UBC Experimental Medicine MEDI 501

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1 UBC Experimental Medicine MEDI 501 Zakaria Hmama, PhD Associate Professor Faculty/Faculty_Hmama.htm October

2 UBC Experimental Medicine MEDI Mtb features 2- TB disease 3- MDR- & XDR-TB 4- Mtb phagosome

3 Tuberculosis (TB) TB is a pulmonary disease that results in ~4,500 deaths every day most of them are vaccinated! TB is the second leading cause of death globally from an infectious disease.

4 Mtb features Mycobacterium tuberculosis (Mtb) is the causative agent of most cases of pulmonary tuberculosis (TB). Discovered in 1882 by Robert Koch

5 Mtb features Dr Stewart Cole Ecole polytechnique Federale de Lausane, Switzerland The genome of Mtb has been sequenced in 1998 and shown to be ~ 4 million base pairs in size. It comprises 3959 protein- coding genes. 52% of these genes can be assigned a funcoon. 376 putaove proteins are unique to Mtb!

6 Mtb features The principal host cell for Mtb is the alveolar macrophage

7 Mtb features Mtb E coli Mtb is a slow growing pathogen. It can survive inside the macrophage. Persists as dormant bacteria in a host cell membrane-derived Vacuole or Phagosome.

8 TB disease Pulmonary TB is an airborne disease

9 TB disease Pulmonary TB is an airborne disease

10 Immune Response to Mtb Minimum Infective Dose: 3-10 bacteria. Dr Stephan Kaufmann Max Planck Institute Berlin, Germany DosR Dormancy gene program 4 Reactivation 5 to 30 yr Rpf Resuscitation gene program

11 Immune Response to Mtb DC Ag cross-priming By mycobacteria-infected Mac Dr Ulrich Schaible Max Planck Institute Berlin, Germany Mac Ag-cross priming of DC T Cells DC Mac

12 Immune Response to Mtb Mouse model Robert J. North and Yu-Jin Jung Ann Rev of Immunol 22, (2004) Mtb infection in the lungs of B6 WT mice and in B6 gene-deleted mice

13 TB treatment First line TB drugs: Standard TB treatment Two months of ethambutol, pyrazinamide, isoniazid and rifampicin Then 4 months of isoniazid and rifampicin alone. MDR-TB is defined as resistance to at least isoniazid and rifampicin

14 TB treatment Why TB treatment needs 6 month combination therapy with four different drugs?

15 TB treatment TB treatment Second line drugs: treatment of MDR-TB Fluoroquinolones, ex: Levofloxacin, Moxifloxacin and Gatifloxacin Aminoglycosides, ex: Capreomycin, Kanamycin and Amikacin XDR-TB is defined as resistance to any fluoroquinolone, and at least one of the aminoglycosides

16 TB treatment Major problems for the success of antibiotherapy in developing countries: 1- Difficulties to access health services for most TB patients. 2- Non adherence of the patients to long term treatment

17 The worst has happened The discovery makes India the third country in which a completely drug-resistant form of the disease has emerged, following cases documented in Italy in 2007 and Iran in Resistance is man-made, caused by exposure to the wrong treatment, the wrong regimen, the wrong treatment duration. Dr Giovanni Migliori WHO director

18 PrevenOve vaccinaoon Preventive vaccination is the best cost-effective strategy to stop the spread of TB, especially in developing countries M. Bovis strain by Calmette and Guerin 1919

19 PrevenOve vaccinaoon BCG represents an attenuated strain of virulent M. bovis. Mtb Common ancestor of the Mtb complex (15,000 yr ago) RD7 RD8 RD9 RD10 RD1 M. bovis M. Bovis BCG 1919 RD1 deletion happened in the lab, following many passages of M. bovis from 1908 to 1919

20 Major Feature of TB Pathogenesis Intracellular survival of Mtb is central to TB pathogenesis Mtb resist to intracellular killing and persists in a host cell membrane-derived Vacuole or Phagosome

21 A turning point for cellular microbiology Philip D Arcy Hart National Institute for Medical Research, Mill Hill, London, NW7 1AA, England Macrophage with ferritin-labeled lysosomes were infected with (1) intact Mtb or (2) coated Mtb with specific antibodies. P: phagosome, L: lysosomes, ETZ: electron transparent zone Similar non fusion has been reported for Chlamydia and toxoplasma gondii

22 Phagolysosome fusion Efficient Ag presentation Late endosomes/ Lysosomes MHC class II

23 Mtb arrest phagolysosome fusion STOP STOP Efficient Ag presentation Late endosomes/ Lysosomes MHC class II

24 A turning point for cellular mycobacteriology Today, many investigators are focusing their efforts on the identification of mycobacterial factors that arrest phagosome maturation. Mycobacterial factors that arrest phagosome maturation represent attractive drug targets. KO of these mycobacterial factors would convert virulent Mtb into protective vaccines.

