Phage protein based therapy for human
pulmonary tuberculosis
Umender Sharma,
GangaGen Biotechnologies, Bangalore.
Project started on Dec 3, 2012
No of FTEs - 2
Desired properties in an anti-Mtb drug
•
Bactericidal
•
Low rates of resistance
•
Intracellular efficacy
•
Should kill non-replicating (NRP) bacteria
•
High safety margin
•
Specific to Mtb
Phage proteins involved in degradation and lysis of
bacterial cell walls
Endolysin / holin
TAME: tail associated muralytic enzyme
TAME
Examples of Enzybiotics tested for efficacy in animals
Fenton M. et al, Bioeng Bugs 2010 Jan-Feb;91):9-16
Enzybiotoics: sites of cleavage
Hermoso JA et al, García JL, García P. Curr Opin Microbiol. 2007 Oct;10(5):461-72.
Enzybiotics: Challenges
• Entry into mycobacterial cell walls
• Protease degradation
• Intracellular penetration
• Half life in vivo
• Immunogenicity
• Delivery
Hypothetical anti-Mtb fusion protein
Catalytic domain (CD)
Mycobacterial permeability
Protein (MPP, e.g., LysB)
Eukaryotic cell permeability
Protein (ECPP, e.g. Mce3A))
Expected outcomes
Active in zymogram
CD
Antibacterial activity in vitro
CD
MPP
Intracellular antibacterial activity
CD
MPP
ECPP
Sources of mycobacterial muralytic proteins
Complete genome sequences of 138 mycobacteriophages known
http://phagesdb.org/
Hatfull GF et al,. J Virol. 2012 Feb;86(4):2382-4.
Development of a phage derived therapeutic protein
(pre-clinical phases)
•
Phase I (proof of concept)
•
Phase IA: demonstration of killing of M. smegmatis/ M. bovis BCG .
•
Phase IB: demonstration of Killing of Mtb and drug combination
studies.
•
Phase II: intracellular efficacy
•
Phase III: animal efficacy
Bioinformatics analysis: candidate
mycobacterial phage lysins
Phages
D29
TM4
L5
Doom
DD5
Gene No.
Function
Size (kDa)
GeneID:3172257/D29_10
Hydrolase
54.8
GeneID:3172258/D29_12
Cutinase
28.5
GeneID:3172257/ D29_11
Holin
14.6
GeneID:3172309/D29_59.2
Hydrolase
29.8
GeneID:932330/TM4_gp5
Phage portal protein
54.65
GeneID:932338/TM4_gp29
Amidase (N-acetylmuramoyl-L-alanine amidase)
58.59
GeneID:932308 /TM4_gp30
Peptidoglycans binding protein
42.37
GeneID:932322/TM4_gp31
Membrane bound hydrolase
13.76
GeneID:932307/TM4_gp36
Calcineurin-like phosphoesterase superfamily
43.69
GeneID:2942930/L5_p14
Phage portal protein
53.77
GI:9625456/L5_p26
Possible tail length determinant
86.41
GI:9625496/L5_p66
Phosphoesterase
23.58
339753563/DOOM_9
D-alanyl-D-alanine carboxypeptidase
54.8
339753564/DOOM_10
Cutinase
35.97
GI:339753638/DOOM_59
Lysis protein S(holin)
2.98
GI:339753627/DOOM_45
LysM: bacterial cell wall degradation
67.72
GeneID:6417318/DD5_11
cutinase
36.92
GeneID:6417340/DD5_13
Holin/portal
53.71
GeneID:6417268/DD5_44
LysM: cell wall degradation protein
67.6
GeneID:6417301/DD5_51
Phosphoestrase
28.14
10
D29 Mycobacteriophage - overview
General characteristics
• Lytic phage
•
Can infect and replicate in the slow-growing pathogenic
strains such as Mycobacterium tuberculosis and
Mycobacterium ulcerans and fast-growing environmental
strains such as Mycobacterium smegmatis.
•
Has a wide host range and will replicate in a wide range of
mycobacteria.
•
Robust phage, widely used in diagnostic applications.
Morphology
•
•
•
Isometric head with a mean diameter of 650 nm
Tail of variable length.
Family – Siphoviridae
11
LysA and LysB proteins of phage D29
LysA
• Is an endolysin protein of 54 kDa
• The enzyme has lysozyme like activities.
• Structure comprises a N-terminal peptidase, a central non-peptidase
catalytic domain and a C-terminal motif involved in cell wall binding.
