Transcript Microbial Pathogenesis-CCMD 793 I Nyles Charon

Intracellular Pathogens
Disease
function of susceptibility
of host
immune
competent/compromised
immunizations
age
trauma
genetics
antimicrobial therapy
relates to mechanism of
bacterial pathogenesis
secretion of factors (toxins)
direct host cell manipulation
(intracellular pathogens)
Evolution of the ‘perfect’ intracellular bacteria
chloroplasts
(cyanobacteria)
mitochondria
(proteobacteria)
CM
rRNA
rRNA
Archaea
halophiles
methanogens
thermophiles
thermoplasma
CW
rRNA
CM
CW
(www.science.mcmaster.ca/ biochem/faculty/gupta…)
CW
CM
OM
Philosophy of an intracellular pathogen
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
Types of intracellular bacterial pathogens
obligate intracellular bacteria
Chlamydia spp. - pneumonia / genital
infections
Rickettsia spp. - typhus / Rocky
Mountain Spotted Fever
Coxiella burnetii - Q fever
Mycobacterium spp. - tuberculosis /
leprosy
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
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
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
I. Bacterial manipulation of host cytoskeleton
a actin filaments (6 nm)
flexible, helical polymer of actin
determine cell shape / movement / division
a 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
bacterial manipulation of host actin
Shigella flexneri
Listeria monocytogenes
actin-mediated membrane ruffling
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
bacterial use of host microtubules
Campylobacter jejuni infection of human embryonic epithelial cells (INT407)
A.
B.
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)
II. Bacterial manipulation of host cell signaling
processes
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
III. Effects on lipid and lipid metabolism
role of host cell lipids in phagocytosis
(From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004)
manipulation of host cell lipids by bacterial pathogens
(From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004)
IV. Bacterial modulation of apoptosis
Induction
Inhibition
(LY Gao, YA Kwaik, Trends in Microbiology, 2000)
Strategies of intracellular bacteria
Bacteria internalized by phagocytosis proliferate in vacuole
Mycobacterium tuberculosis
Mycobacterium tuberculosis
Bacteriology - slim, rod-shaped bacterium
acid-fast (waxy surface excludes Gram-stain)
Disease - tuberculosis - (consumption)
caused by uncontrolled host inflammatory
response => granuloma formation
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
Virulence factors cord factor (waxy surface) induces granuloma
formation
LAM (lipoarabinomannan)
PIM (phosphatidylinositol mannoside)
M. tuberculosis in mouse macrophages
(Courtesy Center for Tuberculosis Research, Johns Hopkins University)
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
• 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
Phagosome maturation
M. tuberculosis phagosome arrest
Mycobacterial phagosomes
(Wilson, McNab, Henderson, Bacterial Disease Mechanisms, 2002)
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
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)
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)
Strategies of intracellular bacteria
Bacteria that induce cellular uptake but invade only transiently
Yersinia spp.
Yersinia spp.
Yersinia enterocolitica - enterocolitis (Yersiniosis)
Yersinia pseudotuberculosis - animal pathogen
Yersinia pestis - bubonic plague
Bacteriology - small, Gram-negative rod
Pathogenesis - invasive organism
(julia.univ.gda.pl/~bioakk/grafika2/yersinia.jpg)
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)
(perso.wanadoo.fr/.../ scrabble/arche_y.html)
Yersinia internalization
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 infectious process
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)
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)
Mechanism of transient invasion by Yersinia
(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.
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.
Strategies of intracellular bacteria
Bacteria internalized but escape from vacuole and
multiply in cytoplasm
Listeria moncytogenes
Listeria moncytogenes
Bacteriology - small Gram-positive rod
motile / facultative anaerobe
growth is enhanced by presence of blood
Disease - food-bourne infection - listeriosis
gastroenteritis / meningitis / abortions
Pathogenesis - invasive organism
(invades non-phagocytic cells)
Virulence factors -
(www.geocities.com/ CapeCanaveral/3504/gallery.htm)
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
(www.ifr.ac.uk/ bacanova/project.html)
Listeria moncytogenes zipper-internalization
(www.diariomedico.com)
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
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
Model - actin assembly induced by Listeria ActA
(P. Cossart, H. Bierne, Current Opinion in Immunology, 2001)
Listeria movement in cytoplasm & dissemination
Listeria move through cytoplasm at a rate of 6-60 mm per minute
(olpaimages.nsf.gov/admin/images/listerias.jpg)
(www.diariomedico.com/.../ 0,2458,69566,00.html)
Experimental analysis of host cell & bacterial
response to intracellular living
Analysis of host cell response to intracellular bacteria
Microarray analysis of host cell mRNA expression
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
Analysis of bacterial response to intracellular environment
Techniques
signature tagged mutagenesis
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
(www.v-max.co.uk/stm.htm)
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
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