25 Work with Mtb (class III pathogen) requires BSL-3 laboratories BCG is used as alternative surrogate pathogen for in vitro studies Although BCG does not cause diseases in human, it mimics many feature of Mtb in vitro: Arrest of phagosome maturation Gatfield J and Pieters J., Science Walburger A et al Science Hmama Z et al J Cell Sci Vergne I et al, PNAS Deghmane AE, J Cell Sci Sun J, J Leuk Biol., 2007 Inhibition of MHC class II-directed Ag presentation Gagliardi MC et al, Vaccine Sendide K et al, Infect Immun Fulton SA et al, Infect Immun Sendide K, J Immunol. 2007

26 David Russell Cornell University Ithaca, NY Phagolysosome fusion results from a process of phagosome remodelling (or maturation) through a series of independent events that culminates in phagosome acidification and complete fusion with lysosomes.

27 Mycobacterial phagosome: 2000 Model Dead Live bacterium Phagosome Early endosomes Late endosomes Rab7 Lysosomes LAMP-1 Rab5 EEA1 ph H + -ATPase ph: 4.5 Cathepsin D Rab5 EEA1 Maturation arrest ph: 6.4

28 Hypothesis: Mtb exports proteins and glycolipids Into host cytosol to interact with and inhibit macrophage regulators of phagosome maturation

29 The Mycobacterial Phagosome Secretion and export of lipoamide dehydrogenase a Diversity of host proteins secreted within the phagosome 0.5 µm b 0.2 µm c 0.2 µm

30 Mycobacterial phagosome: Current Model Cell membrane Cor 1A Phagosome PLCγ1 Rac1 SNARE dependent membrane fusion PknG Vps33-P Vps33? PtpA V-ATPase Subunit H Rab7-GTP Rab7-GDP Rab5-GDP Rab5-GTP Ndk RILP EEA1 Ndk Lysosomes SapM Rac1-GDP Rac1-GTP Lpd? PI[3]P PI? NOX assembly/ activation Coro 1A PLCγ1? Lpd ManLAM Vps34 p150 Coro 1A release PI[4,5]P2 IP3 [Ca 2+ ] CaM CaMKII CaMKII-P SNARE dependent membrane fusion Lysosomes

31 How Mycobacterium blocks phagosome acidification? David Russell Cornell University Ithaca, NY 1: IgG-beads 2: Leishmania 3: Mycobacterium

32 How Mycobacterium blocks phagosome acidification? V-ATPase acidifies intracelluar organelles by coupling the energy of ATP hydrolysis to proton transport across the plasma membrane. V-ATPase acts as proton pump at the phagosomal surface. Does Mtb inactivates V-ATPase? V-ATPase schematic

33 PtpA interacts with the V-ATPase subunit H in vitro Dr Yossef Av-Gay Medicine - UBC Mtb PtpA inhibits phagosome acidification in the macrophages.

34 Mtb PtpA disrupts the interaction between the class C VPS and V-ATPase complexes during infection. Loss of PtpA binding to V-ATPase subunit H impairs Mtb survival within host macrophages.

35 A model for the specific exclusion of V- ATPase and the inhibition of mycobacterial phagosome acidification by PtpA.

36 Early Endosomal Antigen-1 (EEA1) plays an essential role during phagosome maturation Binds to PI3P through C-terminal domain (FYVE) and form a dimer. N-terminal zinc finger domain binds to endosomes via RabGTPase. C Contains also a calmodulin binding domain. N

37 Early Endosomal Antigen-1 (EEA1) plays an essential role during phagosome maturation Latex beads model PI hvps34 hvps34 PI3P EEA-1 Rab5-GTP EEA-1 Late Endosomes Rab5-GDP

38 2 Mtb phagosome EEA-1 Late Endosomes EEA-1 How Mtb excludes EEA1 from its phagosome? Fratti R A et al. J Cell Biol 2001;154:

39 Mtb blocks the increase of cytosolic Ca 2+ Zymosan Dr David Kusner Univ. Iowa Elevation of macrophage cytosolic Ca 2+ results in increased phagosomal maturation Mtb thapsigargin (Tg) was added to evaluate intracellular Ca 2+ stores

