Characterisation of HilD, the Central Activator of Salmonella Pathogenicity, and the Mechanisms Regulating its Activity

Abstract

Anti-virulence drugs present an attractive alternative to traditional antibiotics for the treatment of bacterial infections, such as those caused by the major food-borne pathogen Salmonella enterica. Expression of virulence factors essential for Salmonella infection is tightly controlled by a network of transcription regulators. The AraC-like transcription factor HilD is the main integration point of environmental signals into this regulatory network, and hence is an attractive target for novel anti-virulence drugs. This work investigates the different mechanisms by which HilD is regulated at the protein level to control the expression of virulence genes. Long chain fatty acids (LCFAs) are highly abundant in varying concentrations throughout the intestine and are utilised as environmental cues by Salmonella to coordinate expression of virulence genes. LCFAs bind directly to HilD, in a comparable manner to that reported for the other AraC-like transcription factors ToxT and Rns. HilD can accommodate the binding of a wide range of LCFAs with a chain length of 16 – 24 C-atoms. The binding of LCFAs induces conformational changes in the structure of HilD that disrupt both its dimerisation and DNA-binding ability. The regulatory protein HilE is structurally homologous to hemolysin-coregulated protein (Hcp), which is a structural component of the bacterial type VI secretion system. We found that unlike other Hcp family members, HilE exists as a monomer in solution. HilE forms a stable 1:1 complex with HilD. Using hydrogen-deuterium exchange mass-spectrometry (HDX-MS), we show that HilE directly disrupts HilD dimerisation by binding to the HilD dimerisation interface, thereby also preventing HilD from binding to target DNA. Our results highlight two distinct mechanisms by which HilD activity is repressed, which can be exploited for the development of new antivirulence leads. We previously identified the compound C26 as a novel inhibitor of Salmonella virulence gene expression. Here it is shown that C26 specifically binds to HilD and consequently inhibits its ability to bind to target DNA. HDX-MS indicated that C26 binds to the same binding pocket of HilD as LCFAs, albeit likely with a different binding mode. Unlike LCFAs, C26 does not affect HilD dimerisation nor its repression by HilE. We performed a structure-activity relationship analysis of C26 and have so far identified derivatives with 10-fold higher affinity for HilD. The C26 scaffold appears to be an attractive candidate for the development of new antivirulence compounds against Salmonella and our results provide a starting point for the optimisation of C26 into a lead compound.

Die Dissertation ist gesperrt bis zum 27. November 2024 !