Intracellular Pathogens

1. Intracellular Pathogens

2. function of susceptibility of host relates to mechanism of bacterial pathogenesis secretion of factors (toxins) direct host cell manipulation Disease immune competent/compromised immunizations age trauma genetics antimicrobial therapy (intracellular pathogens)

3. Evolution of the ‘perfect’ intracellular bacteria chloroplasts (cyanobacteria) mitochondria (proteobacteria) CM rRNA rRNA rRNA CW CM Archaea halophiles methanogens thermophiles thermoplasma CM CW CW OM (www.science.mcmaster.ca/ biochem/faculty/gupta…)

4. Cons • must over-come host barriers • resist innate immunity (phagocytic processes) • resist acquired immune responses • adapt to life in bacterially hostile environment Pros • gain access to a protected environment protection from immune response protection from bacterial competitors • nutrient rich environment Philosophy of an intracellular pathogen

5. Types of intracellular bacterial pathogens obligate intracellular bacteria facultative intracellular bacteria Salmonella spp. - typhoid /gastroenteritis Legionella pneumophila - Legionnaires’ disease Brucella spp.- brucellosis Francisella tularensis - tularemia Shigella spp. - dysentery Listeria monocytogenes - listeriosis Yersinia spp. - plague / gastroenteritis Chlamydia spp. - pneumonia / genital infections Rickettsia spp. - typhus / Rocky Mountain Spotted Fever Coxiella burnetii -Q fever Mycobacterium spp. - tuberculosis / leprosy

6. Strategies of intracellular bacteria Internalized by phagocytosis / proliferate in vacuole Mycobacterium tuberculosis Legionella pneumophila Induce cellular uptake - transient invasion Uropathogenic E. coli Yersinia spp. Internalized - reside within vacuole Salmonella enterica Internalized - escape from vacuole - multiply in cytoplasm Shigella flexneri Listeria moncytogenes

7. Bacterial manipulation of host cell function ~ cellular microbiology ~ I. exploitation of cytoskeleton II. manipulation of signaling processes III. effects on lipid and lipid metabolism IV. induction / inhibition of apoptosis

8. actin filaments (6 nm) flexible, helical polymer of actin determine cell shape / movement / division microtubules (23 nm) polymers of tubulin - form long, stiff, hollow tube involved in intracellular movement of - chromosomes / vesicles / organelles intermediate filaments (10 nm) keratins / vimentin / lamins provide cell strength to withstand physical stresses / stretching I. Bacterial manipulation of host cytoskeleton a a

9. bacterial manipulation of host actin Shigella flexneri actin-mediated membrane ruffling Listeria monocytogenes actin-mediated movement (from P. Cossart, Cellular Microbiology, 2000) (www.bio.brandeis.edu/ goodelab/) ~ other bacteria that manipulate host cell actin ~ Yersinia spp. Salmonella spp. Pseudomonas aeruginosa Enteropathogenic E. coil

10. Campylobacter jejuni infection of human embryonic epithelial cells (INT407) A. B. bacterial use of host microtubules A.Confocal fluorescence microscopic image of 1 hour infected INT407 cells. The microtubules (MT) appear as structural skeletons outlining the cells and the FITC-labeled bacteria (arrows) appear as bright white spots along the MTs. B.Immunofluorescent microscopic image of INT407 cells infected for 4 hours, with arrows pointing to numerous bacteria located at perinuclear sites within the host cell. (From: L. Hu, D.J Kopecko, Infect. Immun. 1999)

11. Bacteria Signaling pathway Yersinia Integrin signaling FAK / p130Cas Listeria MAPKK NF-kB E-cadherin VASP / Arp2-Arp3 Salmonella IP3 /Ca2+ Rho / Rac / Cdc42 Shigella Src / cortactin Rho Vinculin / a-Actinin VASP / Arp2-Arp3 II. Bacterial manipulation of host cell signaling processes

12. role of host cell lipids in phagocytosis (From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004) III. Effects on lipid and lipid metabolism

13. manipulation of host cell lipids by bacterial pathogens (From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004)

14. Induction Inhibition (LY Gao, YA Kwaik, Trends in Microbiology, 2000) IV. Bacterial modulation of apoptosis

15. Strategies of intracellular bacteria Bacteria internalized by phagocytosis - proliferate in vacuole Mycobacterium tuberculosis

