Phage protein based therapy for human pulmonary tuberculosis

1. Phage protein based therapy for human pulmonary tuberculosis Umender Sharma, GangaGen Biotechnologies, Bangalore. Project started on Dec 3, 2012 No of FTEs - 2

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

3. Phage proteins involved in degradation and lysis of bacterial cell walls Endolysin / holin TAME TAME: tail associated muralytic enzyme

4. Examples of Enzybiotics tested for efficacy in animals Fenton M. et al, Bioeng Bugs 2010 Jan-Feb;91):9-16

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

6. Enzybiotics: Challenges • Entry into mycobacterial cell walls • Protease degradation • Intracellular penetration • Half life in vivo • Immunogenicity • Delivery

7. 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

8. 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.

9. 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

10. Bioinformatics analysis: candidate mycobacterial phage lysins

11. 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

12. 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

13. Expression and purification of LysA LysA: optimization of protein expression 37°C, 1 mM IPTG 20°C, 250 μM IPTG 20°C, 100 μM IPTG LysA purification S P M S P M S P M kDa 97 66 43 29 20 14 L: Load W: Wash FT: Flow Through E1: Eluate

14. Expression and Purification of LysB Purification of LysB Expression profile of LysB at 37°C, 1mM IPTG W: Wash L: Load FT: Flow through E1-E6: Eluates in 100 mM – 1M imidazole

15. Enzymatic activity of purified LysB Payne K et al, Mol Microbiol. 2009; 73:367-81 LB with Tween and CaCl2 LB without Tween and CaCl2) Assay mixture: LB +1%Tween-20 +1mM CaCl2 +10 µg of enzyme • Lipase activity • 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

16. 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

17. 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.

18. 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. smegmatis • Combination of LysA and LysB gave a ~2 log CFU reduction

19. 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.

20. 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 106cfu/ml Lys B showed activity but in combination gave a 6 log reduction

21. 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) MSKPWLFTVHGTGQPDPLGPGLPADTARDVLDIYRWQPIGNYPAAAFPMWPSVEKGVAELILQIELKLDADPYADFAMAGYSQGAIVVGQVLKHHILPPTGRLHRFLHRLKKVIFWGNPMRQKGFAHSDEWIHPVAAPDTLGILEDRLENLEQYGFEVRDYAHDGDMYASIKEDDLHEYEVAIGRIVMKASGFIGGRDSVVAQLIELGQRPITEGIALAGAIIDALTFFARSRMGDKWPHLYNRYPAVEFLRQI LysB 254 AA Primer for serine to alanine conversion LysB: S-A FP:5’-GATGGCGGGTTACGCGCAGGGAGCCATCG-3’ RF: 5’-CGATGGCTCCCTGCGCGTAACCCGCCATC-3’ Ser82 Asp166 His240 Ala 82 Ala166 His240

22. 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 L FT W E1 E2 M kDa Purification of LysB* using Ni-NTA column Protein expression profile of LysB* 97 66 43 29 20 14 LysB*(S) LysB*(P) LysB(S) LysB(P) M 97 66 43 29 20 14

23. 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 Tween-20 + CaCl2 Only CaCl2 LysB* LysB* LysB LysB • Mutant LysB (S82A) has lost lipase activity

24. 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/mlLysB: 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/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-BFIC-A= MIC of A in combination/ MIC of A alone.FIC-B = MIC of B in combination/ MIC of B alone.

26. 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 <1.0Indifference - 1< x < 4Antagonism - x > 4

27. 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:

28. 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

29. Activity of commercial lipase (Aspergillus niger) Commercial lipase is enzymatically active, but does not inhibit growth of M. smegmatis cells.

30. 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

31. Eth 32 µg/ml MIC- M. bovis BCG LysB 25µg/ml 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 MIC of LysB was determined as 3-6 μg/ml and the synergy studies showed protein gives an additive effect

32. Cfu drop assay of M. bovisBCG 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 • 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. smegmatissuggest that LysB can show • synergy with anti-TB Drugs

34. Thank You

35. Additional info

36. Evolution of phage therapy 1919 Felix d’He´ relle discovered bactereiophages and used phages to treat dysentery 1920- 1950 ‘Historic era’ for phage therapy. Many companies produced phage preps to treat bacterial infections.Cholera trials in India Eastern Europe continued phage therapy West ignores phage therapy 1940 Discovery of antibiotics in the 1940s 1957 Capacity of a purified phage endolysin to kill bacteria was demonstrated Rise in drug resistance , west renews interest in phage therapy 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

37. 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 Antibacterial activity in vitro CD MPP Total duration: 20 months

38. Overcoming the eukaryotic cell (macrophages) barrier: Mycobacterial cell entry protein (Mce3A) • Fluorescent latex beads coated • With the following • GST-Mce3A • No protein • GST • Mce3A • Mce3E Mce3 facilitates intracellular uptake El-Shazly, S. et al, Journal of Medical Microbiology (2007), 56, 1145–1151