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

Tw o functionally distinct Type III Secretion Systems for Salmonella pathogenesis Chiba 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

Type III protein secretion system (TTSS) effector proteins SipB, SipC translocon SipD host cell bacter bact erial al cel cell Outer membrane Inner membrane effector proteins

Salmonella spp important pathogens of humans and animals • Cau ause a w e a w ide v de var ariet ety of of di diseas ases es r rangi nging f ng from om m mild d diarrhoea to to severe system emic i c inf nfect ection ons 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

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

SPI2 function SPI1 function Intracellular grow th Invasion Systemic spreading Macrophage cell death

SPI1-Type III secretion system effect or translocon prot eins SipB, SipC SipD host cell Kubori et al. Scienc e 280:6 02-60 5 (19 98 ) Outer bacterial cell membra ne Inner membra ne effect or prot eins

SPI1-TTSS promotes invasion Effectors induce actin rearrangements and alter vacuole trafficking to trigger invasion, w ithout causing cellular damage SopB PI3P SCV G-actin SipC ADF/cofilin F-actin ,gelsolin SopE/E2 Proteasome Arp2/3 GTP GDP Rac,Cdc42 SopE/E2 F-actin SptP GTP GDP SipA Rac,Cdc42 PI3P: phosphatidylinositol (3,4,5)-3 phosphat e SCV: Salmone lla containing vacuole Arp2/3: actin-relate d protein com plex

SPI1-TTSS-dependent activation of caspase-1, leading to macrophage cell death, pyroptosis flagellin Ipaf Pro Caspase-1 SipB ASC Ions ? inflammasome Pro IL1 β ,Pro IL-18 H 2 O Caspase-1 IL-1 β ,IL-18 Cleavage of substrate Pyroptosis DNA fragmenta tion Forming membrane pore lysis

SPI2-Type III secretion system * SPI2: Salmonella pathogenicity Island at cs31 Cytoplasm of macrophage SseC, SseD SCV membrane SseB OM SsaC SsaJ IM SsaV SsaN （ ATPase) (SifA, SsaB, , SspH-2) SCV : Salmonella containing vacuole

SPI2-mediated SCV membrane dynamics, leading to Salmonella multiplication w ithin host cells SPI1 e ffect or Endocytic pathw ay SPI2 e ffect or ① EEA1 Rab5 Early endosome TR ② Rab11 vATPase Rab7 ③ Man-6PR Lgps Late endosome F-actin Microtubles MTOC Lgps Goldi Lysosome Cathepsin D vATPase Nucleus MTOC: microtube orga nizing cent er

Inverse regulation of expression of SPI-1 and SPI-2 genes Epithelial cell Macrophage Lon protease Inside macrophage cells Gut lumen Osmolarity ↑ Mg 2+ ↓ P i ↓ O 2 ↓ SPI-1 SPI-1 SPI-2 SPI-2 expression expression

Lon is induced as a stress response by Salmonella to hostile environment in macrophages H 2 O 2 stre ss σ 32 Low nutrition σ 32 σ 32 Acidic P( σ 32) P( σ 32) P( σ 70) P( σ 70) clpP clpX lon Lon ClpXP up regulation dow n dow n regulation regulation Coordinated SPI2-TTSS regulation SPI1-TTSS Flagellum

Transcription of SPI1 gene, Levels of SPI1 proteins hilA in macrophage cells ctivity) in macrophage cells 50 50 e a ct ase a 45 45 on ession eras type Δ lon No No Wild t Wi on 40 40 ucifer in infectio ion expres 35 35 MOI=100 100 MOI=10 10 MOI=10 10 e ac tivity/r-luci hilA exp Δ lon mutant 30 30 SipB 25 25 lar hi 20 20 llula dase ac Si SipC I ntrac ellu 15 15 osidas 10 10 ctos gala ct 5 Salmonella w ild type b-gal 0 0 0 2 2 6 10 10 24 24 (b- ti time (hr) Lon is essential for dow n-regulation of SPI1- expression in macrophage cells after phagocytosis

Lon degrades HilC HilC and HilD and HilD t to dow o dow n- n-regulat egulate t e the he expr expres ession of on of S SPI1 1 genes SPI1 at cs63 of Salmonella genome sic sip iac sic spt iag hil prg orghilspravr inv spa 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 Regulatory cascade of SPI1 gene expression + prgHIJK p hilA InvF SicA HilA hilA + p sicA p invF + + + + invFGEABCIJspaOPQRS sicAsipBCDA HilD HilC Degradation p sopE p sigD + sopE + sigDE Lon protease

Salmonella Δ lon mutant induces massive apoptosis in macrophages Δ lon w ild type MOI=10 MOI=10 Δ lon Δ lon Δ SPI1 MOI=1 MOI=10

Over-expression of SPI 1 activates caspase-8-dependent procaspase-3 activation pathw ay 20 Caspase-8 Relative ac tivit y 15 FADD Bid Bax 10 5 Pro mitochondria 0 Δ hilC Δ hilD No hilC + hilD + Caspase-8 infection Δ lon Apaf Cyto. C Caspase-8 Caspase-3 Caspase-9 Pro 6 Caspase-9 Relative activity Pro Caspase-3 5 4 Caspase-9 Caspase-3 3 Cleavage of substrate 2 Apopt osis 1 0 n 9 o O O r , r r i 8 8 8 o o o t S S c - - - o t t t M s s M s i i i e N b b b a a a D D f C i C i C i n h h h i n n n i i i

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 of 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.

Summary � 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

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