HilD induces expression of Salmonella pathogenicity island 2 genes by displacing the global negative regulator H-NS from ssrAB.

Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) have essential roles in the pathogenesis of Salmonella enterica. Previously, we reported transcriptional cross talk between SPI-1 and SPI-2 when the SPI-1 regulator HilD induces expression of the SsrA/B two-component system, the central positive regulator of SPI-2, during the growth of Salmonella to late stationary phase in LB rich medium. Here, we further define the mechanism of the HilD-mediated expression of ssrAB. Expression analysis of cat transcriptional fusions containing different regions of ssrAB revealed the presence of negative regulatory sequences located downstream of the ssrAB promoter. In the absence of these negative cis elements, ssrAB was expressed in a HilD-independent manner and was no longer repressed by the global regulator H-NS. Consistently, when the activity of H-NS was inactivated, the expression of ssrAB also became independent of HilD. Furthermore, electrophoretic mobility shift assays showed that both HilD and H-NS bind to the ssrAB region containing the repressing sequences. Moreover, HilD was able to displace H-NS bound to this region, whereas H-NS did not displace HilD. Our results support a model indicating that HilD displaces H-NS from a region downstream of the promoter of ssrAB by binding to sites overlapping or close to those sites bound by H-NS, which leads to the expression of ssrAB. Although the role of HilD as an antagonist of H-NS has been reported before for other genes, this is the first study showing that HilD is able to effectively displace H-NS from the promoter of one of its target genes.

In silico identification and experimental characterization of regulatory elements controlling the expression of the Salmonella csrB and csrC genes.

The small RNAs CsrB and CsrC of Salmonella indirectly control the expression of numerous genes encoding widespread cellular functions, including virulence. The expression of csrB and csrC genes, which are located in different chromosomal regions, is coordinated by positive transcriptional control mediated by the two-component regulatory system BarA/SirA. Here, we identified by computational analysis an 18-bp inverted repeat (IR) sequence located far upstream from the promoter of Salmonella enterica serovar Typhimurium csrB and csrC genes. Deletion analysis and site-directed mutagenesis of the csrB and csrC regulatory regions revealed that this IR sequence is required for transcriptional activation of both genes. Protein-DNA and protein-protein interaction assays showed that the response regulator SirA specifically binds to the IR sequence and provide evidence that SirA acts as a dimer. Interestingly, whereas the IR sequence was essential for the SirA-mediated expression of csrB, our results revealed that SirA controls the expression of csrC not only by binding to the IR sequence but also by an indirect mode involving the Csr system. Additional computational, biochemical, and genetic analyses demonstrated that the integration host factor (IHF) global regulator positively controls the expression of csrB, but not of csrC, by interacting with a sequence located between the promoter and the SirA-binding site. These findings contribute to the better understanding of the regulatory mechanism controlling the expression of CsrB and CsrC.

A distinct regulatory sequence is essential for the expression of a subset of nle genes in attaching and effacing Escherichia coli.

Enteropathogenic Escherichia coli uses a type III secretion system (T3SS), encoded in the locus of enterocyte effacement (LEE) pathogenicity island, to translocate a wide repertoire of effector proteins into the host cell in order to subvert cell signaling cascades and promote bacterial colonization and survival. Genes encoding type III-secreted effectors are located in the LEE and scattered throughout the chromosome. While LEE gene regulation is better understood, the conditions and factors involved in the expression of effectors encoded outside the LEE are just starting to be elucidated. Here, we identified a highly conserved sequence containing a 13-bp inverted repeat (IR), located upstream of a subset of genes coding for different non-LEE-encoded effectors in A/E pathogens. Site-directed mutagenesis and deletion analysis of the nleH1 and nleB2 regulatory regions revealed that this IR is essential for the transcriptional activation of both genes. Growth conditions that favor the expression of LEE genes also facilitate the activation of nleH1 and nleB2; however, their expression is independent of the LEE-encoded positive regulators Ler and GrlA but is repressed by GrlR and the global regulator H-NS. In contrast, GrlA and Ler are required for nleA expression, while H-NS silences it. Consistent with their role in the regulation of nleA, purified Ler and H-NS bound to the regulatory region of nleA upstream of its promoter. This work shows that at least two modes of regulation control the expression of effector genes in attaching and effacing (A/E) pathogens, suggesting that a subset of effector functions may be coordinately expressed in a particular niche or time during infection.

Integration of a complex regulatory cascade involving the SirA/BarA and Csr global regulatory systems that controls expression of the Salmonella SPI-1 and SPI-2 virulence regulons through HilD.

Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2) play key roles in the pathogenesis of Salmonella enterica. Previously, we showed that when Salmonella grows in Luria-Bertani medium, HilD, encoded in SPI-1, first induces the expression of hilA, located in SPI-1, and subsequently of the ssrAB operon, located in SPI-2. These genes code for HilA and the SsrA/B two-component system, the positive regulators of the SPI-1 and SPI-2 regulons respectively. In this study, we demonstrate that CsrA, a global regulatory RNA binding protein, post-transcriptionally regulates hilD expression by directly binding near the Shine-Dalgarno and translation initiation codon sequences of the hilD mRNA, preventing its translation and leading to its accelerated turnover. Negative regulation is counteracted by the global SirA/BarA two-component system, which directly activates the expression of CsrB and CsrC, two non-coding regulatory RNAs that sequester CsrA, thereby preventing it from binding to its target mRNAs. Our results illustrate the integration of global and specific regulators into a multifactorial regulatory cascade controlling the expression of virulence genes acquired by horizontal transfer events.

PerC and GrlA independently regulate Ler expression in enteropathogenic Escherichia coli.

Ler, encoded by the locus of enterocyte effacement (LEE) of attaching and effacing (A/E) pathogens, induces the expression of LEE genes by counteracting the silencing exerted by H-NS. Ler expression is modulated by several global regulators, and is activated by GrlA, which is also LEE-encoded. Typical enteropathogenic Escherichia coli (EPEC) strains contain the EAF plasmid, which carries the perABC locus encoding PerC. The precise role of PerC in EPEC virulence gene regulation has remained unclear, mainly because EPEC strains lacking the pEAF still express the LEE genes and because PerC is not present in other A/E pathogens such as Citrobacter rodentium. Here, we describe that either PerC or GrlA can independently activate ler expression and, in consequence, of LEE genes depending on the growth conditions. Both PerC and GrlA, with the aid of IHF, counteract the repression exerted by H-NS on ler and can also further increase its activity. Our results substantiate the role of PerC and GrlA in EPEC virulence gene regulation and suggest that these convergent regulatory mechanisms may have represented an evolutionary adaptation in EPEC to co-ordinate the expression of plasmid- and chromosome-encoded virulence factors needed to successfully colonize its intestinal niche.

HilD-mediated transcriptional cross-talk between SPI-1 and SPI-2.

The acquisition of new genetic traits by horizontal gene transfer and their incorporation into preexisting regulatory networks have been essential events in the evolution of bacterial pathogens. An example of successful assimilation of virulence traits is Salmonella enterica, which acquired, at distinct evolutionary times, Salmonella pathogenicity island 1 (SPI-1), required for efficient invasion of the intestinal epithelium and intestinal disease, and SPI-2, essential for Salmonella replication and survival within macrophages and the progression of a systemic infection. A positive regulatory cascade mainly composed of HilD, HilA, and InvF, encoded in SPI-1, controls the expression of SPI-1 genes, whereas the two-component regulatory system SsrA/B, encoded in SPI-2, controls expression of SPI-2 genes. In this study, we report a previously undescribed transcriptional cross-talk between SPI-1 and SPI-2, where the SPI-1-encoded regulator HilD is essential for the activation of both the SPI-1 and SPI-2 regulons but at different times during the stationary phase of growth in Luria-Bertani medium. Our data indicate that HilD counteracts the H-NS-mediated repression exerted on the OmpR-dependent activation of the ssrAB operon by specifically interacting with its regulatory region. In contrast, HilD is not required for SPI-2 regulon expression under the in vitro growth conditions that are thought to resemble the intracellular environment. Our results suggest that two independent SPI-2 activation pathways evolved to take advantage of the SPI-2-encoded information at different niches and, in consequence, in response to different growth conditions.

Mechanisms of post-transcriptional gene regulation in bacterial biofilms.

Biofilms are characterized by a dense multicellular community of microorganisms that can be formed by the attachment of bacteria to an inert surface and to each other. The development of biofilm involves the initial attachment of planktonic bacteria to a surface, followed by replication, cell-to-cell adhesion to form microcolonies, maturation, and detachment. Mature biofilms are embedded in a self-produced extracellular polymeric matrix composed primarily of bacterial-derived exopolysaccharides, specialized proteins, adhesins, and occasionally DNA. Because the synthesis and assembly of biofilm matrix components is an exceptionally complex process, the transition between its different phases requires the coordinate expression and simultaneous regulation of many genes by complex genetic networks involving all levels of gene regulation. The finely controlled intracellular level of the chemical second messenger molecule, cyclic-di-GMP is central to the post-transcriptional mechanisms governing the switch between the motile planktonic lifestyle and the sessile biofilm forming state in many bacteria. Several other post-transcriptional regulatory mechanisms are known to dictate biofilm development and assembly and these include RNA-binding proteins, small non-coding RNAs, toxin-antitoxin systems, riboswitches, and RNases. Post-transcriptional regulation is therefore a powerful molecular mechanism employed by bacteria to rapidly adjust to the changing environment and to fine tune gene expression to the developmental needs of the cell. In this review, we discuss post-transcriptional mechanisms that influence the biofilm developmental cycle in a variety of pathogenic bacteria.