LysB
•
•
•
Is a mycolylarabinogalactan esterase of 29 kDa.
The enzyme cleaves mycolylarabinogalactan bond and releases free
mycolic acids.
LysB structure has a α/β hydrolase organization with a catalytic triad
common to cutinases and also contains a four-helix domain which
helps in binding to lipid substrates
12
Expression and purification of LysA
LysA: optimization of protein expression
37°C, 1 mM IPTG
S
P
M
20°C, 250 μM IPTG
S
P
M
20°C, 100 μM IPTG
S
P
M
LysA purification
kDa
97
66
43
29
20
14
L: Load
W: Wash
FT: Flow Through
E1: Eluate
13
Expression and Purification of LysB
Expression profile of LysB at 37°C, 1mM IPTG
Purification of LysB
W: Wash
L: Load
FT: Flow through
E1-E6: Eluates in 100 mM – 1M imidazole
14
Enzymatic activity of purified LysB
• Lipase activity
LB with Tween and CaCl2
Payne K et al, Mol Microbiol. 2009; 73:367-81
LB without Tween
and CaCl2)
Assay mixture:
LB +1%Tween-20 +1mM CaCl2 +10 µg of enzyme
• PNPB assay
Assay conditions:
200 µl containing purified LysB, 10 mM
substrate, and 25 mM Tris buffer pH 7.2
at RT in dark
•
B
LysB (10μg) LysB (100 μg) BSA (100μg)
Purified D29 LysB showed lipase activity
15
Growth inhibitory activity of LysA and LysB
on M. smegmatis cells
10 µg of LysA or /LysB proteins were spotted on LB agar with M. smegmatis culture
•
Purified LysB protein inhibited growth of M. smegmatis in LB agar
16
Bactericidal activity of LysA and LysB under
non-growing conditions (Tris buffer)
Assay conditions
• Expt set up in 96 well plate_Msm mc2 155
•
•
•
•
•
Plate incubated @ 37C , 100 rpm
Start cell number adjusted to 107cfu/ml in 25mM Tris pH 7.5
Protein concentrations 50 and 100 ug/ml
Plating done after 8 hrs, 24 hrs and 30 hrs on LB agar and incubated at 37ºC for 3
days
In Tris buffer, both LysA and LysB showed bactericidal activity, though
combination of LysA and LysB showed better activity.
17
Bactericidal activity of LysA and LysB under
non-growing conditions (saline)
Assay conditions
•
•
•
•
•
Expt set up in 96 well plate_Msm mc2 155
Plate incubated @ 37C , 100 rpm
Start cell number adjusted to 107cfu/ml in 125 mM Saline
Protein concentrations 50 and 100 μg/ml
Plating done after 8 hrs, 24 hrs and 30 hrs on LB agar and incubated at 37oC for 3
days
• In saline LysA or LysB alone showed no significant kill in M. smegmatis18
•Combination of LysA and LysB gave a ~2 log CFU reduction
Bactericidal activity of LysA and LysB under growing
conditions (7H9 medium)
•
•
In 7H9 medium LysB showed bactericidal activity, whereas LysA was inactive
A combination of LysA and LysB showed better CFU reduction.
