Human-pathogenic Yersinia species Prof. James B. Bliska · Scientific classification 2 Common features • Gram-negative rod-shaped bacteria • Facultative intracellular pathogens

Human-pathogenic Yersinia speciesProf. James B. Bliska

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Human-Pathogenic Yersinia Species

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Prof. James B. Bliska, Ph.D.Department of Molecular Genetics and Microbiology

Stony Brook University

Scientific classification

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Common features

• Gram-negative rod-shaped bacteria• Facultative intracellular pathogens• Adept at circumventing innate immunity (especially Y. pestis)• Single circular chromosome of approximately 4,600,000 nucleotides

– 12 genomes presently sequenced:

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12 genomes presently sequenced:Y. enterocolitica (1)Y. pestis (7)Y. pseudotuberculosis (4)

• Circular plasmid of approximately 70,000 nucleotides– pYV or pCD1– Encodes a type III secretion system



Classification of strains

• Y. enterocolitica

– 6 biogroups (1A, 1B, 2, 3, 4 and 5)

• Y. pseudotuberculosis

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– 21 serogroups based upon variations in O antigen of LPS

• Y. pestis

– 5 groups or biovars (Orientalis, Mediaevalis, Antiqua, Microtus, Pestoides)

5Carniel, E. Microbes and Infection, 3, 2001, 561-569

Overview of Yersinia infections

M cell in small intestine

Ingested

or

6Brendan Wren, 2003

test e

Macrophage

Rare event



Intestinal Yersinia infections

Septicemia

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Mesentericlymphadenitis

Enterocolitis

Adapted from Salyers and Whitt, Bacterial Pathogenesis, ASM Press

Intestinal Yersinia infections: transmission

• Linked to ingestion of contaminated foods, water, and milk; Contaminated foods include pork, tofu, and poultry

• May occur directly from dogs, cats, and swine

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• No role for airborne route of infection or for insect vectors in the transmission of disease

• Reports of person-to-person spread are conflicting and generally are not observed in large outbreaks

Intestinal infections: outcomes

• Diarrhea

– Most common clinical manifestation

– The usual presentation is characterized by diarrhea, low-grade fever, and abdominal pain lasting 1-3 weeks; Diarrhea may be bloody in severe cases

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may be bloody in severe cases

• Vomiting

– Present in approximately 40% of cases

• Mesenteric adenitis, mesenteric ileitis, or acute pseudoappendicitis

– These manifestations are characterized by fever, abdominal pain, tenderness of the right lower quadrant, and leukocytosis



Intestinal infections: outcomes cont.

• Reactive arthritis– Most commonly reported in Scandinavia, polyarticular arthritis

can occur after infection with Y. enterocolitica– The onset of joint symptoms typically occurs 1-2 weeks after

gastrointestinal illness and occurs

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in approximately 2% of patients• Erythemanodosum

– manifests as painful raised red or purple lesions, primarily on the lower extremities

• Septicemia– Most commonly in patients who have predisposing conditions;

Iron overload

Plague: overview• About 10-15 total cases/year in U.S

– Mainly southwestern Unites States

– Bubonic most common form

– Only 1-2 cases per year of pneumonic form

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Transmission modes of Y. pestis

Pneumonic plague

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Bubonic plague

p g

Adapted from Salyers and Whitt, Bacterial Pathogenesis, ASM Press



• Lacks O antigen

• Contains 2 additional plasmids required for unique aspects of disease

– pMT1

Phospholipase: required for survival in flea mid-gut

Unique features of Y. pestis

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Phospholipase: required for survival in flea mid-gut

F1 protein: promotes transmission following flea bite

– pPCP1

Pla: plasminogen activator and adhesin/invasin

Required for dissemination to lymph nodes after flea bite

Promotes bacterial growth in lungs during pneumonic plague

Unique features of Y. pestis cont.

• Pigmentation (pgm) segment

– Genes within (hms) code for extracellular matrix important for biofilm formation in flea

– Pgm segment encoded in Y. pseudotuberculosis

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but hms genes not highly expressed

– Two genes inactivated in Y. pestis

RcsA, a negative regulator of biofilms

The gene for a glycosylhydrolase (NghA) that cleaves beta-linked N-acetylglucosamine residues and reduces biofilm formation

Plague: bubonic

• Inguinal, axillary, or cervical lymph nodes most common

• 80% can become bacteremic

• 60% mortality if untreated

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y



Plague: pneumonic

• From aerosol or septicemic spread to lungs

• Person-to-person transmission by respiratory droplet

• 100% mortality if untreated

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Plague: pneumonic cont.

• Incubation: 1-3 days

• Sudden onset headache, malaise, fever, myalgia, cough

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Pneumonic infiltrateof pneumonic plague

• Pneumonia progresses rapidly to dyspnea, cyanosis, hemoptysis

• Death from respiratory collapse/sepsis

Attributes of a successful bacterial pathogen

1. Adhere to/invade into host cells

2. Regulate gene expression in response to environmental cues

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to environmental cues

3. Avoid, subvert or co-opt host immune responses

4. Acquire nutrients to replicate



Adhesion and invasion determinants• Pili

– Psa/Myf pili

• Afimbrial adhesins

– Ail

Major role in serum resistance

Y dA Lui et al Infect Immun 74

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– YadA

Encoded on plasmid; Gene inactivated in Y. pestis

Binds ECM proteins

• Invasin

– Gene (invA) inactivated in Y. pestis

– Important role in allowing enteropathogenicYersinia to invade M cells overlying Peyer’s patches

Lui et al., Infect. Immun. 74, 5636-44 (2006)

