Signal Transduction & Virulence Low [Mg++] PhoQ pag (+) PhoP P (-) DBD prg

SALMONELLOSIS gastroenteritis and enteric fever caused by nontyphoid Salmonella increased significantly in the last decade (0.8 to 3.7 million cases per yr)

Salmonella enterica (serovar Typhimurium) = S. typhimurium INFECTIOUS AGENT Salmonella enterica (serovar Typhimurium) = S. typhimurium intracellular pathogen usually results in self-limiting gastroenteritis sometimes results in bacteremia, fever & other complications

a severe systemic disease of humans causes a similar disease in mice Typhoid fever a severe systemic disease of humans caused by S. typhi Research Model S. typhimurium causes a similar disease in mice

S. typhimurium to cause disease must survive and propagate in a wide range of tissues to survive and propagate, S. typhimurium must circumvent or hijack host immune system uses macrophages to enter the bloodstream HOW?

Ingestion of contaminated food or water But also, acidic macrophage Infection Ingestion of contaminated food or water must survive lysozyme in saliva acid stomach alkaline bile salts anaerobiosis high osmolarity low iron content But also, acidic macrophage HOW?

INVASION after surviving the trip, cells colonize the small intestine, invading the M cells (specialized epithelial cells that take up luminal antigens) Invasion occurs by Bacterial-mediated Endocytosis Two-steps i) attachment ii) entry

Attachment LUMEN vili M cell

Ruffle formation & phagocytosis phagosome ruffles LUMEN vili epithelial cell M cell macrophages

Entry LUMEN phagosome macrophages

M cell destroyed Entrance to Reticuloendothelial System to bloodstream

How does the pathogen Facilitate entry into M cells? Survival in phagosomes? Survival in macrophages 1. Type III secretory apparatus 2. Type III secretory apparatus 3. Type III secretory apparatus Signal transduction and gene expression

Phenotypic effects of Type III secretion mechanisms on host cells

facilitate entry into M cells? How does the pathogen facilitate entry into M cells? Type 3 secretory apparatus phagosome ruffles LUMEN M cell

How does the pathogen facilitate entry into M cells? Sip Sip inside M cell Sip Sip Sip LPS outer membrane Type III Apparatus periplasm cytoplasmic membrane SPI 1 large pathogenicity island includes inv & spa virulence genes encode a Type III secretory system which exports Sip proteins through bacterial cell surface into the host M cell Sip proteins facilitate invasion e.g. ruffling cytoplasm } invA spa sip (exported) SPI 1

kill bacteria in phagosomes? Why don’t lysosomes kill bacteria in phagosomes? phagosome Type 3 secretory apparatus Secretes proteins into the M cell cytosol Inhibiting phagosome-lysosome fusion LUMEN

} How does the pathogen facilitate entry into macrophages? Inside macrophage EP EP EP LPS outer membrane Type III Apparatus periplasm cytoplasmic membrane cytoplasm } another Type III secretory system (prgHIKL) exports proteins (EPs) through bacterial cell surface into the macrophage Facilitates phagocytosis by macrophage prgHIKL also encoded by SPI 1 prgHIJK EPs SPI 1

Survive in macrophages? How does the pathogen Survive in macrophages? macrophages

How does the pathogen survive the adverse environment once inside the macrophage, S. typhimurium represses genes necessary for entry (e.g., prgHIJK) & activates genes necessary to defeat macrophage defenses How does the pathogen survive the adverse environment of the macrophage?

How does the pathogen “sense” that is inside the macrophage?

(transcription factor) phosphodonor = phosphorylated sensor Two-component Signal Transduction SENSOR (receptor) Histidine autokinase phosphodonor = ATP RESPONSE REGULATOR (transcription factor) Aspartate autokinase phosphodonor = phosphorylated sensor

Two-component Signal Transduction An example

= ATP P P transcription repression ADP sensor phospho-donor RNAP RR DBD

sensor P ADP = ATP phospho-donor transcription activation RNAP RR DBD

Very Short List of VIRULENCE FACTORS CONTROLLED BY 2-COMPONENT SIGNALING CHEMOTAXIS - Escherichia coli & others PERTUSSIS TOXIN - Bordetella pertussis CAPSULE - Pseudomonas aeruginosa PhoPQ regulon - Salmonella typhimurium

{ = ATP P P PhoP prg (PhoP-repressed genes) ADP sensor phospho-donor RNAP P RR DBD { PhoP prg (PhoP-repressed genes)

(PhoP-activated genes) sensor P ADP = ATP phospho-donor RNAP P RR DBD { PhoP pag (PhoP-activated genes)

signal transduction pathway PhoP = response regulator PhoPQ a two-component signal transduction pathway PhoQ = sensor PhoP = response regulator represses prg activates pag Low [Mg++] PhoQ pag (+) P PhoP (-) DBD prg

prg (PhoP-repressed genes) pag (PhoP-activated genes) required for entry into M cells & macrophages (inv, spa, sip, prgHIJK) pag (PhoP-activated genes) survival in macrophages ASPs & anti-defensins [defensins are antimicrobial peptides produced by macrophages]

Evidence that PhoPQ system is required for virulence mutants i) are avirulent in mice ii) do not survive macrophages iii) are sensitive to low pH & defensins