16. Bacteriology - slim, rod-shaped bacterium acid-fast (waxy surface excludes Gram-stain) M. tuberculosis infection of lung - acid-fast staining (pathhsw5m54.ucsf.edu/ overview/tb.html) Pathogenesis -organism invades / lives in macrophages most commonly localized in lungs M. tuberculosis in mouse macrophages (Courtesy Center for Tuberculosis Research, Johns Hopkins University) Mycobacterium tuberculosis Disease -tuberculosis - (consumption) caused by uncontrolled host inflammatory response => granuloma formation Virulence factors - cord factor (waxy surface) induces granuloma formation LAM (lipoarabinomannan) PIM (phosphatidylinositol mannoside)

17. (From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004) Mycobacterium tuberculosis internalization/proliferation Mycobacterium resides / proliferates in vacuoles in phagocytic cells • Prevents acidification of vacuole by excluding proton pump ATPase • LAM - inhibits cytosolic Ca++ release - blocks calmodulin / calmodulin kinase - prevents PI(3)K activation and EEA1 (early endosome associated protein) recruitment to phagosome - EEA1 + syntaxin 6 needed for delivery of transgolgi network hydrolases • PIM - activates Rab5 inducing early endosomal fusion Result - interference of endosomal vacuole maturation

18. Mycobacterial phagosomes Phagosomes containing live mycobacteria isolated by flow cytometry facility and further separated into acidic and non-acidic compartments by staining with LysoTracker (Janisha Patel and Aaron Rae). (www.imperial.ac.uk/cmmi/research/young1.htm) Phagosome maturation M. tuberculosis phagosome arrest (Wilson, McNab, Henderson, Bacterial Disease Mechanisms, 2002)

19. A TB granuloma showing central necrosis and presence of giant cells TB giant cell in the granuloma (www.eastman.ucl.ac.uk/.../ tuberculosis.htm) (www.mrcophth.com/ pathology/granuloma.html) Healed, fibrotic granuloma shows calcification (blue circle). Active inflammation, giant cells and necrosis are absent. Cultures are negative. Necrotizing granuloma shows palisading of epithelioid histiocytes at the margin of the necrosis. Organisms found mainly in the zone of necrosis. (pathhsw5m54.ucsf.edu/ overview/tb.html) (pathhsw5m54.ucsf.edu/ overview/tb.html) M.tuberculosis ~ granuloma

20. Strategies of intracellular bacteria Bacteria that induce cellular uptake - but invade only transiently Yersinia spp.

21. Bacteriology - small, Gram-negative rod (julia.univ.gda.pl/~bioakk/grafika2/yersinia.jpg) (perso.wanadoo.fr/.../ scrabble/arche_y.html) Yersinia spp. Yersinia enterocolitica - enterocolitis (Yersiniosis) Yersinia pseudotuberculosis - animal pathogen Yersinia pestis - bubonic plague Pathogenesis -invasive organism Virulence factors - first step in invasion - adherence invasin -binds host cell b1 integrins Ail - (attachment-invasion locus) YadA - (Yersinia adherence) - binds b1 integrins, fibronectin, collagen, laminin (encoded on 78 kb virulence plasmid)

22. Yersinia spp. initially invade intestine through M cell interaction (Wilson, McNab, Henderson, Bacterial Disease Mechanisms, 2002) Yersinia spp. internalized by ‘zipper’ mechanism (www.ngfn.de/ngfn_en/ inf_tueb.html) Yersinia internalization

23. enters host internalized by host cells / phagocytosed by MF resists phagocytosis (T3S) survives in MF resists phagocytosis (T3S) leave MF replicates extracellularly multiplies in MF Y. pestis Y. pseudotuberculosis INFg activates MF Y. enterocolitica (Pujol, Bliska, Clinical Immunology 2005) Yersinia infectious process

24. Mechanism of Yersinia internalization • Yersinia invasin - binds to b1-integrins • results in cell-spreading over surface • leads to clustering of integrin - tighter binding • induces Rac1 activation - actin polymerization - bacterial engulfment (From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004)

