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The enteric pathogen <jats:italic>Salmonella<\/jats:italic> controls its invasion machinery through the elegant coordination of three AraC\u2010type transcription activators, HilD, HilC, and RtsA. Most environmental signals target HilD to control invasion, whereas HilC and RtsA are known only to augment these effects on HilD. Here we show that a fatty acid found in the murine colon, <jats:italic>cis<\/jats:italic>\u20102\u2010hexadecenoic acid (c2\u2010HDA), represses <jats:italic>Salmonella<\/jats:italic> invasion by directly targeting HilC and RtsA, in addition to HilD. c2\u2010HDA directly binds each of these regulators and inhibits their attachment to DNA targets, repressing invasion even in the absence of HilD. Fatty acid binding, however, does not affect HilC and RtsA protein stability, unlike HilD. Importantly, we show that HilC and RtsA are highly effective in restoring HilD production and invasion gene expression after elimination of the repressive fatty acid c2\u2010HDA. Together, these results illuminate a precise mechanism by which HilC and RtsA may modulate invasion as <jats:italic>Salmonella<\/jats:italic> navigates through different regions of the intestine, contributing to our understanding of how this enteric pathogen senses and adapts to a diverse intestinal environment while maintaining its virulence.<\/jats:p>","DOI":"10.1111\/mmi.14835","type":"journal-article","created":{"date-parts":[[2021,10,23]],"date-time":"2021-10-23T09:51:02Z","timestamp":1634982662000},"page":"1464-1475","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["AraC\u2010type regulators HilC and RtsA are directly controlled by an intestinal fatty acid to regulate <i>Salmonella<\/i> invasion"],"prefix":"10.1111","volume":"116","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-4530-0029","authenticated-orcid":false,"given":"Rimi","family":"Chowdhury","sequence":"first","affiliation":[{"name":"Department of 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