kills Drosophila S2 cells via
apoptosis. Toxicon 56, 1025–1034.
(54) Van Ness, B. G., Howard, J. B., and Bodley, J. W. (1980) ADP-ribosylation of elongation
factor 2 by diphtheria toxin. Isolation and properties of the novel ribosyl-amino acid and its
hydrolysis products. J. Biol. Chem. 255, 10717–20.
(55) Draper, R. K., and Simon, M. I. (1980) The entry of diphtheria toxin into the mammalian cell
cytoplasm: evidence for lysosomal involvement. J. Cell Biol. 87, 849–54.
(56) Fieldhouse, R. J., and Merrill, A. R. (2008) Needle in the haystack: structure-based toxin
discovery. Trends Biochem. Sci. 33, 546–56.
(57) Holbourn, K. P., Shone, C. C., and Acharya, K. R. (2006) A family of killer toxins. Exploring
the mechanism of ADP-ribosylating toxins. FEBS J. 273, 4579–93.
(58) Ng, N., Littler, D., Le Nours, J., Paton, A. W., Paton, J. C., Rossjohn, J., and Beddoe, T. (2013)
Cloning, expression, purification and preliminary X-ray diffraction studies of a novel AB₅
toxin. Acta Crystallogr. Sect. F. Struct. Biol. Cryst. Commun. 69, 912–5.
(59) Tsuge, H., Nagahama, M., Oda, M., Iwamoto, S., Utsunomiya, H., Marquez, V. E., Katunuma,
N., Nishizawa, M., and Sakurai, J. (2008) Structural basis of actin recognition and arginine
ADP-ribosylation by Clostridium perfringens iota-toxin. Proc. Natl. Acad. Sci. U. S. A. 105,
7399–404.
(60) Aktories, K., Geipel, U., Wille, M., and Just, I. (1990) Characterization of the ADPribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota
toxin. J. Physiol. (Paris). 84, 262–6.
(61) Aktories, K., Schmidt, G., and Lang, A. E. (2014) Photorhabdus luminescens Toxins TccC3
and TccC5: Insecticidal ADP-Ribosyltransferases that Modify Threonine and Glutamine.
Curr. Top. Microbiol. Immunol.
(62) Pautsch, A., Vogelsgesang, M., Tränkle, J., Herrmann, C., and Aktories, K. (2005) Crystal
structure of the C3bot-RalA complex reveals a novel type of action of a bacterial
exoenzyme. EMBO J. 24, 3670–80.
(63) Chardin, P., Boquet, P., Madaule, P., Popoff, M. R., Rubin, E. J., and Gill, D. M. (1989) The
mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3
and affects actin microfilaments in Vero cells. EMBO J. 8, 1087–92.
(64) Fahrer, J., Kuban, J., Heine, K., Rupps, G., Kaiser, E., Felder, E., Benz, R., and Barth, H.
(2010) Selective and specific internalization of clostridial C3 ADP-ribosyltransferases into
macrophages and monocytes. Cell. Microbiol. 12, 233–47.
(65) Hueck, C. J. (1998) Type III protein secretion systems in bacterial pathogens of animals and
plants. Microbiol. Mol. Biol. Rev. 62, 379–433.
(66) Radics, J., Königsmaier, L., and Marlovits, T. C. (2014) Structure of a pathogenic type 3
secretion system in action. Nat. Struct. Mol. Biol. 21, 82–7.
(67) Aktories, K., and Frevert, J. (1987) ADP-ribosylation of a 21-24 kDa eukaryotic protein(s) by
C3, a novel botulinum ADP-ribosyltransferase, is regulated by guanine nucleotide. Biochem.
J. 247, 363–8.
(68) Fu, Z. Q., Guo, M., Jeong, B., Tian, F., Elthon, T. E., Cerny, R. L., Staiger, D., and Alfano, J.
R. (2007) A type III effector ADP-ribosylates RNA-binding proteins and quells plant
100
immunity. Nature 447, 284–8.
(69) Just, I., Schallehn, G., and Aktories, K. (1992) ADP-ribosylation of small GTP-binding
proteins by Bacillus cereus. Biochem. Biophys. Res. Commun. 183, 931–936.