Mechanism of Yersinia invasion

Role in the disease process is to allow bacterial penetration of the gut-associated lymphoid tissue

20Adapted from Salyers and Whitt, Bacterial Pathogenesis, ASM Press

Small intestinal mucosa (ileum)

Invasin

• 986 amino acid outer-membrane protein

• D4-D5 domains bind

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5 do a s b dwith high affinity to several β1 integrins (α3, α4, α5, α6 and αV)

Hamburger Z. A. et al., Science, 286, 291-5, (1999)



Mechanism of invasin-promoted uptake

• Integrin clustering activates signaling pathway

• Actin rearrangements lead to zippering of membrane around bacterium

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Systemic infection in mice may bypass lymph node colonization

Septicemia

23Adapted from Salyers and Whitt, Bacterial Pathogenesis, ASM Press

Mesentericlymphadenitis

Enterocolitis

Regulate gene expression • Transcription factors

– RovA, member of the MarR/SlyA family of winged-helix transcription factors

Regulates expression of invasin and Psa

Important for virulence in intestinal and bubonicYersinia infections

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– PhoP, two-component regulator

Required for survival in macrophages

– VirF, AraC-like activator of the ysc-yop virulon encoded on pYV/pCD1



Avoid, subvert or co-opt host immune responses

After translocation across M cells, the bacteria encounter macrophages or dendritic cells

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Localization of Y. pseudotuberculosis in rabbit M cell and macrophage 4 hours post infection

26Fujimura et al. J. Clin. Electron Microscopy 1992

Extracellular location of Y. enterocolitica in rabbit Peyer’s patches 24 hr post infection

27Lian et al., J. Med. Microbiol. 1987



NaiveMφ

PMN

Extracellular replication

Resist phagocytosis

Simplified Yersinia pathogenesis model

28Apoptosis

EscapeAvoid

intracellular killing

Release?

PMN

Summary of vacuole trafficking studies

• Y. pestis-containing vacuoles acquire markers of late endosomes/lysosomes (between 1.5 to 8 hr post infection)

• PhoP-regulated genes required for survival in phagosomes

2920h post infection in a mouse bone-marrow-derived macrophage

for survival in phagosomes

• Spacious phagosome formation associated with replication at 8 hr post infection

• Does not require other known virulence factors (pCD1, Pla)

Grabenstein et al., Infect. Immun. 2006

..........

..

. ...

. . ..

.

.

.

Model of “YCV” trafficking

Pinocytosis

Earlyendosome

Spaciousphagosome

Phagocytosis

pH 7

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Nucleus

............. ...

......... ...

...

.. ..

.Lateendosome

Lysosomes

Golgi

?

Grabenstein et al., Infect. Immun. 2006Pujol et al., Infect. Immun. 2009



Simplified Yersinia pathogenesis model

NaiveMf

PMN

Extracellular replication

31Apoptosis

EscapeAvoid

intracellular killing

Release?

PMN

pCD1 encodes type III protein secretion system

Yops, LcrV

37°C

32Adapted from: Cornelis, Nature Reviews, 2002Thanassi and Hultgren, Curr. Opin. Cell. Biol., 2000

Yersinia

Yersinia type III secretion system

YopB/YopD

Yersinia37°C

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Host cell

EffectorsYopHPTPase

YopOGDI

YopJAcetyl transferase

YopTCys protease

YopMLRR

YopEGAP



Overview of Yop effectors: antagonize phagocytosis, inhibit cytokine production, induce apoptosis

34Navarro et al., 2005

PTP

Actin cytoskeleton rearrangement target Rho GTPases

35Viboud and Bliska, 2005

PTPasecounteracts phagocytosis Acetyl Transferase

inhibits MAPK and NF-κB pathways

Function unknown

The type III “injectisome”

36From Cornelis, G.R., Nature Reviews, 2002



LcrV

37Mueller et al., Science 2005

Acquire nutrients to replicate

38Carniel, E. Microbes and Infection, 3, 2001, 561-569

The Yersinia HPI comprises genes involved in uptake of iron

• HPI encodes genes involved in the synthesis and transport of the siderophore yersiniabactin

• The siderophore-Fe3+ complex recognizes a specific bacterial outer membrane receptor;

Proteins located in the periplasm and the inner membrane

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Proteins located in the periplasm and the inner membrane of the cell wall transport the complex into cytosol

• HPI is found in other enterobacteria such as Escherichia coli, Klebsiella, Citrobacter and Salmonella

Miller, M.C. et al., Journal of Inorganic Biochemistry, 100, 2006, 1495-1500



Some open questions in Yersinia pathogenesis

• Other than pMT1 and pPCP1, what are the critical genetic differences between Y. pestis and Y. pseudotuberculosisthat account for differences in virulence?

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• How does Yersinia block phagosome acidification

• How does translocation of type III effector proteins occur?

Thank you

• Members of the Yersinia research community who provided data discussed in this presentation

• Listeners

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References

• pMT1– Phospholipase: Hinnebusch B.J., et al., Science, 269, 733-5 (2002)– F1 protein: Sebbane F., et al., Infect Immun. 77, 1222-9 (2009)

• Hms genes and biofilm productionPMID 18523005

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– PMID: 18523005– PMID: 18931111

• Invasin– PMID: 10514372

• Dissemination of Y. pseudotuberculosis from intestine to blood stream in mice– PMID: 16754724– RovA

PMID: 15165608



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Human-pathogenic Yersinia species Prof. James B. Bliska · Scientific classification 2 Common features • Gram-negative rod-shaped bacteria • Facultative intracellular pathogens