Cfu drop assay for proteins in Msm ATCC 607
under non growing conditions
Assay conditions
• Experiment set up in 96 well plate _ Msm ATCC 607
•
•
•
•
Plate incubated @ 37C , 100 rpm
Start cell number adjusted to 106cfu/ml in 25mM Tris pH 7.5
Protein concentrations 100 and 200 µg/ml
Plating done after 8 hrs, 24 hrs and 30 hrs on LB agar and
incubated at 37ºC for 3 days
cfu drop in Msm ATCC 607 in 25mM Tris pH 7.5
8
A_100
log10 cfu/ml
7
A_200
6
5
B_100
4
B_200
3
A+B_100
2
A+B_200
1
CC
0
0 hrs
24 hrs
30 hrs
•In Tris at cell number 106 cfu/ml Lys B showed activity but in combination
gave a 6 log reduction
Active Site mutant of LysB (S82A)
The catalytic triad Ser82-Asp166-His240 is located at the edge of the central β-sheet in LysB protein structure
(Payne et al. Mol. Microbiol. 2009)
LysB
254 AA
Ser82
Asp166
His240
Ala 82
Ala166
His240
MSKPWLFTVHGTGQPDPLGPGLPADTARDVLDIYRWQPIGNYPAAAFPMWPS
VEKGVAELILQIELKLDADPYADFAMAGYSQGAIVVGQVLKHHILPPTGRLHRFL
HRLKKVIFWGNPMRQKGFAHSDEWIHPVAAPDTLGILEDRLENLEQYGFEVRD
YAHDGDMYASIKEDDLHEYEVAIGRIVMKASGFIGGRDSVVAQLIELGQRPITEG
IALAGAIIDALTFFARSRMGDKWPHLYNRYPAVEFLRQI
Primer for serine to alanine conversion
LysB: S-A FP:5’-GATGGCGGGTTACGCGCAGGGAGCCATCG-3’
RF: 5’-CGATGGCTCCCTGCGCGTAACCCGCCATC-3’
21
Active Site mutant of LysB (S82A)
•Stratagene kit was used for SDM following standard protocol
•Randomly five colonies were picked up for SDM screening
•10 μl of crude protein was spotted on LB agar + 1% Tween-20 + 1mM CaCl2 plate
Purification of LysB* using Ni-NTA column
Protein expression profile of LysB*
L
LysB*(S) LysB*(P) LysB(S) LysB(P)
M
FT
W
E1
E2
M
kDa
97
66
97
66
43
43
29
29
20
20
14
14
22
Enzymatic activity of mutant (S82A) LysB
• Lipase assay:
•
Tween-20 + CaCl2
Only CaCl2
LB + 1%Tween-20 + 1mM CaCl2 + 10 µg of LysB
LysB*
LysB*
LysB
LysB
•
PNPB Assay :
•
200 µl containing purified LysB, 10 mM
substrate, and 25 mM Tris buffer pH 7.2 at
RT in dark
•
Mutant LysB (S82A) has lost lipase activity
23
Bactericidal activity of mutant LysB: CFU drop
assay on M. smegmatis
Assay conditions:
M. smegmatis in 7H9 medium (OD600~ 0.6) in well plate
LysA: 100 μg/ml LysB: 100 μg/ml
37 °C with shaking, CFU was measured at 12 hrs interval.
•
Mutant LysB does not show bactericidal activity on M. smegmatis
24
Synergy of LysA and LysB with anti-TB drugs
Assay
•MIC was done in combination with proteins and frontline drugs for TB
•Strain used was Msm mc2 155, Media: 7H9 Broth
•Drugs used were Rif, Inh and Eth
•Starting conc. of drugs
Rif: 32 µg/ml
Inh:16 µg/ml
Eth:16 µg/ml
•Protein conc.- LysA and LysB –50µg/ml
•Start cell number: 105 cfu/ml, Plate incubated at 37ºC for 3 days
•Color development:
•Addition of dye: 0.02% Resazurine dye+10% Tween80, incubated at 37ºC for 3 hours
•Read in spectramax at 575nm and 610nm
•Synergy
•Synergy is observed where there is a shift in MIC compared to drug or protein alone
•FIC value is calculated (Fractional Inhibitory concentration)
FIC index= FIC-A + FIC-B
FIC-A= MIC of A in combination/ MIC of A alone.
FIC-B = MIC of B in combination/ MIC of B alone.
25
Results
•LysA did not give any shift in MIC in combination
•LysB alone gave a MIC of 3-6 µg/ml
•LysB gave a evident shift in MIC with all the 3 drugs used
•Fractional Inhibitory concentration is:
•Interpretation
Synergism - x < 0.5
Additive 0.5 <x <1.0
Indifference - 1< x < 4
Antagonism - x > 4
Drug
MIC (µg/ml
FIC Index
Rif
8
0.06229
Inh
8
0.1323
Eth
4
0.06229
LysB
3.125
26
MBC of lysB against M. smegmatis
MBC was set up for M. smegmatis
Media:7H9
Start cell number:106 cfu/ml
Incubation time :72 hours (static)
Start conc: Rif:64 µg/ml, Eth:32 µg/ml, LysB: 25 µg/ml
Results:
Drug
MBC(µg/ml)
Rif
32
Eth
32
LysB
12.5
27
Haemolysis assay for LysA/ LysB
•Assay done in a 96 well plate format with appropriate controls
•Proteins are serially diluted in 1X PBS
•RBC added at 10 % Haematocrit (Human RBCs)
•Plate incubated at 37 ºC for 1 hour
•Plate was centrifuged @3000rpm for 15 min.
