Virulence: Mechanisms of Gene Regulation II Regulating the Pathogenesis of Cholera CTX phage ToxT ctxA ctxB + CtxA CtxB

Cholera Pathogenesis Causative Agent life-threatening diarrheal disease can rapidly attain epidemic proportions through inadequate sanitation Causative Agent Vibrio cholerae extracellular pathogen highly motile, uniflagellated, Gram-negative curved rod

survive passage through the gastric acid barrier of the stomach To cause disease V. cholerae must: be ingested survive passage through the gastric acid barrier of the stomach colonize the upper small intestine produce & excrete toxin disseminate in a watery diarrhea

synthesizes virulence factors that help it reach, adhere to, & V. cholerae synthesizes virulence factors that help it reach, adhere to, & colonize the intestinal epithelial layer the genes that encode these virulence factors are regulated by environmental stimuli unique to the host

mutants lacking toxin genes CHOLERA TOXIN main virulence factor very potent PROOF mutants lacking toxin genes are less virulent

} CHOLERA TOXIN CTX phage A2 B ctxA ctxB A1 CtxA CtxB CtxB CtxB CtxB

A1 subunit enters host membrane epithelial cell cytoplasm CHOLERA TOXIN: binds host GM1 receptor A1 subunit enters host membrane A2 A1 B A2 B intestinal lumen Receptor A1 epithelial cell cytoplasm

a G (GTP-binding) protein epithelial cell cytoplasm A1 subunit modifies (ADP-ribosylation) a G (GTP-binding) protein A2 B intestinal lumen Receptor A1 GS GS ADPR epithelial cell cytoplasm

activates Adenylate cyclase modified G protein activates Adenylate cyclase A1 GS ADPR cAMP AC AC cAMP cAMP active inactive AC AC AC ATP ATP ATP

activates protein kinase A, which activates transporters, cAMP activates protein kinase A, which activates transporters, causing efflux of ions and water i.e., diarrhea HCO3- K+ Na+ H2O intestinal lumen H2O Na+ K+ K+ Na+ H2O H2O HCO3- P cAMP protein kinase A AC AC cAMP cAMP active inactive AC AC AC ATP ATP ATP

Virulence Factors involved in Colonization i) single polar flagellum nonmotile strains are less virulent flagellar genes expressed when toxin genes are not motility used to reach site of colonization motility genes turned off colonization and toxigenic genes turned on

Virulence Factors involved in Colonization (cont’d) ii) TCP (toxin colonization pilus) iii) ACF (accessory colonization factors) mutations in tcp or acf reduce colonization tcp & acf located on large pathogenicity island called TCP-ACF element

the synthesis of virulence factors activation of the ToxR regulon permits the synthesis of virulence factors

The ToxR Regulon Signal + + Cholera toxin tcp toxT acf ctxA ctxB toxR ToxS toxR toxS ToxR + ToxT acf ctxA ctxB toxT + tcp CtxA Cholera toxin CtxB

outside host low temp vibrio

toxR toxS transcription is turned on at low temperature Thus, ToxR & ToxS proteins are synthesized ToxR ToxS Lo ToC + toxR toxS

inserted into membrane ToxS ToxR ToxRS proteins inserted into membrane

host ingests vibrio high temp not yet in intestine

toxR toxS transcription off are no longer synthesized at high temperature Hi ToC - toxR toxS ToxR & ToxS proteins are no longer synthesized

ToxRS is still in membrane However no signal is received Thus ToxS ToxR ToxRS is still in membrane However no signal is received Thus ToxR regulon off

once inside the host intestine an intestinal (lumenal) signal However once inside the host intestine an intestinal (lumenal) signal is sent to ToxR & ToxS which activates the ToxR regulon vibrio

Lumenal signal received ToxRS activate ToxT synthesis ToxS ToxR toxT + ToxT ToxT autoregulates increases toxT transcription ToxS ToxR ToxT toxT + + ToxT ToxT ToxT ToxT Lots of ToxT synthesized

transcription of a pathogenicity island Signal TCP-ACF element ToxS ToxR ToxT tcp toxT ToxT acf + + + ToxT ToxT also activates transcription of a pathogenicity island the TCP-ACF element

ToxT also regulates transcription the lysogenic CTX prophage ctxA ToxT + CtxA ctxB CtxB CTX phage ToxT also regulates transcription of the ctxA ctxB operon carried by the lysogenic CTX prophage

- lumenal TCP-ACF signal pathogenicity island CTX prophage + ToxS ToxR ToxT tcp toxT ToxT acf + + + ToxT CTX prophage ToxR ToxS ToxT Lo ToC ctxA ctxB + + toxR toxS - Hi ToC CtxA CtxB chromosomal toxR toxS operon

The ToxR Regulon Signal + + Cholera toxin tcp toxT acf ctxA ctxB toxR ToxS toxR toxS ToxR + ToxT acf ctxA ctxB toxT + tcp CtxA Cholera toxin CtxB

The ToxR regulon stimulus = unknown (lumenal in nature) sensor = ToxS regulator = ToxR member genes: toxT tcp acf ctxAB NOTE: ToxT (a member of the ToxR regulon) is itself a regulator that activates many ToxR regulon promoters