Proteomic analysis reveals the global effect of the BarA/SirA-Csr regulatory cascade in Salmonella Typhimurium grown in conditions that favor the expression of invasion genes.

In many bacteria, the BarA/SirA and Csr regulatory systems control expression of genes encoding a wide variety of cellular functions. The BarA/SirA two-component system induces the expression of CsrB and CsrC, two small non-coding RNAs that sequester CsrA, a protein that binds to target mRNAs and thus negatively or positively regulates their expression. BarA/SirA and CsrB/C induce expression of the Salmonella Pathogenicity Island 1 (SPI-1) genes required for Salmonella invasion of host cells. To further investigate the regulatory role of the BarA/SirA and Csr systems in Salmonella, we performed LC-MS/MS proteomic analysis using the WT S. Typhimurium strain and its derived ΔsirA and ΔcsrB ΔcsrC mutants grown in SPI-1-inducing conditions. The expression of 164 proteins with a wide diversity, or unknown, functions was significantly affected positively or negatively by the absence of SirA and/or CsrB/C. Interestingly, 19 proteins were identified as new targets for SirA-CsrB/C. Our results support that SirA and CsrB/C act in a cascade fashion to regulate gene expression in S. Typhimurium in the conditions tested. Notably, our results show that SirA-CsrB/C-CsrA controls expression of proteins required for the replication of Salmonella in the intestinal lumen, in an opposite way to its control exerted on the SPI-1 proteins. SIGNIFICANCE: The BarA/SirA and Csr global regulatory systems control a wide range of cellular processes, including the expression of virulence genes. For instance, in Salmonella, BarA/SirA and CsrB/C positively regulate expression of the SPI-1 genes, which are required for Salmonella invasion to host cells. In this study, by performing a proteomic analysis, we identified 164 proteins whose expression was positively or negatively controlled by SirA and CsrB/C in SPI-1-inducing conditions, including 19 new possible targets of these systems. Our results support the action of SirA and CsrB/C in a cascade fashion to control different cellular processes in Salmonella. Interestingly, our data indicate that SirA-CsrB/C-CsrA controls inversely the expression of proteins required for invasion of the intestinal epithelium and for replication in the intestinal lumen, which suggests a role for this regulatory cascade as a molecular switch for Salmonella virulence. Thus, our study further expands the insight into the regulatory mechanisms governing the virulence and physiology of an important pathogen.

CsrA Positively and Directly Regulates the Expression of the pdu, pocR, and eut Genes Required for the Luminal Replication of Salmonella Typhimurium.

Enteric pathogens, such as Salmonella, have evolved to thrive in the inflamed gut. Genes located within the Salmonella pathogenicity island 1 (SPI-1) mediate the invasion of cells from the intestinal epithelium and the induction of an intestinal inflammatory response. Alternative electron acceptors become available in the inflamed gut and are utilized by Salmonella for luminal replication through the metabolism of propanediol and ethanolamine, using the enzymes encoded by the pdu and eut genes. The RNA-binding protein CsrA inhibits the expression of HilD, which is the central transcriptional regulator of the SPI-1 genes. Previous studies suggest that CsrA also regulates the expression of the pdu and eut genes, but the mechanism for this regulation is unknown. In this work, we show that CsrA positively regulates the pdu genes by binding to the pocR and pduA transcripts as well as the eut genes by binding to the eutS transcript. Furthermore, our results show that the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the pdu and eut genes mediated by PocR or EutR, which are the positive AraC-like transcriptional regulators for the pdu and eut genes, respectively. By oppositely regulating the expression of genes for invasion and for luminal replication, the SirA-CsrB/CsrC-CsrA regulatory cascade could be involved in the generation of two Salmonella populations that cooperate for intestinal colonization and transmission. Our study provides new insight into the regulatory mechanisms that govern Salmonella virulence. IMPORTANCE The regulatory mechanisms that control the expression of virulence genes are essential for bacteria to infect hosts. Salmonella has developed diverse regulatory mechanisms to colonize the host gut. For instance, the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the SPI-1 genes, which are required for this bacterium to invade intestinal epithelium cells and for the induction of an intestinal inflammatory response. In this study, we determine the mechanisms by which the SirA-CsrB/CsrC-CsrA regulatory cascade controls the expression of the pdu and eut genes, which are necessary for the replication of Salmonella in the intestinal lumen. Thus, our data, together with the results of previous reports, indicate that the SirA-CsrB/CsrC-CsrA regulatory cascade has an important role in the intestinal colonization by Salmonella.