(70) Ottmann, C., Yasmin, L., Weyand, M., Veesenmeyer, J. L., Diaz, M. H., Palmer, R. H.,
Francis, M. S., Hauser, A. R., Wittinghofer, A., and Hallberg, B. (2007) Phosphorylationindependent interaction between 14-3-3 and exoenzyme S: from structure to pathogenesis.
EMBO J. 26, 902–13.
(71) Goehring, U. M., Schmidt, G., Pederson, K. J., Aktories, K., and Barbieri, J. T. (1999) The Nterminal domain of Pseudomonas aeruginosa exoenzyme S is a GTPase-activating protein
for Rho GTPases. J. Biol. Chem. 274, 36369–72.
(72) Knight, D. A., Finck-Barbançon, V., Kulich, S. M., and Barbieri, J. T. (1995) Functional
domains of Pseudomonas aeruginosa exoenzyme S. Infect. Immun. 63, 3182–6.
(73) Burr, S. E., Stuber, K., and Frey, J. (2003) The ADP-ribosylating toxin, AexT, from
Aeromonas salmonicida subsp. salmonicida is translocated via a type III secretion pathway.
J. Bacteriol. 185, 6583–91.
(74) Coburn, J., Kane, A. V, Feig, L., and Gill, D. M. (1991) Pseudomonas aeruginosa exoenzyme
S requires a eukaryotic protein for ADP-ribosyltransferase activity. J. Biol. Chem. 266,
6438–46.
(75) Braun, M., Stuber, K., Schlatter, Y., Wahli, T., Kuhnert, P., and Frey, J. (2002)
Characterization of an ADP-ribosyltransferase toxin (AexT) from Aeromonas salmonicida
subsp. salmonicida. J. Bacteriol. 184, 1851–8.
(76) Fehr, D., Burr, S. E., Gibert, M., d’Alayer, J., Frey, J., and Popoff, M. R. (2007) Aeromonas
exoenzyme T of Aeromonas salmonicida is a bifunctional protein that targets the host
cytoskeleton. J. Biol. Chem. 282, 28843–52.
(77) Barbieri, J. T., and Sun, J. (2004) Pseudomonas aeruginosa ExoS and ExoT. Rev. Physiol.
Biochem. Pharmacol. 152, 79–92.
(78) Vilches, S., Wilhelms, M., Yu, H. B., Leung, K. Y., Tomás, J. M., and Merino, S. (2008)
Aeromonas hydrophila AH-3 AexT is an ADP-ribosylating toxin secreted through the type
III secretion system. Microb. Pathog. 44, 1–12.
(79) Sun, J., and Barbieri, J. T. (2003) Pseudomonas aeruginosa ExoT ADP-ribosylates CT10
regulator of kinase (Crk) proteins. J. Biol. Chem. 278, 32794–800.
(80) Lyons, B., Ravulapalli, R., Lanoue, J., Lugo, M. R., Dutta, D., Carlin, S., and Merrill, A. R.
(2016) Scabin, a novel DNA-acting ADP-ribosyltransferase from Streptomyces scabies. J.
Biol. Chem. 291, 11198–11215.
(81) Shniffer, A., Visschedyk, D. D., Ravulapalli, R., Suarez, G., Turgeon, Z. J., Petrie, A. A.,
Chopra, A. K., and Merrill, A. R. (2012) Characterization of an actin-targeting ADPribosyltransferase from Aeromonas hydrophila. J. Biol. Chem. 287, 37030–41.
(82) Ravulapalli, R., Lugo, M. R., Pfoh, R., Visschedyk, D., Poole, A., Fieldhouse, R. J., Pai, E. F.,
and Merrill, A. R. (2015) Characterization of Vis toxin, a novel ADP-ribosyltransferase
from Vibrio splendidus. Biochemistry 54, 5920–5936.
(83) Visschedyk, D., Rochon, A., Tempel, W., Dimov, S., Park, H.-W., and Merrill, A. R. (2012)
Certhrax toxin, an anthrax-related ADP-ribosyltransferase from Bacillus cereus. J. Biol.
Chem. 287, 41089–102.
