Systematic analysis of the regulation of type three secreted effectors in Salmonella enterica serovar Typhimurium

BMC Microbiology Systematic analysis of the regulation of type three secreted effectors in Salmonella enterica serovar Typhimurium Yakhya Dieye 1 Jessica L Dyszel 0 Rebin Kader 1 Brian MM Ahmer 0 0 Department of Microbiology, The Ohio State University , Columbus, OH 43210 USA 1 Biodesign Institute, Arizona State University , Tempe, AZ 85287 USA Background: The type III secretion system (TTSS) is an important virulence determinant of Gram-negative bacterial pathogens. It enables the injection of effector proteins into the cytosol of eukaryotic cells. These effectors ultimately manipulate the cellular functions of the infected organism. Salmonella enterica serovar Typhimurium encodes two virulence associated TTSSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2 that are required for the intestinal and systemic phases of the infection, respectively. However, recent studies suggest that the roles of these TTSSs are not restricted to these compartments. The regulation of TTSSs in Salmonella is very complex with several regulators operating to activate or to repress expression depending on the environmental conditions. Results: We performed a systematic analysis of the regulation of type III effectors during growth in vitro. We have tested the ability of seven regulatory genes to regulate ten effector genes. Each regulator was expressed in the absence of the other six to avoid cascade effects. Our results confirm and extend the previously reported regulation of TTSS1 and TTSS2 effectors by InvFSicA and SsrB respectively. Conclusion: The set of strains constructed for this study can be used to quickly and systematically study the regulation of newly identified effector genes of Salmonella enterica. The approach we have used can also be applied to study complex regulatory cascades in other bacterial species. - Background The type III secretion system (TTSS) is a complex molecular machine found in numerous Gram-negative bacterial pathogens of animals and plants [1]. This secretion system encodes a syringe-like organelle that injects effector proteins directly into the cytosol of eukaryotic cells. The effectors ultimately affect host cell physiology. S. enterica serovar Typhimurium (hereafter referred to simply as Typhimurium) possesses two virulence associated TTSSs encoded by the Salmonella Pathogenicity Islands (SPI) 1 and 2 [2]. TTSS1 (encoded by SPI1) delivers effectors that mediate the invasion of epithelial cells and the intestinal events of Typhimurium infection [3-5]. TTSS2 is required during the systemic phase of Typhimurium infection [6]. It secretes effectors that allow the survival and multiplication of the pathogen within macrophages [7]. The regulation of the Typhimurium TTSSs is complex with several regulatory proteins involved (Figure 1). SPI1 encodes five regulators, four of which are known to be involved in regulatory cascades that lead to the expression of genes inside and outside of SPI1 [8]. The central regulator of SPI1 gene expression is HilA, a member of the OmpR/ToxR family [5,8]. HilA directly activates the expression of two SPI1 operons that encode TTSS1 apparatus components [5,9]. One of these operons begins with the invF gene, which encodes a transcriptional activator of the AraC family. InvF activates the expression of TTSS1 effectors encoded both inside and outside of SPI1 [10,11]. The activity of InvF requires the SicA co-regulator which is also encoded within SPI1 [12,13]. The third and fourth regulators are HilC and HilD, both of which belong to the AraC family. Both can directly activate the expression of hilA [14,15]. They can also activate the expression of the invF operon independently of HilA [16,17]. The fifth regulator encoded within SPI1, SprB, contains a LuxR/UhpA helix-turn-helix motif, however no target genes for SprB have been identified [18]. SPI1 gene expression is also regulated by factors encoded outside of SPI1 (Figure 1A). The SirA/BarA two-component system is an ancient regulatory system with orthologs found throughout the -proteobacteria that is involved in virulence gene expression, exoenzyme and antibiotic production, motility, and biofilm formation [19,20]. In Typhimurium, SirA has been shown to bind and activate the promoters of hilC and hilA [21], although an alternate model has also been proposed in which SirA acts through hilD rather than through hilA and hilC [22]. Additional regulators of SPI1 gene expression encoded outside of SPI1 include the two-component regulatory systems PhoPQ [23], PhoBR [24], and OmpR/EnvZ [24], and the AraC-like transcriptional activator RtsA [25]. How these regulatory inputs are integrated is not yet known. The central regulator of SPI2, and some TTSS2 effector genes located outside of SPI2, is the SsrAB two-component system [26]. SsrB has been shown to bind and activate the promoter of the srfH/sseI gene that encodes a TTSS2 effector [27]. Similarly, SsrB is thought to directly activate the expression of SPI2 operons as well as other A- SPI1 Effectors B- SPI2 Effectors effector genes located outside of SPI2 [28,29]. The expression of ssrA and ssrB is activated by factors encoded outside of SPI2 (Figure 1B). These include the twocomponent systems OmpR/EnvZ [30], PhoPQ [31], and the slyA gene [32]. Recently the ydgT gene was identified as a negative regulator of SPI2 gene expression [33]. The effectors secreted by TTSS1 are required for the invasion of intestinal epithelial cells [4,34]. In contrast, the SPI2 genes are induced after Typhimurium has invaded or is phagocytized by eukaryotic cells [29,31,35-37]. These observations led to the hypothesis that TTSS1 is needed to invade intestinal cells, but is not required during the subsequent phases of Typhimurium pathogenesis, while TTSS2 is expressed only when the bacteria reside within eukaryotic cells. Several recent reports suggest a more complicated role of the Typhimurium TTSSs. For example, SPI1 mutants have a replication defect and are unable to synthesize a normal SCV inside epithelial cells suggesting that SPI1 genes are involved in these functions [38]. Conversely, SPI2 genes have been shown to be involved in the induction of the inflammatory response caused by S. enterica serovar Dublin in a bovine ligated-ileal-loop model [39] and to be essential for the full virulence of Typhimurium in murine infectious enterocolitis [40,41]. These results suggest that each TTSS might be expressed in different compartments within the host, and that at least some effectors might be involved in more than one phase of the infection. Given the complexity of these regulatory hierarchies, we took a systematic in vitro approach to categorizing the regulatory inputs of effector genes in Typhimurium. Besides examining the effects of individual regulatory mutations on individual effectors, we wanted to test each regulatory gene in the absence of other regulators. For example, the regulatory hierarchy for SPI1 is complex in that (...truncated)