25. (www.nature.com/.../ fig_tab/nature01603_F5.html) Binding of Yersinia to host-cell receptors triggers phagocytic pathways that result in bacterial uptake. The rapid translocation of several effectors by Yersinia disarms these pathways, facilitating bacterial avoidance of phagocytosis.YopHdephosphorylates a number of tyrosine-phosphorylated signaling proteins including Fyb, SKAP-HOM and p130cas, thereby disrupting their abilities to mediate further downstream signaling events in the cytoskeletal pathway.YopEdisrupts actin filaments by acting as a GTPase-activating protein for the GTPases Rac1, Rho and Cdc42. YopTproteolytically cleaves this family of GTPases, resulting in their release from the membrane. YopO blocks the activation of Rho through a mechanism that is not fully understood. Mechanism of transient invasion by Yersinia

26. Strategies of intracellular bacteria Bacteria internalized but escape from vacuole and multiply in cytoplasm Listeria moncytogenes

27. Bacteriology - small Gram-positive rod motile / facultative anaerobe growth is enhanced by presence of blood (www.geocities.com/ CapeCanaveral/3504/gallery.htm) (www.ifr.ac.uk/ bacanova/project.html) Listeria moncytogenes Disease - food-bourne infection - listeriosis gastroenteritis / meningitis / abortions Pathogenesis -invasive organism (invades non-phagocytic cells) Virulence factors - Adherence - first step in invasion InlA - adheres to E-cadherin InlB - adheres to HGF receptor Met Vacuole membrane lysis - PLC Adherence -leads to actin polymerization bacterial engulfment into vacuole by “zipper” - mechanism

28. Listeria moncytogenes zipper-internalization (www.diariomedico.com)

29. (From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004) Mechanism - Listeria monocytogenes internalization • Listeria InlB binds hepatic growth factor receptor - Met • Induces PI(3)K recruitment • Leads to: activation of Rac1 (controls actin dynamics) activation of Akt - cell survival (anti-apoptotic) • After invasion - Listeria resides in EEA1 / Rab5 enriched vacuole • Favors fusion with early endosome - delays phagosomal maturation

30. Intracellular movement - using ActA Cell-to-cell spread Entry and formation of the phagocytic vacuole - Listeria escape from vacuole using listeriolysin/ PLC (makes pores) - results in rise in pH- prevents further maturation of vacuole - allows bacteria to rupture membrane - escape and replicate in cytosol (From: Cossart P, Lecuit M: EMBO J 1998) Formation and lysis of the two-membrane vacuole - Intervening membranes lysed using PLC & Mp1 Listeria escape from vacuole, grow, disseminate

31. Model - actin assembly induced by Listeria ActA (P. Cossart, H. Bierne, Current Opinion in Immunology, 2001)

32. (olpaimages.nsf.gov/admin/images/listerias.jpg) Listeria movement in cytoplasm & dissemination Listeria move through cytoplasm at a rate of 6-60 mm per minute (www.diariomedico.com/.../ 0,2458,69566,00.html)

33. Experimental analysis of host cell & bacterial response to intracellular living

34. Microarray analysis of host cell mRNA expression Analysis of host cell response to intracellular bacteria induction pro-inflammatory cytokines IL-8 MCP-1(monocyte chemotatic protein 1) GMCSF(granulocyte-macrophage stimulating factor) TNFa prostaglandin release Cox-2(cyclo-oxygenase) PGE2 / PGF2 neutrophil adhesion molecules ICAM-1(intercellular adhesion molecule) LFA 1(leukocyte function associated antigen) induction of apoptosis

35. Techniques directed mutagenesis random mutagenesis STM -(signature tagged mutagenesis) - identifies genes essential for survival in vivo IVET-(in vivo expression technology) - examines promoter expression in vivo proteomics -compare proteome patterns under intra- and extra-cellular growth conditions microarray - subtractive and differential analysis of mRNA signature tagged mutagenesis (www.v-max.co.uk/stm.htm) Analysis of bacterial response to intracellular environment

36. Immune protection against intracellular bacteria interrupt infection anti-bacterial antibodies anti-bacterial vaccines antibiotics innate immune response cellular or humoral immune response eliminate infection cellular immune response opsonic humoral immune response

37. Concepts - intracellular bacteria • evolution of intracellular bacteria (pros / cons) • types / strategies of intracellular pathogens • mechanisms of host cell manipulation • different strategies of individual pathogens • experimental analysis of host and bacterial cell response to intracellular living • immune protection / response to intracellular bacteria