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Disease
function of susceptibilityof host
relates to mechanism ofbacterial pathogenesis
immune competent/compromised
immunizationsage
trauma genetics
antimicrobial therapy
(intracellular pathogens)
secretion of factors (toxins) direct host cell manipulation
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)
Archaeahalophiles
methanogensthermophilesthermoplasma
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
Types of intracellular bacterial pathogens
obligate intracellular bacteriafacultative intracellular bacteria
Chlamydia spp. - pneumonia / genital infections
Rickettsia spp. - typhus / Rocky Mountain Spotted Fever
Coxiella burnetii - Q feverMycobacterium spp. - tuberculosis /
leprosy
Salmonella spp. - typhoid /gastroenteritisLegionella pneumophila - Legionnaires’
diseaseBrucella spp. - brucellosis
Francisella tularensis - tularemia
Shigella spp. - dysentery
Listeria monocytogenes - listeriosis
Yersinia spp. - plague / gastroenteritis
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 tuberculosisLegionella pneumophila
Uropathogenic E. coliYersinia spp.
Salmonella enterica
Shigella flexneriListeria moncytogenes
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 ~
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
(from P. Cossart, Cellular Microbiology, 2000)
Shigella flexneriactin-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 aeruginosaEnteropathogenic E. coil
(www.bio.brandeis.edu/ goodelab/)
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.
II. Bacterial manipulation of host cell signaling processes
BacteriaSignaling pathway
Yersinia Integrin signalingFAK / p130Cas
Listeria MAPKKNF-BE-cadherinVASP / Arp2-Arp3
Salmonella IP3 / Ca2+
Rho / Rac / Cdc42
Shigella Src / cortactinRhoVinculin / -ActininVASP / Arp2-Arp3
(From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004)
role of host cell lipids in phagocytosis
III. Effects on lipid and lipid metabolism
(From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004)
manipulation of host cell lipids by bacterial pathogens
(LY Gao, YA Kwaik, Trends in Microbiology, 2000)
IV. Bacterial modulation of apoptosisInhibitionInduction
Bacteria internalized by phagocytosis - proliferate in vacuole
Mycobacterium tuberculosis
Strategies of intracellular bacteria
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)
Mycobacterium tuberculosis internalization/proliferation
(From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004)
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
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)
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)
Bacteria that induce cellular uptake -but invade only transiently
Yersinia spp.
Strategies of intracellular bacteria
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.
(julia.univ.gda.pl/~bioakk/grafika2/yersinia.jpg)
Bacteriology - small, Gram-negative rod
Pathogenesis - invasive organism
(perso.wanadoo.fr/.../ scrabble/arche_y.html)
(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
Yersinia internalization
enters host internalized by host cells /phagocytosed by M
resists phagocytosis (T3S)
survives in M
multiplies in M
INF activates M
leave Mreplicates extracellularly
resists phagocytosis (T3S)
Y. pestisY. pseudotuberculosis
Y. enterocolitica
(Pujol, Bliska, Clinical Immunology 2005)
Yersinia infectious process
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)
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 p130cas, 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)
Bacteria internalized but escape from vacuole andmultiply in cytoplasm
Listeria moncytogenes
Strategies of intracellular bacteria
Listeria moncytogenes
Pathogenesis - invasive organism (invades non-phagocytic cells)
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)
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
Mechanism - Listeria monocytogenes internalization
(From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004)
• 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
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)
(P. Cossart, H. Bierne, Current Opinion in Immunology, 2001)
Model - actin assembly induced by Listeria ActA
(olpaimages.nsf.gov/admin/images/listerias.jpg)
(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
Analysis of host cell response to intracellular bacteria
induction pro-inflammatory cytokinesIL-8
MCP-1 (monocyte chemotatic protein 1)GMCSF (granulocyte-macrophage stimulating factor)
TNF
Microarray analysis of host cell mRNA expression
prostaglandin releaseCox-2 (cyclo-oxygenase)
PGE2 / PGF2
neutrophil adhesion moleculesICAM-1 (intercellular adhesion molecule)
LFA 1 (leukocyte function associated antigen)
induction of apoptosis
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)
Immune protection against intracellular bacteria
interrupt infectionanti-bacterial antibodiesanti-bacterial vaccines
antibioticsinnate immune response
cellular or humoral immune response
eliminate infectioncellular immune response
opsonic humoral immune response
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