Two functionally distinct Type III Secretion Systems for Salmonella

pathogenesis

Tomoko YamamotoDepartment of Microbiology and Molecular Genetics,

Graduate School of Pharmaceutical Sciences,Chiba University

Chiba

SipB, SipCSipDtranslocon

host cell

effector proteins

Outer membrane

Type III protein secretion system (TTSS)

bacterial cell

Inner membrane

effector proteins

•Cause a wide variety of diseases ranging from mild diarrhoea to severe systemic infections like typhoid fever

•Estimated 16 million cases of typhoid fever per year occur with about 600,000 fatal outcomes

an interesting model organism•For the study of host-pathogen interaction

-able to enter into non-phagocytic cells (e.g. epithelial cells) and grow within phagocytic cells (e.g. macrophages)

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

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 within macrophages

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

Outline

Systemic spreading

SPI2 function

Invasion

SPI1 function

Macrophage cell death

Intracellular growth

SipB, SipCSipD

translocon

host cell

effector proteins

Outer membrane

Kubori et al. Science280:602-605 (1998)

SPI1-Type III secretion system

bacterial cell

Inner membrane

effector proteins

SPI1-TTSS promotes invasion

SopE/E2

Arp2/3

F-actin

G-actin

F-actin

SipA

ADF/cofilin,gelsolin

SipC

Rac,Cdc42GTP GDP

Effectors induce actin rearrangements and alter vacuole trafficking to trigger invasion, without causing cellular damage

SptP

SopE/E2Proteasome

SopB

GTP GDPRac,Cdc42

PI3P

PI3P: phosphatidylinositol (3,4,5)-3phosphate

SCV: Salmonella containing vacuole

SCV

Arp2/3: actin-related protein complex

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

H2O

Cleavage of substrate

lysis

inflammasome

Pyroptosis

SipB

?

flagellin

Pro Caspase-1

DNA fragmentationForming 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:Salmonellacontaining vacuole

Nucleus

Goldi

①

②

③

Microtubles

Endocytic pathway

Early endosome

Late endosome

MTOC

F-actin

Lysosome

EEA1Rab5TRRab11

vATPaseRab7Lgps Man-6PR

LgpsCathepsin DvATPase

SPI1 effector

SPI2 effector

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

MTOC: microtube organizing center

Inverse regulation of expressionof SPI-1 and SPI-2 genes

Gut lumen

SPI-1

SPI-2expression

Osmolarity↑ O2↓

Epithelial cell

Inside macrophage cells

SPI-1

SPI-2expression

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 Salmonellato hostile environment in macrophages

stressH2O2

Lownutrition

Acidic

ClpXP

Flagellum

down regulation

Coordinatedregulation

SPI1-TTSS

down regulation

SPI2-TTSS

up regulation

time(hr)

50

45

40

35

30

25

20

15

10

5

00 2 6 10 24

Intr

acel

lula

r hilA

exp

ress

ion

(b-g

ala

ctos

idas

e ac

tivi

ty/r

-luc

ifer

ase

act

ivit

y)

Transcription of SPI1 gene, hilA in macrophage cells

Δlon mutant

Salmonella wild type

Wild type Δlon No infection

MOI=100MOI=10 MOI=10

SipC

SipB

Levels of SPI1 proteins in macrophage cells

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

prginv sic sip iac sic spt iag hil org hil spravr

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

HilAhilA

philA

Lon degrades HilC and HilD to down-regulate the expression of SPI1 genes

prgHIJK+

invFGEABCIJspaOPQRSpinvF

InvF

+psicA

SicA

sicAsipBCDA

sigDE

psigDpsopE

sopE

++

++

HilC HilD

+ +

Lon protease

Degradation

wild type MOI=10

Δlon ΔSPI1 MOI=10

ΔlonMOI=1

Salmonella Δlon mutant induces massive apoptosis in macrophages

ΔlonMOI=10

Apoptosis

Cleavage of substrate

Pro Caspase-3

Pro Caspase-9

Apaf Cyto. C

BaxBid

Caspase-8

FADD

mitochondriaPro Caspase-8

Over-expression of SPI1 activates caspase-8-dependent procaspase-3 activation pathway

Caspase-9Caspase-3

0

5

10

15

20

Δlon

No infection

hilC+hilD+ ΔhilCΔhilD

Rel

ativ

e ac

tivi

ty

Caspase-8

01

2

3

4

5

DMSO

6

Rel

ativ

e ac

tivi

ty

Caspase-3 Caspase-9

No infe

ction

Cas-8

inhibito

rCas-8

, 9

inhibito

r

DMSOCas-8

inhi

bitor

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 would be detrimental to the pathogen because Salmonella resides in macrophage cells.

It would 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 which is induced in response to the hostile environment in macrophage cells would be essential for the suppression of apoptosis through the control of caspase-8 activity in the macrophage cells after Salmonella infection.

Two functionally distinct TTSSSPI1 • 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 toreplication and spatial distribution

Inverse regulation of Two TTSSsLon which is induced in Salmonella growing in

macrophage cells after phagocytosis controls negatively SPI1 expression and positively SPI2 expression

Summary

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