Phage protein based therapy for human pulmonary tuberculosis

Umender Sharma,

GangaGen Biotechnologies, Bangalore.

Project started on Dec 3, 2012No of FTEs - 2

• Bactericidal

• Low rates of resistance

• Intracellular efficacy

• Should kill non-replicating (NRP) bacteria

• High safety margin

• Specific to Mtb

Desired properties in an anti-Mtb drug

TAMEEndolysin / holin

TAME: tail associated muralytic enzyme

Phage proteins involved in degradation and lysis of bacterial cell walls

Examples of Enzybiotics tested for efficacy in animals

Fenton M. et al, Bioeng Bugs 2010 Jan-Feb;91):9-16

Hermoso JA et al, García JL, García P. Curr Opin Microbiol. 2007 Oct;10(5):461-72.

Enzybiotoics: sites of cleavage

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))

CD MPP ECPP

CD MPP

CD

Expected outcomes

Active in zymogram

Antibacterial activity in vitro

Intracellular antibacterial activity

http://phagesdb.org/ Hatfull GF et al,. J Virol. 2012 Feb;86(4):2382-4.

Complete genome sequences of 138 mycobacteriophages known

Sources of mycobacterial muralytic proteins

• 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

Development of a phage derived therapeutic protein(pre-clinical phases)

Bioinformatics analysis: candidate mycobacterial phage lysins

10

Phages Gene No. Function Size (kDa)

D29 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

TM4 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

L5 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

Doom 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

DD5 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

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

12

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

LysA and LysB proteins of phage D29

Expression and purification of LysA

13

LysA: optimization of protein expression

37°C, 1 mM IPTG 20°C, 250 μM IPTG 20°C, 100 μM IPTG

L: LoadW: WashFT: Flow ThroughE1: Eluate

S P MS P M S P M kDa

9766

43

29

20

14

LysA purification

Expression and Purification of LysB

14

W: WashL: LoadFT: Flow throughE1-E6: Eluates in 100 mM – 1M imidazole

Expression profile of LysB at 37°C, 1mM IPTG Purification of LysB

Enzymatic activity of purified LysB

• Lipase activity

15

Assay mixture:LB +1%Tween-20 +1mM CaCl2 +10 µg of enzyme

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)

LB with Tween and CaCl2 LB without Tween and CaCl2)

• PNPB assay

• Purified D29 LysB showed lipase activity

Payne K et al, Mol Microbiol. 2009; 73:367-81

Growth inhibitory activity of LysA and LysB on M. smegmatis cells

16

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

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

17• In Tris buffer, both LysA and LysB showed bactericidal activity, though combination of LysA and LysB showed better activity.

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

18• In saline LysA or LysB alone showed no significant kill in M. smegmatis•Combination of LysA and LysB gave a ~2 log CFU reduction

Bactericidal activity of LysA and LysB under non-growing conditions (saline)

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

•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)

21

Ser82 Asp166 His240

Ala 82 Ala166 His240

254 AALysB

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)

MSKPWLFTVHGTGQPDPLGPGLPADTARDVLDIYRWQPIGNYPAAAFPMWPSVEKGVAELILQIELKLDADPYADFAMAGYSQGAIVVGQVLKHHILPPTGRLHRFLHRLKKVIFWGNPMRQKGFAHSDEWIHPVAAPDTLGILEDRLENLEQYGFEVRDYAHDGDMYASIKEDDLHEYEVAIGRIVMKASGFIGGRDSVVAQLIELGQRPI

TEGIALAGAIIDALTFFARSRMGDKWPHLYNRYPAVEFLRQI

Primer for serine to alanine conversion

LysB: S-A FP:5’-GATGGCGGGTTACGCGCAGGGAGCCATCG-3’

RF: 5’-CGATGGCTCCCTGCGCGTAACCCGCCATC-3’

Active Site mutant of LysB (S82A)

22

Purification of LysB* using Ni-NTA columnProtein expression profile of LysB* L FT W E1 E2 M