(84) Visschedyk, D. D., Perieteanu, A. A., Turgeon, Z. J., Fieldhouse, R. J., Dawson, J. F., and
Merrill, A. R. (2010) Photox, a novel actin-targeting mono-ADP-ribosyltransferase from
Photorhabdus luminescens. J. Biol. Chem. 285, 13525–34.
(85) Tsuge, H., Nagahama, M., Nishimura, H., Hisatsune, J., Sakaguchi, Y., Itogawa, Y.,
101
Katunuma, N., and Sakurai, J. (2003) Crystal structure and site-directed mutagenesis of
enzymatic components from Clostridium perfringens iota-toxin. J. Mol. Biol. 325, 471–83.
(86) Domenighini, M., Magagnoli, C., Pizza, M., and Rappuoli, R. (1994) Common features of the
NAD-binding and catalytic site of ADP-ribosylating toxins. Mol. Microbiol. 14, 41–50.
(87) Masignani, V., Balducci, E., Serruto, D., Veggi, D., Aricò, B., Comanducci, M., Pizza, M., and
Rappuoli, R. (2004) In silico identification of novel bacterial ADP-ribosyltransferases. Int.
J. Med. Microbiol. 293, 471–8.
(88) Wilson, B. A., Reich, K. A., Weinstein, B. R., and Collier, R. J. (1990) Active-site mutations of
diphtheria toxin: effects of replacing glutamic acid-148 with aspartic acid, glutamine, or
serine. Biochemistry 29, 8643–51.
(89) Wilde, C., Vogelsgesang, M., and Aktories, K. (2003) Rho-specific Bacillus cereus ADPribosyltransferase C3cer cloning and characterization. Biochemistry 42, 9694–702.
(90) Laing, S., Unger, M., Koch-Nolte, F., and Haag, F. (2011) ADP-ribosylation of arginine.
Amino Acids 41, 257–69.
(91) Perelle, S., Domenighini, M., and Popoff, M. R. (1996) Evidence that Arg-295, Glu-378, and
Glu-380 are active-site residues of the ADP-ribosyltransferase activity of iota toxin. FEBS
Lett. 395, 191–4.
(92) Ménétrey, J., Flatau, G., Boquet, P., Ménez, A., and Stura, E. A. (2008) Structural basis for the
NAD-hydrolysis mechanism and the ARTT-loop plasticity of C3 exoenzymes. Protein Sci.
17, 878–86.
(93) Krska, D., Ravulapalli, R., Fieldhouse, R. J., Lugo, M. R., and Merrill, A. R. (2015) C3larvin
toxin, an ADP-ribosyltransferase from Paenibacillus larvae. J. Biol. Chem. 290, 1639–53.
(94) Toda, A., Tsurumura, T., Yoshida, T., Tsumori, Y., and Tsuge, H. (2015) Rho GTPase
recognition by C3 exoenzyme based on C3-RhoA complex structure. J. Biol. Chem. 290,
19423–19432.
(95) Lugo, M. R., and Merrill, A. R. (2015) A comparative structure-function analysis of active-site
inhibitors of Vibrio cholerae cholix toxin. J. Mol. Recognit. 28, 539–52.
(96) Lugo, M. R., Ravulapalli, R., Dutta, D., and Rod Merrill, A. (2016) Structural variability of
C3larvin toxin. Intrinsic dynamics of the α/β fold of the C3-like group of mono-ADPribosyltransferase toxins. J. Biomol. Struct. Dyn. 1–24.
(97) Beattie, B. K., Prentice, G. A., and Merrill, A. R. (1996) Investigation into the catalytic role for
the tryptophan residues within domain III of Pseudomonas aeruginosa exotoxin A.
Biochemistry 35, 15134–42.
(98) Han, S., Craig, J. A., Putnam, C. D., Carozzi, N. B., and Tainer, J. A. (1999) Evolution and
mechanism from structures of an ADP-ribosylating toxin and NAD complex. Nat. Struct.
Biol. 6, 932–6.
(99) Bell, C. E., and Eisenberg, D. (1996) Crystal structure of diphtheria toxin bound to
nicotinamide adenine dinucleotide. Biochemistry 35, 1137–49.
(100) Nagahama, M., Sakaguchi, Y., Kobayashi, K., Ochi, S., and Sakurai, J. (2000)
Characterization of the enzymatic component of Clostridium perfringens iota-toxin. J.
