Stringent response interacts with the ToxR regulon to regulate Vibrio cholerae virulence factor expression

The epidemic diarrheal disease cholera is caused by the Gram-negative bacterium Vibrio cholerae. V. cholerae virulence factors include the toxin-coregulated pilus (TCP) and cholera toxin, which are major factors responsible for host colonization and production of diarrhea. Expression of cholera toxin and TCP genes is controlled by the ToxR regulon. The ToxR regulon includes the transcriptional activators ToxR, TcpP, and ToxT. ToxT directly initiates transcription of the cholera toxin and TCP genes. TcpP and ToxR are necessary for expression of toxT. TcpP and ToxR activity requires TcpH and ToxS, respectively. Additionally, ToxR is able to directly initiate transcription of the cholera toxin genes independent of TcpP and ToxT. TCP is required early in infection to colonize the small intestine, then cholera toxin is expressed later in infection to produce diarrhea. We tested whether stringent response, the low nutrient stress response, was involved in regulation of virulence genes. Using an infant mouse model, we found that V. cholerae strains with deletions of the stringent response genes were unable to colonize the small intestine. We further tested these stringent response-null mutants and found that stringent response was necessary for TCP expression, although effects on cholera toxin expression were not significant. We then tested whether stringent response regulation of TCP occurred through the ToxR regulon. We found that stringent response induced toxT and tcpPH expression, while repressing toxRS. This differential regulation of ToxR and TcpP may explain the differential expression of TCP and cholera toxin in vivo.