SBM 2044: Lecture 3
Weapons delivery & deployment
Secretion & targeting
of protein virulence factors
Protein secretion in bacteria
• Membranes act as a barrier to the movement of large
molecules into or out of the cell
• Gram-positive and Gram-negative bacteria have many
important structures which are located outside the wall
• So how are the large molecules from which some of
these structures are made transported out of the cell for
the assembly?
• How about exoenzymes and other proteins? How are
they released through the membrane?
• Mechanisms of protein secretion are important and can
be exploited for vaccine development.
Protein secretion in Gram-Negative
Bacteria
• Different cell layers for Gram + and Gram –
bacteria
• For Gram +, the secreted proteins must be
transported across a single membrane. Then
through a relatively porous peptidoglycan into
either:
– the external environment
– become embedded /attached to the peptidoglycan
• For Gram –, the secreted protein must be
transported across the IM; escape proteindegrading enzymes in the periplasmic space; and
finally across the OM
How are the large molecules being transported
out across the plasma membrane?
• General secretory pathway (GSP) is a protein
translocation mechanism
• GSP consists of cytosolic chaperones, an integral
membrane translocase consisting of several
proteins operating cooperatively and signal
peptidase
• Require energy from hydrolysis of ATP or GTP,
and sometimes by proton motive force
• Exported proteins are recognised by having a
signal sequence at their N-terminus, which is
cleaved by signal peptidase.
General Secretion Pathway (GSP)
SecB = chaperon: maintains protein in secretion-competent
state by preventing premature folding in cytoplasm
GSP: Sec-dependant secretion
Gram-positive bacteria
Sufficient to get protein
out of the cell
Gram-negative bacteria
Proteins reach periplasm, but
OM is additional barrier need other mechansims to
get protein thro’ OM.
OM
sec
IM
Signal-peptide
sec
How do Gram-neg. bacteria get proteins thro’ OM ??
• > 5 quite different mechanisms identified to date
- any particular protein excreted by one of these
‘overall’ mechanisms
Sec-dependent
Type II
Type IV
Type V
+ various others
– e.g. fimbrial systems
Sec-independent
Type I
Type III
Proteins secreted first to
periplasm by GSP (Sec)
and then thro’ OM
Secreted proteins get directly from
cytoplasm to outside without entering
the periplasm
Tat-Pathway
• Twin-arginine translocation pathway
• Tat translocase is composed of the membrane
proteins TatABC
• Translocate folded proteins across membrane
• Optional Reading:
– Palmer & Berks (2003). Moving folded proteins
across the bacterial cell membrane. Microbiology
149, 547–556
Type II protein secretion
• Present in pathogens such as Klebsiella pneumoniae,
Pseudomonas aeruginosa and Vibrio cholerae
• Secrete degradative enzymes pullulanases, cellulases,
pectinases, proteases and lipases
• Secrete cholera toxin and pili proteins
• Complex pathway with 12-14 proteins for translocation
through OM
• May also use a different plasma membrane transportation
system, the Tat pathway (for folded proteins)
Type IV protein secretion
• Sec-independent
• Secrete protein and transfer DNA from
donor bacterium to a recipient during
bacterial conjugation
Type IV: Conjugal transfer in Agrobacterium tumerfaciens
DNA transfer is sec-independent, but sec-dependant Pertussis
toxin is secreted from periplasm using homologous of
many (not all) of the Agrobacterium Type IV components
Type V protein secretion
• In periplasmic space, many proteins may
are able to form channel in the OM, through
which they transport themselves
Type V secretion
Essentially ‘autosecretion’ thro’ OM.
• relatively rare
• Example: IgA proteases secreted by Neisseria gonorrhoeae
Mature protease released
by autocatalytic cleavage
OM
Very few proteins can do this
sec
N-terminal
signal-peptide
C-terminal g, a and b domains
• b domain = OM-spanning sequence
• a + g domains – chaperon sequences??
How do Gram-neg. bacteria get proteins thro’ OM ??
• > 5 quite different mechanisms identified to date
- any particular protein excreted by one of these
‘overall’ mechanisms
Sec-dependent
Type II
Type IV
Type V
+ various others (e.g. fimbriae)
Sec-independent
Type I
Type III
Proteins secreted first to
periplasm by GSP (Sec)
and then thro’ OM
Secreted proteins get directly from
cytoplasm to outside without entering
the periplasm
Type I secretion pathways
Discovered in studying E. coli a-haemolysin (HlyA)
• HlyA lacks an N-terminal secretion signal-peptide, but
is nonetheless secreted efficiently
secretion involves a sec-independent pathway
Employed by various Gram-neg. species
Each pathway specific for a single protein - although
can be > 1 Type I pathway in cell to secrete different
proteins.