Bacteriol. 182, 2096–103.
(101) Barth, H., Preiss, J. C., Hofmann, F., and Aktories, K. (1998) Characterization of the catalytic
site of the ADP-ribosyltransferase Clostridium botulinum C2 toxin by site-directed
mutagenesis. J. Biol. Chem. 273, 29506–11.
(102) Tsurumura, T., Tsumori, Y., Qiu, H., Oda, M., Sakurai, J., Nagahama, M., and Tsuge, H.
(2013) Arginine ADP-ribosylation mechanism based on structural snapshots of iota-toxin
and actin complex. Proc. Natl. Acad. Sci. U. S. A. 110, 4267–72.
102
(103) Collier, R. J. (2001) Understanding the mode of action of diphtheria toxin: a perspective on
progress during the 20th century. Toxicon 39, 1793–803.
(104) Iglewski, B. H., and Kabat, D. (1975) NAD-dependent inhibition of protein synthesis by
Pseudomonas aeruginosa toxin. Proc. Natl. Acad. Sci. U. S. A. 72, 2284–8.
(105) Yates, S. P., Taylor, P. L., Jørgensen, R., Ferraris, D., Zhang, J., Andersen, G. R., and Merrill,
A. R. (2005) Structure-function analysis of water-soluble inhibitors of the catalytic domain
of exotoxin A from Pseudomonas aeruginosa. Biochem. J. 385, 667–75.
(106) Park, K.-S., Ono, T., Rokuda, M., Jang, M.-H., Okada, K., Iida, T., and Honda, T. (2004)
Functional characterization of two type III secretion systems of Vibrio parahaemolyticus.
Infect. Immun. 72, 6659–65.
(107) Makino, K., Oshima, K., Kurokawa, K., Yokoyama, K., Uda, T., Tagomori, K., Iijima, Y.,
Najima, M., Nakano, M., Yamashita, A., Kubota, Y., Kimura, S., Yasunaga, T., Honda, T.,
Shinagawa, H., Hattori, M., and Iida, T. (2003) Genome sequence of Vibrio
parahaemolyticus: a pathogenic mechanism distinct from that of Vibrio cholerae. Lancet
361, 743–9.
(108) Koh, M., Yong, H.-Y., Kim, E.-S., Son, H., Jeon, Y. R., Hwang, J.-S., Kim, M.-O., Cha, Y.,
Choi, W. S., Noh, D.-Y., Lee, K.-M., Kim, K.-B., Lee, J.-S., Kim, H. J., Kim, H., Kim, H.H., Kim, E. J., Park, S. Y., Kim, H. S., Moon, W. K., Choi Kim, H.-R., and Moon, A.
(2016) A novel role for flotillin-1 in H-Ras-regulated breast cancer aggressiveness. Int. J.
cancer 138, 1232–45.
(109) Coleman, M. L., Marshall, C. J., and Olson, M. F. (2004) RAS and RHO GTPases in G1phase cell-cycle regulation. Nat. Rev. Mol. Cell Biol. 5, 355–366.
(110) Liu, Y., Liu, T., Sun, Q., Niu, M., Jiang, Y., and Pang, D. (2015) Downregulation of Ras
GTPase‑activating protein 1 is associated with poor survival of breast invasive ductal
carcinoma patients. Oncol. Rep. 33, 119–24.
(111) Adjei, A. A. (2001) Blocking oncogenic Ras signaling for cancer therapy. J. Natl. Cancer
Inst. 93, 1062–74.
(112) Bele, A., Mirza, S., Zhang, Y., Ahmad Mir, R., Lin, S., Kim, J. H., Gurumurthy, C. B., West,
W., Qiu, F., Band, H., and Band, V. (2015) The cell cycle regulator ecdysoneless cooperates
with H-Ras to promote oncogenic transformation of human mammary epithelial cells. Cell
Cycle 14, 990–1000.
(113) Thirukkumaran, C., and Morris, D. G. (2009) Oncolytic viral therapy using reovirus. Methods
Mol. Biol. 542, 607–34.
(114) Fu, H., Coburn, J., and Collier, R. J. (1993) The eukaryotic host factor that activates
exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family. Proc.
