Diffusible Signal Factors Act through AraC-Type Transcriptional Regulators as Chemical Cues To Repress Virulence of Enteric Pathogens

Abstract

Successful colonization by enteric pathogens is contingent upon effective interactions with the host and the resident microbiota. These pathogens thus respond to and integrate myriad signals to control virulence. Long-chain fatty acids repress the virulence of the important enteric pathogens Salmonella enterica and Vibrio cholerae by repressing AraC-type transcriptional regulators in pathogenicity islands. While several fatty acids are known to be repressive, we show here that cis-2-unsaturated fatty acids, a rare chemical class used as diffusible signal factors (DSFs), are highly potent inhibitors of virulence functions. We found that DSFs repressed virulence-gene expression of enteric pathogens by interacting with transcriptional regulators of the AraC family. In S. Typhimurium, DSFs repress the activity of HilD, an AraC-type activator essential to the induction of epithelial cell invasion, by both preventing its interaction with target DNA and inducing its rapid degradation by Lon protease. cis-2-hexadecenoic acid (c2-HDA), a DSF produced by Xylella fastidiosa, was the most potent among those tested, repressing the HilD-dependent transcriptional regulator hilA and the type III secretion effector sopB by >200- and 68-fold, respectively. Further, c2-HDA attenuated the transcription of the ToxT-dependent cholera toxin synthesis genes of V. cholerae. c2-HDA significantly repressed invasion-gene expression by Salmonella in the murine colitis model, indicating that the HilD-dependent signaling pathway functions within the complex milieu of the animal intestine. These data argue that enteric pathogens respond to DSFs as interspecies signals to identify appropriate niches in the gut for virulence activation, which could be exploited to control the virulence of enteric pathogens.

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

“…LCFAs harboring a cis- oriented unsaturation at their second carbon strongly inhibit Salmonella invasion by preventing the binding of the invasion activator HilD to its DNA targets and inducing its rapid degradation [ 36 ]. As molecules of this class are produced by the Gammaproteobacteria and employed in quorum sensing, we sought to determine whether they exist as chemical signals within the mammalian intestine, and thus constitute a bona fide means by which Salmonella invasion might be modulated.…”

“…We have previously shown that LCFAs reduce invasion gene expression by inducing the rapid degradation of HilD by Lon protease [ 36 ]. To investigate if the binding of c2-HDA to specific residues of HilD targets the protein for degradation, we measured wild type and mutant HilD levels in Salmonella grown in the presence of LCFAs.…”

“…Finally, we have shown that the fatty acid interacting site of HilD is able to discern and preferentially respond to the DSF c2-HDA over LCFAs without the cis -2 conformation. Furthermore, Bosire et al [ 36 ] demonstrated that c2-HDA is superior amongst cis -2 unsaturated fatty acids of various carbon lengths in its control of SPI-1 gene expression. These findings suggest that the 16-carbon tail with a cis -2 unsaturation is the optimal LCFA to be accommodated into the binding pocket in an energetically favorable conformation, which may explain the increased potency of repression by c2-HDA over other cis -2 unsaturated LCFAs.…”

“…Invasion was quantified by gentamicin protection assay [ 36 , 51 , 52 ]. HEp-2 cells were seeded into 24-well cell culture plates (~10 5 /well) and allowed to attach overnight at 37° C with 5% CO 2 ).…”

“…These fatty acids are exported into the extracellular milieu for perception by other bacteria. RpfF of plant pathogen Xyllela fastidiosa produces the DSF cis -2-hexadecenoic acid (c2-HDA) that has been shown to drastically reduce invasion gene expression in Salmonella by reducing the half-life of HilD and preventing it from binding to the hilC promoter [ 36 ].…”