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  • 1. Intracellular Pathogens
  • 2. Disease immune competent/compromised immunizations age trauma genetics antimicrobial therapy (intracellular pathogens) function of susceptibility of host relates to mechanism of bacterial pathogenesis secretion of factors (toxins) direct host cell manipulation
  • 3. CM CW CM OM CW CM CW (www.science.mcmaster.ca/ biochem/faculty/gupta…) rRNA rRNA rRNA Evolution of the ‘perfect’ intracellular bacteria chloroplasts (cyanobacteria) mitochondria (proteobacteria) Archaea halophiles methanogens thermophiles thermoplasma
  • 4. Philosophy of an intracellular pathogen
    • must over-come host barriers
    • resist innate immunity (phagocytic processes)
    • resist acquired immune responses
    • adapt to life in bacterially hostile environment
    Cons Pros
    • gain access to a protected environment
    • protection from immune response
    • protection from bacterial competitors
    • nutrient rich environment
  • 5. Types of intracellular bacterial pathogens obligate intracellular bacteria facultative intracellular bacteria Chlamydia spp. - pneumonia / genital infections Rickettsia spp. - typhus / Rocky Mountain Spotted Fever Coxiella burnetii - Q fever Mycobacterium spp. - tuberculosis / leprosy Salmonella spp. - typhoid /gastroenteritis Legionella pneumophila - Legionnaires’ disease Brucella spp. - brucellosis Francisella tularensis - tularemia Shigella spp. - dysentery Listeria monocytogenes - listeriosis Yersinia spp. - plague / gastroenteritis
  • 6. Strategies of intracellular bacteria Internalized by phagocytosis / proliferate in vacuole Induce cellular uptake - transient invasion Internalized - reside within vacuole Internalized - escape from vacuole - multiply in cytoplasm Mycobacterium tuberculosis Legionella pneumophila Uropathogenic E. coli Yersinia spp. Salmonella enterica Shigella flexneri Listeria moncytogenes
  • 7. Bacterial manipulation of host cell function I. exploitation of cytoskeleton II. manipulation of signaling processes III. effects on lipid and lipid metabolism IV. induction / inhibition of apoptosis ~ cellular microbiology ~
  • 8. I. Bacterial manipulation of host cytoskeleton   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
  • 9. (from P. Cossart, Cellular Microbiology, 2000) Shigella flexneri actin-mediated membrane ruffling Listeria monocytogenes actin-mediated movement bacterial manipulation of host actin ~ other bacteria that manipulate host cell actin ~ Yersinia spp. Salmonella spp. Pseudomonas aeruginosa Enteropathogenic E. coil (www.bio.brandeis.edu/ goodelab/)
  • 10. 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) Campylobacter jejuni infection of human embryonic epithelial cells (INT407) A. B.
  • 11. II. Bacterial manipulation of host cell signaling processes Bacteria Signaling pathway Yersinia Integrin signaling FAK / p130 Cas Listeria MAPKK NF-  B E-cadherin VASP / Arp2-Arp3 Salmonella IP 3 / Ca 2+ Rho / Rac / Cdc42 Shigella Src / cortactin Rho Vinculin /  -Actinin VASP / Arp2-Arp3
  • 12. III. Effects on lipid and lipid metabolism (From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004) role of host cell lipids in phagocytosis
  • 13. (From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004) manipulation of host cell lipids by bacterial pathogens
  • 14. IV. Bacterial modulation of apoptosis (LY Gao, YA Kwaik, Trends in Microbiology, 2000) Inhibition Induction
  • 15. Bacteria internalized by phagocytosis - proliferate in vacuole Mycobacterium tuberculosis Strategies of intracellular bacteria
  • 16. Mycobacterium tuberculosis Virulence factors - cord factor (waxy surface) induces granuloma formation LAM (lipoarabinomannan) PIM (phosphatidylinositol mannoside) Disease - tuberculosis - (consumption) caused by uncontrolled host inflammatory response => granuloma formation Pathogenesis - organism invades / lives in macrophages most commonly localized in lungs M. tuberculosis in mouse macrophages ( Courtesy Center for Tuberculosis Research, Johns Hopkins University) 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)
  • 17. Mycobacterium tuberculosis internalization/proliferation Mycobacterium resides / proliferates in vacuoles in phagocytic cells
    • Prevents acidification of vacuole by excluding proton pump ATPase
    Result - interference of endosomal vacuole maturation
    • 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
    (From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004)
