Tw o functionally distinct Type III Secretion Systems for Salmonella - - PowerPoint PPT Presentation

Tw o functionally distinct Type III Secretion Systems for Salmonella pathogenesis

Tomoko Yamamoto Tomoko Yamamoto Department of Microbiology and Molecular Genetics, Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Graduate School of Pharmaceutical Sciences, Chiba University Chiba University

Chiba

SipB, SipC SipD translocon host cell effector proteins Outer membrane

Type III protein secretion system (TTSS)

bact bacter erial al cel cell Inner membrane effector proteins

• Cau

ause a w e a w ide v de var ariet ety of

• f di

diseas ases es r rangi nging f ng from

• m m

mild d diarrhoea to to severe system emic i c inf nfect ection

• ns l

like t ke typhoi phoid f d fev ever er

• Estimated 16 million cases of typhoid fever per

year occur w ith about 600,000 fatal out comes

an interesting model organism

• For the study of host-pat hogen interaction
• able to enter into non-phagocytic cells (e.g. epithelial cells)

and grow w ithin phagocytic cells (e.g. macrophages)

Salmonella Pathogenicity island (SPI): SPI1 and SPI2 encoding Type III protein secretion syst em

Salmonella spp

important pathogens of humans and animals

Functions of SPI-1 and SPI-2 Type III secretion systems (TTSSs) on Salmonella pathogenesis Inverse regulation of SPI1-TTSS and SPI2-TTSS w ithin macrophages Control

• f

host macrophage cell death, pyroptosis and apoptosis, by SPI-1 effectors

Outline

Systemic spreading

SPI2 function

Invasion

SPI1 function

Macrophage cell death Intracellular grow th

SipB, SipC SipD

translocon host cell

effect or prot eins Outer membra ne Kubori et al. Scienc e 280:6 02-60 5 (19 98 )

SPI1-Type III secretion system

bacterial cell

Inner membra ne effect or prot eins

SPI1-TTSS promotes invasion

SopE/E2 Arp2/3 F-actin G-actin F-actin SipA ADF/cofilin ,gelsolin SipC Rac,Cdc42 GTP GDP

Effectors induce actin rearrangements and alter vacuole trafficking to trigger invasion, w ithout causing cellular damage

SptP SopE/E2 Proteasome SopB GTP GDP Rac,Cdc42 PI3P

PI3P: phosphatidylinositol (3,4,5)-3 phosphat e SCV: Salmone lla containing vacuole

SCV

Arp2/3: actin-relate d protein com plex

SPI1-TTSS-dependent activation of caspase-1, leading to macrophage cell death, pyroptosis

Caspase-1

Ipaf ASC Pro IL1β,Pro IL-18 IL-1β,IL-18 Ions H 2O Cleavage of substrate lysis inflammasome Pyroptosis

SipB

? flagellin Pro Caspase-1

DNA fragmenta tion Forming membrane pore

* SPI2:Salmonella pathogenicity Island at cs31

Cytoplasm of macrophage SCV membrane

SsaN（ATPase) SsaC SsaJ SsaV SseB SseC, SseD (SifA, SsaB, , SspH-2)

IM OM

SPI2-Type III secretion system

SCV :Salmonella containing vacuole

Nucleus Goldi ① ② ③ Microtubles Endocytic pathw ay Early endosome Late endosome MTOC

F-actin

Lysosome

EEA1 Rab5 TR Rab11 vATPase Rab7 Lgps Man-6PR Lgps Cathepsin D vATPase

SPI1 e ffect or SPI2 e ffect or

SPI2-mediated SCV membrane dynamics, leading to Salmonella multiplication w ithin host cells

MTOC: microtube orga nizing cent er

Inverse regulation of expression

• f SPI-1 and SPI-2 genes

Gut lumen

SPI-1 SPI-2 expression

Osmolarity↑ O2↓

Epithelial cell Inside macrophage cells

SPI-1 SPI-2 expression

Mg2+↓ Pi↓

Macrophage Lon protease

lon clpP clpX

P(σ70) P(σ32) P(σ32) P(σ70)

