Intracellular Pathogens

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

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OM

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(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

(www.diariomedico.com)

Listeria moncytogenes zipper-internalization

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

Experimental analysis of host cell & bacterial response to intracellular living

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