The increasing use of polymyxins1 in addition to the dissemination of plasmid-borne colistin resistance threatens to cause a serious breach in our last line of defense against multidrug resistant Gram-negative pathogens, and heralds the emergence of truly pan-resistant infections. Colistin resistance often arises through covalent modification of lipid A with cationic residues such as phosphoethanolamine (PEtN) – as is mediated by Mcr-12 – which reduce the affinity of polymyxins for lipopolysaccharide (LPS)3. Thus, new strategies are needed to address the rapidly diminishing number of treatment options for Gram-negative infections4. The difficulty in eradicating Gram-negative bacteria is largely due to a highly impermeable outer membrane, which serves as a barrier to many otherwise effective antibiotics5. Here, we describe an unconventional screening platform designed to enrich for non-lethal, outer membrane-active compounds with potential as adjuvants for conventional antibiotics. This approach identified the antiprotozoal drug pentamidine6 as an effective perturbant of the Gram-negative outer membrane through its interaction with LPS. Pentamidine displayed synergy with antibiotics typically restricted to Gram-positive bacteria, yielding effective drug combinations with activity against a wide range of Gram-negative pathogens in vitro, and against systemic Acinetobacter baumannii infections in mice. Notably, the adjuvant activity of pentamidine persisted in polymyxin resistant bacteria in vitro and in vivo. Overall, pentamidine and structural analogs represent unexploited molecules for the treatment of Gram-negative infections, particularly those having acquired polymyxin resistance determinants.

Salmonella Typhimurium has a broad arsenal of genes that are tightly regulated and coordinated to facilitate adaptation to the various host environments it colonizes. The genome of Salmonella Typhimurium has undergone multiple gene acquisition events and has accrued changes in non-coding DNA that have undergone selection by regulatory evolution. Together, at least 17 horizontally acquired pathogenicity islands (SPIs), prophage-associated genes, and changes in core genome regulation contribute to the virulence program of Salmonella. Here, we review the latest understanding of these elements and their contributions to pathogenesis, emphasizing the regulatory circuitry that controls niche-specific gene expression. In addition to an overview of the importance of SPI-1 and SPI-2 to host invasion and colonization, we describe the recently characterized contributions of other SPIs, including the antibacterial activity of SPI-6 and adhesion and invasion mediated by SPI-4. We further discuss how these fitness traits have been integrated into the regulatory circuitry of the bacterial cell through cis-regulatory evolution and by a careful balance of silencing and counter-silencing by regulatory proteins. Detailed understanding of regulatory evolution within Salmonella is uncovering novel aspects of infection biology that relate to host-pathogen interactions and evasion of host immunity.

The evolution of bacterial pathogenicity, heavily influenced by horizontal gene transfer, provides new virulence factors and regulatory connections that alter bacterial phenotypes. Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) are chromosomal regions that were acquired at different evolutionary times and are essential for Salmonella virulence. In the intestine of mammalian hosts, Salmonella expresses the SPI-1 genes that mediate its invasion to the gut epithelium. Once inside the cells, Salmonella down-regulates the SPI-1 genes and induces the expression of the SPI-2 genes, which favor its intracellular replication. The mechanism by which the invasion machinery is deactivated following successful invasion of host cells is not known. Here, we show that the SPI-2 encoded transcriptional regulator SsrB, which positively controls SPI-2, acts as a dual regulator that represses expression of SPI-1 during intracellular stages of infection. The mechanism of this SPI-1 repression by SsrB was direct and acts upon the hilD and hilA regulatory genes. The phenotypic effect of this molecular switch activity was a significant reduction in invasion ability of S. enterica serovar Typhimurium while promoting the expression of genes required for intracellular survival. During mouse infections, Salmonella mutants lacking SsrB had high levels of hilA (SPI-1) transcriptional activity whereas introducing a constitutively active SsrB led to significant hilA repression. Thus, our results reveal a novel SsrB-mediated mechanism of transcriptional crosstalk between SPI-1 and SPI-2 that helps Salmonella transition to the intracellular lifestyle.

Bacteria have evolved to sense and respond to their environment by altering gene expression and metabolism to promote growth and survival. In this work we demonstrate that Salmonella displays an extensive (>30 hour) lag in growth when subcultured into media where dicarboxylates such as succinate are the sole carbon source. This growth lag is regulated in part by RpoS, the RssB anti-adaptor IraP, translation elongation factor P, and to a lesser degree the stringent response. We also show that small amounts of proline or citrate can trigger early growth in succinate media and that, at least for proline, this effect requires the multifunctional enzyme/regulator PutA. We demonstrate that activation of RpoS results in the repression of dctA, encoding the primary dicarboxylate importer, and that constitutive expression of dctA induced growth. This dicarboxylate growth lag phenotype is far more severe across multiple Salmonella isolates than in its close relative E. coli. Replacing 200 nt of the Salmonella dctA promoter region with that of E. coli was sufficient to eliminate the observed lag in growth. We hypothesized that this cis-regulatory divergence might be an adaptation to Salmonella’s virulent lifestyle where levels of phagocyte-produced succinate increase in response to bacterial LPS, however we found that impairing dctA repression had no effect on Salmonella’s survival in acidified succinate or in macrophages. Importance Bacteria have evolved to sense and respond to their environment to maximize their chance of survival. By studying differences in the responses of pathogenic bacteria and closely related non-pathogens, we can gain insight into what environments they encounter inside of an infected host. Here we demonstrate that Salmonella diverges from its close relative E. coli in its response to dicarboxylates such as the metabolite succinate. We show that this is regulated by stress response proteins and ultimately can be attributed to Salmonella repressing its import of dicarboxylates. Understanding this phenomenon may reveal a novel aspect of the Salmonella virulence cycle, and our characterization of its regulation yields a number of mutant strains that can be used to further study it.

Highlights d Horizontal acquisition of SsrB rewired the regulatory landscape of S. enterica d SsrB divergently regulates flagellar motility in S. enterica and S. bongori d An evolved SsrB-binding region upstream of flhDC drives repression in S. enterica d SsrB motility repression promotes evasion of the host inflammasome during infection