HilE represses the activity of HilD via a mechanism distinct from that of intestinal long-chain fatty acids

Abstract

The expression of virulence factors essential for the invasion of host cells by Salmonella enterica is tightly controlled by a network of transcription regulators. The AraC/XylS transcription factor HilD is the main integration point of environmental signals into this regulatory network, with many factors affecting HilD activity. Long chain fatty acids (LCFAs), which are highly abundant throughout the host intestine directly bind to, and repress HilD, acting as environmental cues to coordinate virulence gene expression. The regulatory protein HilE also negatively regulates HilD activity, through a protein-protein interaction. Both of these regulators inhibit HilD dimerisation, preventing HilD from binding to target DNA. We investigated the structural basis of these mechanisms of HilD repression. LCFAs bind to a conserved pocket in HilD, in a comparable manner to that reported for other AraC/XylS regulators, whereas HilE forms a stable heterodimer with HilD by binding to the HilD dimerisation interface. Our results highlight two distinct mechanisms by which HilD activity is repressed, which could be exploited for the development of new antivirulence leads.

Discovery of a synthetic small molecule targeting the central regulator ofSalmonellapathogenicity

“…No activity of CDCA was observed at the highest tested concentration of 100 µM. These data are in line with the reported activities of the FAs 39 and CDCA 35 , and therefore confirm the reliability of the assay to quantify HilD transcriptional activity.…”

“…The bile acid CDCA has been shown to interfere with HilD dimerization and DNA binding 35 . Similarly, long chain fatty acids (LCFAs) have been shown to bind to HilD and block its dimerization 32,39 . We showed that C26 shares the same binding pocket as CDCA and LCFAs, however, its binding mode is suggested to be different since the inhibition occurs without interfering with the dimerization of the protein.…”

“…Taken together, our data suggest that C26 inhibits HilD binding to P hilA without disrupting its dimerization, a mechanism distinct from that of other inhibitors of HilD. We further investigated the effect of C26 on the formation of HilD-HilE heterodimers using a microscale thermophoresis (MST) dimerization assay that we previously described 39 . In contrast to oleic acid, which disrupted the binding of HilE to HilD, no effect on heterodimerization was observed for C26 (Extended data Fig.…”

“…4a). We previously identified a pocket involving the cupin barrel and α-helices 7 and 10 as the oleic acid binding site 39 . Assuming the same pocket to bind C26, we identified potential key amino acid positions involved in HilD-ligand interaction using simulations of protonated C26 within this pocket (Fig.…”

“…CDCA and oleic acid have been shown to inhibit the binding of HilD to DNA by disrupting HilD homodimerization 35,39 . We used multi-angle light scattering coupled to size-exclusion chromatography (SEC-MALS) to investigate whether C26 had a similar effect.…”