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.

Although nanoparticle-tagged antimicrobal peptides have gained considerable importance in recent years, their structure-function correlation has not yet been explored. Here, we have studied the mechanism of action of a designed antimicrobial peptide, VG16KRKP (VARGWKRKCPLFGKGG), delivered via gold nanoparticle tagging against Salmonella infection by combining biological experiments with high- and low-resolution spectroscopic techniques. In comparison with the free VG16KRKP peptide or gold nanoparticle alone, the conjugated variant, Au-VG16KRKP, is non-cytotoxic to eukaryotic cells, but exhibits strong bacteriolytic activity in culture. Au-VG16KRKP can penetrate host epithelial and macrophage cells as well as interact with intracellular S. Typhi LPS under both in vitro and in vivo conditions. Treatment of mice with Au-VG16KRKP post-infection with S. Typhi resulted in reduced intracellular bacterial recovery and highly enhanced protection against S. Typhi challenge. The three-dimensional high resolution structure of nanoparticle conjugated VG16KRKP depicted the generation of a well-separated amphipathic structure with slight aggregation, responsible for the increase of the local concentration of the peptide, thus leading to potent activity. This is the first report on the structural and functional characterization of a nanoparticle conjugated synthetic antimicrobial peptide that can kill intracellular pathogens and eventually protect against S. Typhi challenge in vivo.

SummaryAdhesion and invasion of Intestinal Epithelial Cells (IECs) are critical for the pathogenesis of Salmonella Typhi, the aetiological agent of human typhoid fever. While type three secretion system-1 (T3SS-1) is a major invasion apparatus of Salmonella, independent invasion mechanisms were described for non-typhoidal Salmonellae. Here, we show that T2942, an AIL-like protein of S. Typhi Ty2 strain, is required for adhesion and invasion of cultured IECs. That invasion was T3SS-1 independent was proved by ectopic expression of T2942 in the non-invasive E. coli BL21 and double-mutant Ty2 (Ty2Δt2942ΔinvG) strains. Laminin and fibronectin were identified as the host-binding partners of T2942 with higher affinity for laminin. Standalone function of T2942 was confirmed by cell adhesion of the recombinant protein, while the protein or anti-T2942 antiserum blocked adhesion/ invasion of S. Typhi, indicating specificity. A 20-amino acid extracellular loop was required for invasion, while several loop regions of T2942 contributed to adhesion. Further, T2942 cooperates with laminin-binding T2544 for adhesion and T3SS-1 for invasion. Finally, T2942 was required and synergistically worked with T3SS-1 for pathogenesis of S. Typhi in mice. Considering wide distribution of T2942 among clinical strains, the protein or the 20-mer peptide may be suitable for vaccine development.

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