40 Ca 2+ -CAMKII Cascade Dr David Kusner Univ. Iowa Ca 2+ Calmodulin Ca 2+ Calmodulin T 286 Calmodulin Kinse II Ca 2+ Calmodulin T 286 Calmodulin Kinse II Ca 2+ Calmodulin P- T 286 Calmodulin Kinse II

41 Latex beads Vojo Deretic Univ New Mexico LAM inhibits [Ca 2+ ]c rise Block of Ca 2+ /calmodulin interaction inhibits phagosomal recruitment of EEA-1 W7 is a specific inhibitor of Ca 2+ /calmodulin interaction

42 Incubation of liposomes with macrophage cytosol (source of EEA1 & PI3K) Incubation of calmodulin-coated beads with macrophage cytosol (source of PI3K) PI3 Kinase + Ca 2+ Interaction of PI3K with calmodulin is Ca 2+ -dependent PI3K and calmodulin regulate EEA1 recruitment

43 The study revealed a link between Ca 2+ signaling, PI3K and EEA1 recruitment, and suggested that LAM interferes with EEA1- deppendent phagosome maturation by blocking cytosolic Ca 2+ rise. [Ca 2+ ] Ca-CAM CAMKII LAM PI hvps34 hvps34 PI3P EEA-1 Rab5-GTP EEA-1 Late Endosomes Rab5-GDP

44 Lipoarabinomannan (LAM) inhibits P-L fusion LAM is a mycobacterial lipoglycan, which represents a major component of the cell wall A virulence factor that inactivate macrophages

45 Lipoarabinomannan (LAM) inhibits P-L fusion Two type of LAM: ManLAM: Mannose caped Lipoarabinomannan (pathogenic mycobacteria) AraLAM: Arabinose caped Lipoarabinomannan (non-pathogenic mycobacteria) Ex: M. smegmatis

46 PI3P is retained on phagosomes harboring dead mycobacteria but is eliminated from phagosomes with live bacilli. Vojo Deretic Univ New Mexico Live bacteria secretes a lipid phosphatase, SapM, that hydrolyzes PI3P,

47 Mechanism of phagosomal exclusion of EEA-1 by Mtb Phagocytosis LAM [Ca 2+ ] CaM Ca 2+ -CaM CaMKII P-CaMKII Vps34/p150 Late Endosomes LAM + SapM PI PI[3]P = EEA1 = EEA1 = = SapM Rab5 PI

48 Rab GTPases and mycobacterial phagosome Rab5 and Rab7 GTPases contribute to vesicular trafficking. They have a direct impact on phagosome maturation.

49 Rab5 and Rab7 GTPases contribute to vesicular trafficking, They have a direct impact on phagosome maturation Rab GTPases recruitment and activation Prenylation: Rab RGGTase Rab targeted to membrane of endosome GTP GDP Rab5-GTP interacts with EEA1 Activation: Rab-GDP GEF Rab-GTP Rab7-GTP interacts with RILP Pi Inactivation: Rab-GTP GAP Rab-GDP GDI Recycling: Rab-GDP GDI Rab-GDI (GDP) Cannot dock to membrane with GDI bound form GDF Rab-GDP

50 NDK inactivates Rho GTPases?

51 Mycobacterial NDK expresses GAP activities in vitro Does NDK GAP activity extend to Rab GTPases? Does NDK contribute to the pathogenesis of mycobacterium?

52 Ndk binds to Rab5, Jim Sun Ndk-coated beads exclude EEA1 from their phagosomes Bright Field EEA1-GFP Zoom BSA and catalyzes the switch GTP/GDP - Mtb Ndk Smg Ndk % cpm: [γ-32p]-gtp Mtb Ndk D Rab5

53 Bright Field WT BCG/Dextran Ndk knock is associated with a phenotype of increased P-L fusion and reduced intracellular survival BCG 55 kda WT S-Ndk AS-Ndk LpdC S-Ndk 17 kda Ndk * * AS-Ndk 0 h 24 h 48 h

54 Mechanism of phagosomal exclusion of EEA-1 by Mtb Phagocytosis LAM [Ca 2+ ] Late Endosomes Vps34/p150 SapM PI[3]P = EEA1 = Rab5-GTP PI NDK Rab5-GDP LAM + SapM + NDK = EEA1 = EEA1

55 GAP in other organisms Several pathogenic bacteria secrete proteins that act as GAP for members of Rho-GTPases, and eventually facilitate their pathogenesis: Secreted cytotoxin, ExoS of Pseudomonas aeruginosa, disrupts the actin cytoskeleton Yersinia pseudotuberculosis secretes a cytotoxic factor, YopE, which depolymerizes the actin stress fiber