Intracellular pathogens
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Intracellular Pathogens. 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.

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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)


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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…)


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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


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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


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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


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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


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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


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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


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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)


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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


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role of host cell lipids in phagocytosis

(From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004)

III. Effects on lipid and lipid metabolism


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manipulation of host cell lipids by bacterial pathogens

(From J. Pizarro-Cerda, P. Cossart, Nature Cell Biology, 2004)


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Induction

Inhibition

(LY Gao, YA Kwaik, Trends in Microbiology, 2000)

IV. Bacterial modulation of apoptosis


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Strategies of intracellular bacteria

Bacteria internalized by phagocytosis -

proliferate in vacuole

Mycobacterium tuberculosis


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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)


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(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


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Mycobacterial 2004)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)


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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


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Strategies of intracellular bacteria giant cells

Bacteria that induce cellular uptake -

but invade only transiently

Yersinia spp.


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Bacteriology - giant cellssmall, 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)


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Yersinia spp. giant cellsinitially 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


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enters host giant cells

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


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Mechanism of giant cells 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)


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(www.nature.com/.../ fig_tab/nature01603_F5.html) giant cells

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


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Strategies of intracellular bacteria giant cells

Bacteria internalized but escape from vacuole and

multiply in cytoplasm

Listeria moncytogenes


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Bacteriology - giant cellssmall 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


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Listeria moncytogenes giant cellszipper-internalization

(www.diariomedico.com)


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(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


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Intracellular movement - using ActA 2004)

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


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Model - actin assembly induced by 2004)Listeria ActA

(P. Cossart, H. Bierne, Current Opinion in Immunology, 2001)


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(olpaimages.nsf.gov/admin/images/listerias.jpg) 2004)

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)



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Microarray analysis of host cell mRNA expression intracellular living

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


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Techniques intracellular living

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


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Immune protection against intracellular bacteria intracellular living

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


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Concepts - intracellular bacteria intracellular living

  • 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


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