•100 µL Supernatant transferred to fresh plate and the plate is read at 540nm using
spectramax
% Heamolysis: Absorbance of sample - Absorbance of blank X 100
Absorbance of positive control
% Haemolysis:
800 µg/ml
Lys A
0.70
1.73
1 mg/ml
Lys B
-0.34
-0.24
400 µg/ml 200 µg/ml 100 µg/ml
4.27
2.33
1.28
0.61
1.38
1.30
0.5 mg/ml 0.25 mg/ml 0.125 mg/ml
-0.19
-0.37
-0.60
-0.46
-0.55
-0.51
•LysA at 800 µg/ml and LysB at 1 mg/ml does not show any lysis of RBC
28
Activity of commercial lipase (Aspergillus niger)
Commercial lipase is enzymatically active, but does not inhibit growth of
M. smegmatis cells.
29
OD fall and cfu drop assay with A. niger lipase and LysB
Strain: M. smegmatis MC2 (OD600 ~ 0.5)
Medium: 7H9 medium at 37oC at 100 RPM on 96 well plate format
LysB and Lipase conc.: 100 μg/ml
30
MIC- M. bovis BCG
MIC done in 96 well plate format
M. bovis BCG_10^5 cfu/ml start
Media: 7H9 broth + 10 % ADC
Start conc: Rif:0.25 μg/ml Inh: 0.5 μg/ml
Eth: 32 μg/ml, LysB: 25 μg/ml
LysB 25µg/ml
Eth 32 µg/ml
MIC of LysB was determined as 3-6 μg/ml and the synergy studies showed protein
gives an additive effect
Drug
MIC(µg/ml)
FIC Index
Rif
0.015
0.56
Inh
0.03
0.63
Eth
16
0.615
LysB
12.5
Cfu drop assay of M. bovis BCG
Cfu drop assay was set up in a 96 well plate incubated at 37 @100 rpm
start cell number 10^7 cfu/ml
Media:7H9 + ADC
Plated after 18 hrs and 30 hrs duration on 7H9 agar with 10 % ADC + Malachite
green
• LysB showed an inhibitory effect at highest concentration
used at the end of 30 hours
Summary
•
A number of candidate muralytic enzymes identified in genomes of
Mycobacteriophages.
•
LysA and LysB of D29 phages were expressed in E. coli and the
recombinant proteins were purified.
•
Purified LysB was shown to have lipase activity.
•
Though LysB alone showed bactericidal activity under some assay
conditions, a combination of LysA and LysB showed better activity
•
An active site mutant of LysB (S82A) had lost the lipase activity and was
inactive In CFU reduction as well.
•
Drug combination studies in M. smegmatis suggest that LysB can show
synergy with anti-TB Drugs
33
Thank You
Additional info
35
19201950
‘Historic era’ for phage therapy.
Many companies produced phage
preps to treat bacterial infections.
Cholera trials in India
1940
Discovery of antibiotics in the 1940s
1957
Capacity of a purified phage endolysin
to kill bacteria was demonstrated
2001
Fischetti and co-workers demonstrated in vivo
efficacy of purified recombinant endolysin
against group A streptococci in mice
2006
Use of phages for killing pathogenic bacteria
In meat products approved by US FDA
Rise in drug resistance ,
west renews interest
in phage therapy
Felix d’He´ relle discovered bactereiophages
and used phages to treat dysentery
West ignores phage therapy
1919
Eastern Europe continued phage
therapy
Evolution of phage therapy
Activities and timelines (Phase 1A)
•
Identification of putative muralytic proteins in a Mtb phage
genome by bioinformatics analysis
•
Expression and purification of the full length and truncated
proteins in E. coli and demonstration of their enzymatic
activity by zymogram analysis / OD drop assay in surrogate
organisms
•
2 mo
•
6 mo
•
Expression and purification of fusion proteins (e.g. for
enhancing mycobacterial permeability, lipolytic) in E. coli
•
3 mo
•
Optimization of the construct for desirable anti-mycobacterial
properties, confirmation of MIC and bactericidal properties of
the protein
•
6 mo
•
In vitro kill kinetics on replicating and non-replicating
surrogate mycobacteria (Msm, BCG)
•
3 mo
CD
Total duration: 20 months
MPP
Antibacterial activity in vitro
Overcoming the eukaryotic cell (macrophages) barrier:
Mycobacterial cell entry protein (Mce3A)
Fluorescent latex beads coated
With the following
a.
b.
c.
d.
e.
GST-Mce3A
No protein
GST
Mce3A
Mce3E
Mce3 facilitates intracellular uptake
El-Shazly, S. et al, Journal of Medical Microbiology (2007), 56, 1145–1151