Each involves 3 ‘accessory’ proteins, one being an ‘ABC’
(ATP-binding cassette) transporter (e.g. E. coli HlyB)
Type III protein secretion
• Sec-independent
• Inject virulence factors directly into host
cells
• Secrete (inject) toxins, phagocytosis
inhibitors, stimulators for cytoskeleton
reorganisation in the host cell.
Type III Secretion
• Involves sets of ~ 20 genes - many share homology
between different species, suggesting common ancestors
& functions
• In all cases, genes involved are clustered together:
- on virulence plasmids in Yersiniae, Shigella, & EIEC
- in ‘Pathogenicity islands’: LEE in EPEC & EHEC
SPI-I & SPI-II in Salmonella
Probably ‘acquired’ by horizontal transfer & ‘adapted’ by different
species to secrete different sets of ‘effector’ (virulence) proteins
Type III Secretion - some examples
• Differences mainly in the nature & function of the
‘effector’ proteins - at least some of the proteins involved
in secretion ‘apparatus’ very similar in diff species
Pathogen
secreted
effector proteins
Function
Yersiniae sp.
YOPs
killing phagocytes
Shigella sp.
IpaA-D
Bacterial invasion
Salmonella
SIPs + SOPs
Bacterial invasion
EPEC & EHEC
Tir
A/E Lesions
Type III secretion system and other
virulence genes of Yersinia are
encoded on the pYV plasmid
Note the similar basal body
structures in both the TTSS
injectisome and the flagella
Euk cell
membrane
Pore
Yersiniae Type III
secretion apparatus
Needle
OM
Peptidoglycan
Periplasm
Basal body
IM
Scanning tunneling electron
microscopy shows
injectisome tip - lock
EM of purified Type III secretion complexes
S. typhimurium Type III ‘needle complex’
Note: ‘Needles’ very much thinner & shorter than EPEC ‘filaments’,
but apparatus spanning IM & OM probably very similar
Type III Secretion Systems
Unlike other systems, proteins not secreted as soon as they
are translated, but can accumulate in cytoplasmic ‘pools’.
Infers need for a signal
to trigger secretion
Shigella sp. secrete invasion proteins called IpaA - D. Found
> 90% remained cell-associated in broth cultures (small
quantities released - possible ‘leakage’ rather than secretion).
However, rapidly secreted in presence of mammalian cells
Activation of Type III secretion
Studies on several pathogens (Yersiniae, Shigella, EPEC)
have shown that Type III secretion activated in proximity
to host cells
What is the trigger ?
• Various studies suggested that adhesion to host cells is
the activation trigger
‘contact-dependant secretion’
• However, may not be that simple - evidence that some
Type III secretion systems can be activated by ‘soluble’
signalling molecules
e.g. EPEC in tissue culture medium, but not L-broth
Quorum sensing recently implicated
Quorum sensing
Remarkable ability of bacteria to sense their own cell
population density & respond by activating and/or
repressing appropriate sets of genes
Prototype system:
Bioluminescence in Vibrio fischeri - emits light
at very high cell densities of light in organ of host but not
when free in sea -
AHL = N-acetylated-homoserine lactone
• Small molecules that diffuse freely through cell membrane
• Concentrations inside and outside cell equilibrate
Shading reflects
[AHL] in media
Low cell density
Low cytoplasmic [AHL]
No induction
High cell density
High cytoplasmic [AHL]
‘Auto-induction’ of lux operon
AHL often called an ‘AI’ (auto-inducer)
Similarities + Differences
Type I
Type III
Sec-independent - secretion
apparatus spans IM + OM
3 ‘accessory’ secretion proteins
Sec-independent - secretion
apparatus spans IM + OM
~ 20 ‘accessory’ secretion proteins,
(identified by isolating mutants)
Single protein secreted
Multiple proteins secreted, tho’ all
for similar ‘end’ (e.g. invasion)
Target protein secreted rapidly
upon translation
Secreted proteins can ‘accumulate’
in bacterial cell before secretion in
response to ‘external’ signal
Secreted protein released into the
Secreted proteins injected directly
bacterial cell environment – before into host cell - appears to be main
any interactions with host cells
function of Type III systems
• Any QUESTIONS so far?
Sec-dependant General secretion pathway (GSP)
Gram-negative bacteria
Proteins reach periplasm, but
OM is additional barrier need other mechanisms to
get protein out thro’ OM.