Lon σ32 σ32 σ32

Lon is induced as a stress response by Salmonella to hostile environment in macrophages

stre ss H 2O2 Low nutrition Acidic

ClpXP

Flagellum

dow n regulation Coordinated regulation

SPI1-TTSS

dow n regulation

SPI2-TTSS

up regulation

ti time (hr)

50 50 45 45 40 40 35 35 30 30 25 25 20 20 15 15 10 10 5 2 2 6 10 10 24 24

I ntrac ellu llula lar hi hilA exp expres ession

• n

(b- b-gal gala ct ctos

• sidas

dase ac e ac tivity/r-luci ucifer eras ase a e a ct ctivity)

Transcription of SPI1 gene, hilA in macrophage cells

Δlon mutant Salmonella w ild type

Wi Wild t type Δlon

• n

No No in infectio ion

MOI=100 100 MOI=10 10 MOI=10 10

Si SipC SipB

Levels of SPI1 proteins in macrophage cells

Lon is essential for dow n-regulation of SPI1- expression in macrophage cells after phagocytosis

prg inv sic sip iac sic spt iag hil

• rghilspravr

H F G E A B C I J O P Q R S A B C D A P P P B A D H I J K A C B A spa

SPI1 at cs63 of Salmonella genome Regulatory cascade of SPI1 gene expression

HilA hilA philA

Lon degrades HilC HilC and HilD and HilD t to dow

• dow n-

n-regulat egulate t e the he expr expres ession of

• n of S

SPI1 1 genes

prgHIJK + invFGEABCIJspaOPQRS pinvF InvF + psicA SicA sicAsipBCDA sigDE psigD psopE sopE + + + + HilC HilD + +

Lon protease Degradation

w ild type MOI=10 Δlon ΔSPI1 MOI=10 Δlon MOI=1

Salmonella Δlon mutant induces massive apoptosis in macrophages

Δlon MOI=10

Apopt osis Cleavage of substrate Pro Caspase-3 Pro Caspase-9 Apaf

• Cyto. C

Bax Bid Caspase-8 FADD mitochondria Pro Caspase-8

Over-expression of SPI 1 activates caspase-8-dependent procaspase-3 activation pathw ay

Caspase-9 Caspase-3

5 10 15 20 Δlon No infection hilC+hilD+ ΔhilCΔhilD Relative ac tivit y

Caspase-8 1 2 3 4 5

D M S O

6 Relative activity Caspase-3 Caspase-9

N

• i

n f e c t i

• n

C a s

• 8

i n h i b i t

• r

C a s

• 8

, 9 i n h i b i t

• r

D M S O C a s

• 8

i n h i b i t

• r

Physiological significance of control of cell death via negative regulation of SPI1 expression

At the initial stage of infection (intestinal phase of infection), Salmonella escape the macrophage killing mechanism by induction

• f flagellin-dependent and SPI1-dependent cell death, pyroptosis.

Once Salmonella has established a systemic infection, excess cell death like apoptosis w ould be detrimental to the pathogen because Salmonella resides in macrophage cells. It w ould be required to suppress apoptosis to allow time for the bacterium to replicate, escape and invade new macrophages for systemic infection. Therefore, negative regulation of SPI1-TTSS expression by Lon w hich is induced in response to the hostile environment in macrophage cells w ould be essential for the suppression of apoptosis through the control of caspase-8 activity in the macrophage cells after Salmonella infection.

Tw o functionally distinct TTSS SPI1 • invasion of epithelial cells

• release of inflammatory cytokines
• induction of cell death, pyroptosis
• induction of caspase-8, leading to apoptosis

SPI-2 • SCV membrane dynamics leading to replication and spatial distribution Inverse regulation of Tw o TTSSs Lon w hich is induced in Salmonella grow ing in macrophage cells after phagocytosis controls negatively SPI1 expression and positively SPI2 expression

Summary

Department of Microbiology and Molecular Genetics, Department of Microbiology and Molecular Genetics, Graduate School of Pharmaceutical Sciences, Chiba University Graduate School of Pharmaceutical Sciences, Chiba University