A Salmonella Typhi RNA thermosensor regulates virulence factors and innate immune evasion in response to host temperature

Sensing and responding to environmental signals is critical for bacterial pathogens to successfully infect and persist within hosts. Many bacterial pathogens sense temperature as an indication they have entered a new host and must alter their virulence factor expression to evade immune detection. Using secondary structure prediction, we identified an RNA thermosensor (RNAT) in the 5' untranslated region (UTR) of tviA encoded by the typhoid fever-causing bacterium Salmonella enterica serovar Typhi (S. Typhi). Importantly, tviA is a transcriptional regulator of the critical virulence factors Vi capsule, flagellin, and type III secretion system-1 expression. By introducing point mutations to alter the mRNA secondary structure, we demonstrate that the 5' UTR of tviA contains a functional RNAT using in vitro expression, structure probing, and ribosome binding methods. Mutational inhibition of the RNAT in S. Typhi causes aberrant virulence factor expression, leading to enhanced innate immune responses during infection. In conclusion, we show that S. Typhi regulates virulence factor expression through an RNAT in the 5' UTR of tviA. Our findings demonstrate that limiting inflammation through RNAT-dependent regulation in response to host body temperature is important for S. Typhi's "stealthy" pathogenesis. Author summary Salmonella enterica serovar Typhi (S. Typhi) is a human-restricted bacterial pathogen that causes 11-21 million new cases of typhoid fever worldwide each year. During acute infection, S. Typhi evades immune detection and rapidly spreads systemically, which has earned it the nickname of a "stealth" pathogen. The transcriptional regulator TviA is unique to S. Typhi and critical for this stealthy phenotype because it represses the expression of major pathogen-associated molecular patterns and induces the important virulence factor Vi capsule, which enable S. Typhi to evade immune detection. We show that S. Typhi regulates virulence factor expression in response to host body temperature via an RNA thermosensor (RNAT) located in the 5' untranslated region of tviA, which controls production of TviA protein in response to temperature fluctuations. Mutational perturbation of the tviA RNAT causes aberrant virulence factor expression in response to temperature and reveals that a functional RNAT is required for S. Typhi to evade innate immune detection and activation. Our work is the first to describe an RNAT in a critical virulence factor transcriptional regulator in S. Typhi and demonstrates the important role that temperature-sensing plays in S. Typhi pathogenesis.