Natl. Acad. Sci. U. S. A. 90, 2320–4.
(115) Gómez-Suárez, M., Gutiérrez-Martínez, I. Z., Hernández-Trejo, J. A., Hernández-Ruiz, M.,
Suárez-Pérez, D., Candelario, A., Kamekura, R., Medina-Contreras, O., Schnoor, M., OrtizNavarrete, V., Villegas-Sepúlveda, N., Parkos, C., Nusrat, A., and Nava, P. (2016) 14-3-3
Proteins regulate Akt Thr308 phosphorylation in intestinal epithelial cells. Cell Death
Differ. 23, 1060–72.
(116) Zhao, J., Du, Y., Horton, J. R., Upadhyay, A. K., Lou, B., Bai, Y., Zhang, X., Du, L., Li, M.,
Wang, B., Zhang, L., Barbieri, J. T., Khuri, F. R., Cheng, X., and Fu, H. (2011) Discovery
and structural characterization of a small molecule 14-3-3 protein-protein interaction
inhibitor. Proc. Natl. Acad. Sci. U. S. A. 108, 16212–6.
(117) Liu, Q., Zhang, S., and Liu, B. (2016) 14-3-3 proteins: Macro-regulators with great potential
for improving abiotic stress tolerance in plants. Biochem. Biophys. Res. Commun.
103
(118) Yasmin, L., Veesenmeyer, J. L., Diaz, M. H., Francis, M. S., Ottmann, C., Palmer, R. H.,
Hauser, A. R., and Hallberg, B. (2010) Electrostatic interactions play a minor role in the
binding of ExoS to 14-3-3 proteins. Biochem. J. 427, 217–24.
(119) Masters, S. C., Pederson, K. J., Zhang, L., Barbieri, J. T., and Fu, H. (1999) Interaction of 143-3 with a nonphosphorylated protein ligand, exoenzyme S of Pseudomonas aeruginosa.
Biochemistry 38, 5216–21.
(120) Zhang, L., Wang, H., Masters, S. C., Wang, B., Barbieri, J. T., and Fu, H. (1999) Residues of
14-3-3 zeta required for activation of exoenzyme S of Pseudomonas aeruginosa.
Biochemistry 38, 12159–64.
(121) Pinto, A. F., Ebrahimi, M., Saleeb, M., Forsberg, Å., Elofsson, M., and Schüler, H. (2016)
Identification of inhibitors of Pseudomonas aeruginosa exotoxin-S ADP-ribosyltransferase
activity. J. Biomol. Screen. 21, 590–5.
(122) Turgeon, Z., White, D., Jørgensen, R., Visschedyk, D., Fieldhouse, R. J., Mangroo, D., and
Merrill, A. R. (2009) Yeast as a tool for characterizing mono-ADP-ribosyltransferase toxins.
FEMS Microbiol. Lett. 300, 97–106.
(123) Sambrook, J., Fritsch, E., and Maniatis, T. (1989) Molecular cloning.
(124) Barrio, J. R., Secrist, J. A., and Leonard, N. J. (1972) A fluorescent analog of nicotinamide
adenine dinucleotide. Proc. Natl. Acad. Sci. 69, 2039–2042.
(125) Simeonov, A. (2013) Recent developments in the use of differential scanning fluorometry in
protein and small molecule discovery and characterization. Expert Opin. Drug Discov. 8,
1071–82.
(126) Arnoldo, A., Curak, J., Kittanakom, S., Chevelev, I., Lee, V. T., Sahebol-Amri, M., Koscik,
B., Ljuma, L., Roy, P. J., Bedalov, A., Giaever, G., Nislow, C., Merrill, A. R., Merrill, R. A.,
Lory, S., and Stagljar, I. (2008) Identification of small molecule inhibitors of Pseudomonas
aeruginosa exoenzyme S using a yeast phenotypic screen. PLoS Genet. 4, e1000005.
(127) Radke, J., Pederson, K. J., and Barbieri, J. T. (1999) Pseudomonas aeruginosa exoenzyme S
is a biglutamic acid ADP-ribosyltransferase. Infect. Immun. 67, 1508–10.