  • 18. Phagosome maturation (Wilson, McNab, Henderson, Bacterial Disease Mechanisms, 2002) M. tuberculosis phagosome arrest 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)
  • 19. M.tuberculosis ~ granuloma 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) Healed, fibrotic granuloma shows calcification (blue circle). Active inflammation, giant cells and necrosis are absent. Cultures are negative. (pathhsw5m54.ucsf.edu/ overview/tb.html) TB giant cell in the granuloma (www.eastman.ucl.ac.uk/.../ tuberculosis.htm) A TB granuloma showing central necrosis and presence of giant cells (www.mrcophth.com/ pathology/granuloma.html)
  • 20. Bacteria that induce cellular uptake - but invade only transiently Yersinia spp. Strategies of intracellular bacteria
  • 21. Yersinia enterocolitica - enterocolitis (Yersiniosis) Yersinia pseudotuberculosis - animal pathogen Yersinia pestis - bubonic plague Virulence factors - first step in invasion - adherence invasin - binds host cell  1 integrins Ail - (attachment-invasion locus) YadA - ( Yersinia adherence) - binds  1 integrins, fibronectin, collagen, laminin (encoded on 78 kb virulence plasmid) Yersinia spp. Pathogenesis - invasive organism (julia.univ.gda.pl/~bioakk/grafika2/yersinia.jpg) Bacteriology - small, Gram-negative rod (perso.wanadoo.fr/.../ scrabble/arche_y.html)
  • 22. Yersinia internalization (Wilson, McNab, Henderson, Bacterial Disease Mechanisms, 2002) Yersinia spp. initially invade intestine through M cell interaction (www.ngfn.de/ngfn_en/ inf_tueb.html) Yersinia spp. internalized by ‘zipper’ mechanism
  • 23. Yersinia infectious process enters host internalized by host cells / phagocytosed by M  resists phagocytosis (T3S) survives in M  multiplies in M  INF  activates M  leave M  replicates extracellularly resists phagocytosis (T3S) Y. pestis Y. pseudotuberculosis Y. enterocolitica (Pujol, Bliska, Clinical Immunology 2005)
  • 24. Mechanism of Yersinia internalization
    • Yersinia invasin - binds to  1-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. Mechanism of transient invasion by Yersinia 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. YopH dephosphorylates a number of tyrosine-phosphorylated signaling proteins including Fyb, SKAP-HOM and p130 cas , thereby disrupting their abilities to mediate further downstream signaling events in the cytoskeletal pathway. YopE disrupts actin filaments by acting as a GTPase-activating protein for the GTPases Rac1, Rho and Cdc42. YopT proteolytically 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. (www.nature.com/.../ fig_tab/nature01603_F5.html)
  • 26. Bacteria internalized but escape from vacuole and multiply in cytoplasm Listeria moncytogenes Strategies of intracellular bacteria
  • 27. Listeria moncytogenes Pathogenesis - invasive organism (invades non-phagocytic cells) Adherence - leads to actin polymerization bacterial engulfment into vacuole by “ zipper” - mechanism Disease - food-bourne infection - listeriosis gastroenteritis / meningitis / abortions Virulence factors - Adherence - first step in invasion InlA - adheres to E-cadherin InlB - adheres to HGF receptor Met Vacuole membrane lysis - PLC 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)
  • 28. (www.diariomedico.com) Listeria moncytogenes zipper-internalization
  • 29. 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
    (From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004)
  • 30. Listeria escape from vacuole, grow, disseminate Intracellular movement - using ActA Cell-to-cell spread Formation and lysis of the two-membrane vacuole - Intervening membranes lysed using PLC & Mp1 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)
  • 31. (P. Cossart, H. Bierne, Current Opinion in Immunology, 2001) Model - actin assembly induced by Listeria ActA
  • 32. ( www.diariomedico.com/.../ 0,2458,69566,00.html) Listeria movement in cytoplasm & dissemination Listeria move through cytoplasm at a rate of 6-60  m per minute (olpaimages.nsf.gov/admin/images/listerias.jpg)
  • 33. Experimental analysis of host cell & bacterial response to intracellular living
  • 34. 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) TNF  prostaglandin release Cox-2 (cyclo-oxygenase) PGE 2 / PGF 2 neutrophil adhesion molecules ICAM-1 (intercellular adhesion molecule) LFA 1 (leukocyte function associated antigen) induction of apoptosis Microarray analysis of host cell mRNA expression
  • 35. Analysis of bacterial response to intracellular environment 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)
  • 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