9766

43

29

20

14

kDa LysB*(S) LysB*(P) LysB(S) LysB(P) M

97 66

43

29

20

14

•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

Enzymatic activity of mutant (S82A) LysB

• Lipase assay: • LB + 1%Tween-20 + 1mM CaCl2 + 10 µg of LysB

• PNPB Assay :• 200 µl containing purified LysB, 10 mM

substrate, and 25 mM Tris buffer pH 7.2 at

RT in dark

23

Only CaCl2

LysB

LysB*

Tween-20 + CaCl2

LysB

LysB*

• Mutant LysB (S82A) has lost lipase activity

Bactericidal activity of mutant LysB: CFU drop assay on M. smegmatis

24

Assay conditions: M. smegmatis in 7H9 medium (OD600~ 0.6) in well plateLysA: 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

25

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/mlEth: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.

 

26

•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 <1.0Indifference - 1< x < 4Antagonism - x > 4

Results

Drug MIC (µg/ml FIC Index

Rif 8 0.06229

Inh 8 0.1323

Eth 4 0.06229

LysB 3.125

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

27

Drug MBC(µg/ml)

Rif 32

Eth 32

LysB 12.5

Results:

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:

 

•LysA at 800 µg/ml and LysB at 1 mg/ml does not show any lysis of RBC

800 µg/ml 400 µg/ml 200 µg/ml 100 µg/ml

0.70 4.27 1.28 1.381.73 2.33 0.61 1.30

1 mg/ml 0.5 mg/ml 0.25 mg/ml 0.125 mg/ml

-0.34 -0.19 -0.60 -0.55-0.24 -0.37 -0.46 -0.51

Lys A

Lys B

28

Activity of commercial lipase (Aspergillus niger)

29

Commercial lipase is enzymatically active, but does not inhibit growth of M. smegmatis cells.

OD fall and cfu drop assay with A. niger lipase and LysB

30

Strain: M. smegmatis MC2 (OD600 ~ 0.5)Medium: 7H9 medium at 37oC at 100 RPM on 96 well plate formatLysB and Lipase conc.: 100 μg/ml

MIC- M. bovis BCGMIC 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

MIC of LysB was determined as 3-6 μg/ml and the synergy studies showed protein gives an additive effect

Eth 32 µg/ml

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 was set up in a 96 well plate incubated at 37 @100 rpmstart 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

Cfu drop assay of M. bovis BCG

• LysB showed an inhibitory effect at highest concentration used at the end of 30 hours

33

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

Thank You

35

Additional info

Evolution of phage therapy

Felix d’He´ relle discovered bactereiophages and used phages to treat dysentery

‘Historic era’ for phage therapy. Many companies produced phage preps to treat bacterial infections.

Cholera trials in India

Discovery of antibiotics in the 1940s

Capacity of a purified phage endolysinto kill bacteria was demonstrated

1919

1920-1950

1940

1957

2001 Fischetti and co-workers demonstrated in vivo efficacy of purified recombinant endolysin

against group A streptococci in mice

Use of phages for killing pathogenic bacteria In meat products approved by US FDA

2006

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

• Expression and purification of fusion proteins (e.g. for enhancing mycobacterial permeability, lipolytic) in E. coli

• Optimization of the construct for desirable anti-mycobacterial properties, confirmation of MIC and bactericidal properties of the protein

• In vitro kill kinetics on replicating and non-replicating surrogate mycobacteria (Msm, BCG)

• 2 mo

• 6 mo

• 3 mo

• 6 mo

• 3 mo

Total duration: 20 months

Antibacterial activity in vitro CD MPP

El-Shazly, S. et al, Journal of Medical Microbiology (2007), 56, 1145–1151

Overcoming the eukaryotic cell (macrophages) barrier: Mycobacterial cell entry protein (Mce3A)

Fluorescent latex beads coatedWith the following

a. GST-Mce3Ab. No proteinc. GSTd. Mce3Ae. Mce3E

Mce3 facilitates intracellular uptake