(Types I - V secretion)
Gram-positive bacteria
Sufficient to get protein
out. In this case, other
mechanisms needed to
retain wall - associated
proteins
OM
IM
Type II
secretion
sec
sec
Signal-peptide
Targeting secreted proteins to Gram-positive cell walls
Four distinct mechanisms identified to date:
Rare:
•
Binding to wall teichoic acid
•
Binding to membrane anchored LTA
More widespread:
•
Lipoprotein ‘anchors’
• C-terminal wall-associating signals
1. Binding to cell-wall teichoic acid
Streptococcus pneumoniae and Streptococcus suis
Pneumococcal surface protein A (PspA)
Pneumococcal autolysin (LytA)
S. suis autolysin- [homologous to pneumococcal LytA]
C-terminal ends share homologous choline-binding
domains – enable binding to TA of these species
Reminder of the structure of teichoic acid:
Polymer of either Glycerol phosphate or Ribitol phosphate, with
various substituents (R)
poly-ribitol phosphate
O
O
P
O
O
H
H
H
H
H
C
C
C
C
C
H
O
OH O
H
R
O
O
P
O
R’
H
O
C
H
n
In most species studied to date
R = D-alanine
R’ = N-acetylglucosamine
In S. pneumoniae and S. suis
R = phosphodiester linked choline - chemically more stable than
ester-linked D-Ala
2. Binding to membrane anchored LTA
Single example recognised only recently
- InlB of Listeria monocytogenes – has C-terminal
domain that ‘targets’ LTA – mechanism??
3. Lipoproteins
• attached at outer surface of cytoplasmic membrane by a
lipid anchor
Examples include penicillinase in S. aureus
• Similar mechanisms used in both Gram-pos. & Gram-neg.
Distinctive N-terminal signal peptides
recognized by
distinct Sec apparatus with specialized signal
peptidase (called signal peptidase II)
Lipoprotein signal peptides
NShort
hydrophobic
1-3 positively
sequence
charged a.a.
Signal peptidase II
cleavage site
-Leu-x-y- Cysx and y usually
small, uncharged
residues
A diglyceride is attached
to the N-terminal Cys of
the mature protein
Diglyceride
Contrast with ‘typical’ GSP secretion signal-peptide ( Lecture 3 )
4. ‘Sorting’ via C-terminal wall-associating signals
Vast majority of Gram-pos. wall-associated proteins share
structurally similar C-terminal wall-associating signals
Hydrophobic /Charged ‘tail’
membrane ‘anchor’
-C
Pro-rich region
LPxTG
motif
15 - 20 hydrophobic
residues
5 - 10
mostly
charged
C-terminal wall-associating signals
Studies of S. aureus Protein A,
showed that membrane ‘anchor’
plays a transient role in a more
complex wall-associating pathway
Pro-rich
‘flexible’
wall-spanning
Hydrophobic
Charged ‘tail’
Membrane ‘anchor’
+
+
Care: do not be misled by some textbooks/reviews which say proteins
anchored in membrane.
C
N-terminal signal peptide
N
Wall-associating
signal
Signal
peptidase
wall-associated
‘Sortase’
Cleavage at
LPxTG
N
mRNA
G
Cross-linked
to cell-wall
Some, but not
C
Majority
Minority
necessarily all,
‘cleaved’
simply
covalently
‘anchored’? at LPxTG
linked to wall
(e.g. ActA in Listeria)
(e.g. InaA, Prot. A)
Retaining secreted proteins in Gram-positive cell walls
1. Binding to wall teichoic acid
Limited to a very few species (e.g. S. pneumoniae, S. suis)
2. Binding to membrane anchored LTA
Single example recognised only recently (InlB of Listeria
monocytogenes)
3. Lipoprotein ‘anchors’
A minority of wall-associated proteins in many species anchored
to outer surface of cell membrane via an N-terminal lipid anchor
4. C-terminal wall-associating signals
Vast majority of wall-associated proteins studied to date
share structurally similar C-terminal wall-associating signals
Retaining proteins at Gram-negative cell-surfaces
First step: Sec-dependent secretion to periplasm (GSP)
Then:
• Targeting of integral OM proteins - OM-interacting
‘surfaces’ result from folding in periplasm
(may involve periplasmic Dsb and Ppi enzymes)
OR
• Individual biogenesis pathways – e.g. fimbriae
E. coli fimbrial adhesins: > 40 distinct adhesins identified
• Most are variations on common theme - common ‘ancestor’
• Each encoded by a cluster of genes encoding regulators of
expression, structural components and additional proteins
for fimbrial biogenesis
Type I (common) fim genes
B
E
A
Regulators
(in cytoplasm)
I
C
D
Major subunit
Minor subunits
F
G
H
Chaperone
‘Usher’ (OM)
Type I fimbrial biogenesis
Minor subunits:
‘Tip’ structure FimH = adhesin
FimF + G
Fim G - also regulates fimbriae length?
Fim A
major
subunit
OM
Fim C periplasmic chaperone
Fim D ‘Usher’
assembly &
attachment
IM
Sec
Secreted thro’ IM
by Sec-apparatus
All components
References
• Prescott’s Microbiology Chapter 3,
Paragraph 3.8 ONLY: Prokaryotic Cell
Structure and Function
Optional
• Sherris Medical Microbiology Chapter 3 p37-40
ONLY
– and some relevant paragraphs in Chapter 10.