(128) Pallen, M. J., Beatson, S. A., and Bailey, C. M. (2005) Bioinformatics, genomics and
evolution of non-flagellar type-III secretion systems: A Darwinian perpective. FEMS
Microbiol. Rev. 29, 201–229.
(129) Liu, S., Kulich, S. M., and Barbieri, J. T. (1996) Identification of glutamic acid 381 as a
candidate active site residue of Pseudomonas aeruginosa exoenzyme S. Biochemistry 35,
2754–8.
(130) Koehler, C., Carlier, L., Veggi, D., Balducci, E., Di Marcello, F., Ferrer-Navarro, M., Pizza,
M., Daura, X., Soriani, M., Boelens, R., and Bonvin, A. M. J. J. (2011) Structural and
biochemical characterization of NarE, an iron-containing ADP-ribosyltransferase from
Neisseria meningitidis. J. Biol. Chem. 286, 14842–51.
(131) Nyborg, J. K., and Peersen, O. B. (2004) That zincing feeling: the effects of EDTA on the
behaviour of zinc-binding transcriptional regulators. Biochem. J. 381, e3–4.
(132) Ravulapalli, R., Lugo, M. R., Pfoh, R., Visschedyk, D. D., Poole, A., Fieldhouse, R. J., Pai, E.
F., and Merrill, A. R. (2015) Structure and function of Vis toxin, an ADP-ribosyltransferase
from Vibrio splendidus. Biochemistry 150909173946006.
(133) Kumar, S., Tsai, C. J., and Nussinov, R. (2000) Factors enhancing protein thermostability.
Protein Eng. 13, 179–91.
(134) Beattie, B. K., and Merrill, A. R. (1999) A fluorescence investigation of the active site of
Pseudomonas aeruginosa exotoxin A. J. Biol. Chem. 274, 15646–54.
(135) Adam, P. R., Patil, M. K., Dickenson, N. E., Choudhari, S., Barta, M., Geisbrecht, B. V.,
104
Picking, W. L., and Picking, W. D. (2012) Binding affects the tertiary and quaternary
structures of the Shigella translocator protein IpaB and Its chaperone IpgC. Biochemistry 51,
4062–4071.
(136) Banerjee, A., Dey, S., Chakraborty, A., Datta, A., Basu, A., Chakrabarti, S., and Datta, S.
(2014) Binding mode analysis of a major T3SS translocator protein PopB with its chaperone
PcrH from Pseudomonas aeruginosa. Proteins 82, 3273–85.
(137) Vogelaar, N. J., Jing, X., Robinson, H. H., and Schubot, F. D. (2010) Analysis of the crystal
structure of the ExsC.ExsE complex reveals distinctive binding interactions of the
Pseudomonas aeruginosa type III secretion chaperone ExsC with ExsE and ExsD.
Biochemistry 49, 5870–9.
(138) Muslin, A. J., Tanner, J. W., Allen, P. M., and Shaw, A. S. (1996) Interaction of 14-3-3 with
Signaling Proteins Is Mediated by the Recognition of Phosphoserine. Cell 84, 889–897.
(139) Coblitz, B., Wu, M., Shikano, S., and Li, M. (2006) C-terminal binding: An expanded
repertoire and function of 14-3-3 proteins. FEBS Lett. 580, 1531–1535.
(140) Petosa, C., Masters, S. C., Bankston, L. A., Pohl, J., Wang, B., Fu, H., and Liddington, R. C.
(1998) 14-3-3 binds a phosphorylated Raf peptide and an unphosphorylated peptide via its
conserved amphipathic groove. J. Biol. Chem. 273, 16305–16310.
(141) Patel, A., Cummings, N., Batchelor, M., Hill, P. J., Dubois, T., Mellits, K. H., Frankel, G., and
Connerton, I. (2006) Host protein interactions with enteropathogenic Escherichia coli
(EPEC): 14-3-3tau binds Tir and has a role in EPEC-induced actin polymerization. Cell.
Microbiol. 8, 55–71.
(142) Henriksson, M. L., Sundin, C., Jansson, A. L., Forsberg, A., Palmer, R. H., and Hallberg, B.
(2002) Exoenzyme S shows selective ADP-ribosylation and GTPase-activating protein
(GAP) activities towards small GTPases in vivo. Biochem. J. 367, 617–28.
105