A diffusible signal factor of the intestine dictates Salmonella invasion through its direct control of the virulence activator HilD

Abstract

Successful intestinal infection by Salmonella requires optimized invasion of the gut epithelium, a function that is energetically costly. Salmonella have therefore evolved to intricately regulate the expression of their virulence determinants by utilizing specific environmental cues. Here we show that a powerful repressor of Salmonella invasion, a cis-2 unsaturated long chain fatty acid, is present in the murine large intestine. Originally identified in Xylella fastidiosa as a diffusible signal factor for quorum sensing, this fatty acid directly interacts with HilD, the master transcriptional regulator of Salmonella, and prevents hilA activation, thus inhibiting Salmonella invasion. We further identify the fatty acid binding region of HilD and show it to be selective and biased in favour of signal factors with a cis-2 unsaturation over other intestinal fatty acids. Single mutation of specific HilD amino acids to alanine prevented fatty acid binding, thereby alleviating their repressive effect on invasion. Together, these results highlight an exceedingly sensitive mechanism used by Salmonella to colonize its host by detecting and exploiting specific molecules present within the complex intestinal environment.

AraC‐type regulators HilC and RtsA are directly controlled by an intestinal fatty acid to regulate Salmonella invasion

“…HilD, HilC, and RtsA constitute a positive feed‐forward regulatory loop that controls SPI‐1. We previously showed that the intestinal long‐chain fatty acid c2‐HDA (Figure 1a) is a potent repressive fatty acid of SPI‐1 expression that binds directly to HilD (K d ~3.5 µM) (Bosire et al., 2020; Chowdhury et al., 2021). c2‐HDA can repress SPI‐1 expression in Salmonella Δ hilD mutants and diminish hilA expression that is driven individually by HilC or RtsA (Bosire et al., 2020), suggesting that these regulators may also be targets of c2‐HDA.…”

“…They are involved in intraspecies, interspecies, and interkingdom communication, by which they control motility, biofilm formation, antibiotic tolerance, persistence, and virulence (An & Tang, 2018; Beaulieu et al., 2013; Chandrasekaran et al., 2018; Ryan et al., 2015; Saffarian et al., 2019; Suppiger et al., 2016; Twomey et al., 2012). The DSF c2‐HDA is an exceptionally potent repressive compound of SPI‐1 expression and is present in the murine large intestine (Bosire et al., 2020; Chowdhury et al., 2021). It binds to HilD with high affinity and prevents it from binding to hilC and hilA promoters.…”

“…It binds to HilD with high affinity and prevents it from binding to hilC and hilA promoters. It also destabilizes HilD, leading to the rapid degradation of this activator by Lon protease (Bosire et al., 2020; Chowdhury et al., 2021). However, it is not known whether c2‐HDA represses Salmonella invasion exclusively through its targeting of HilD.…”

Modulation of Quorum Sensing and Biofilms in Less Investigated Gram-Negative ESKAPE Pathogens

“…Recent studies showed that DSFs and other fatty acids inhibit key virulence mechanisms, such as planktonic growth, capsule production, and cell adhesion and induce biofilm dispersal in K. pneumoniae ( Rahmani-Badi et al, 2014 ; Gupta et al, 2020 ; Kumar et al, 2020 ). Chowdhury et al (2021) reported a cis -2-hexadecenoic acid (c2-HAD) DSF homolog encoding gene ( rpfF ) in Enterobacter sp. This DSF was proven to control the intestinal invasion of Salmonella sp.…”

“…This DSF was proven to control the intestinal invasion of Salmonella sp. and control the main virulence regulon in this microorganism ( Chowdhury et al, 2021 ). c2-HAD is supposed to interfere also with the virulence of other intestinal gram-negative bacteria, including Enterobacter species.…”