Two Additional Connections between the Transcriptional Programs Controlling Invasion and Intracellular Replication of Salmonella: HilD-SprB Positively Regulates phoP and slyA.

Salmonella virulence relies on the ability of this bacterium to invade the intestinal epithelium and to replicate inside macrophages, which are functions mainly encoded in Salmonella pathogenicity islands 1 and 2 (SPI-1 and SPI-2), respectively. Complex regulatory programs control the expression of SPI-1 and SPI-2 and functionally related genes, involving the integration of ancestral regulators and regulators that Salmonella has acquired during its evolution. Interestingly, some previous studies have revealed cross talk between the regulatory programs for SPI-1 and SPI-2. Here, we report two additional connections between the regulatory programs controlling the expression of genes for invasion and intracellular replication. Our results show that the acquired regulators HilD and SprB, both encoded in SPI-1, induce, in a cascade fashion, the expression of PhoP and SlyA, two ancestral regulators that activate the expression of SPI-2 and other genes required for intracellular replication. We provide evidence supporting that the regulation of phoP and slyA by HilD-SprB was adapted during the divergence of Salmonella from its closer species, Escherichia coli, with the acquisition of SPI-1 and thus the gain of HilD and SprB, as well as through cis-regulatory evolution of phoP and slyA. Therefore, our study further expands the knowledge about the intricate regulatory network controlling the expression of virulence genes in Salmonella. IMPORTANCE Bacteria have developed diverse regulatory mechanisms to control genetic expression, in the case of pathogenic bacteria, to induce the expression of virulence genes in particular niches during host infection. In Salmonella, an intricate regulatory network has been determined, which controls the spatiotemporal expression of the SPI-1 and SPI-2 gene clusters that mediate the invasion to and the replication inside host cells, respectively. In this study, we report two additional pathways of cross talk between the transcriptional programs for SPI-1 and SPI-2. Additionally, our results support that these additional regulatory pathways were adapted during the divergence of Salmonella from its closer species, Escherichia coli. This study further expands the knowledge about the mechanisms determining the Salmonella virulence.

The Two-Component System CpxRA Represses Salmonella Pathogenicity Island 2 by Directly Acting on the ssrAB Regulatory Operon.

The acquisition of Salmonella pathogenicity island 2 (SPI-2) conferred on Salmonella the ability to survive and replicate within host cells. The ssrAB bicistronic operon, located in SPI-2, encodes the SsrAB two-component system (TCS), which is the central positive regulator that induces the expression of SPI-2 genes as well as other genes located outside this island. On the other hand, CpxRA is a two-component system that regulates expression of virulence genes in many bacteria in response to different stimuli that perturb the cell envelope. We previously reported that the CpxRA system represses the expression of SPI-1 and SPI-2 genes under SPI-1-inducing conditions by decreasing the stability of the SPI-1 regulator HilD. Here, we show that under SPI-2-inducing conditions, which mimic the intracellular environment, CpxRA represses the expression of SPI-2 genes by the direct action of phosphorylated CpxR (CpxR-P) on the ssrAB regulatory operon. CpxR-P recognized two sites located proximal and distal from the promoter located upstream of ssrA. Consistently, we found that CpxRA reduces the replication of Salmonella enterica serovar Typhimurium inside murine macrophages. Therefore, our results reveal CpxRA as an additional regulator involved in the intracellular lifestyle of Salmonella, which in turn adds a new layer to the intricate regulatory network controlling the expression of Salmonella virulence genes. IMPORTANCE SPI-2 encodes a type III secretion system (T3SS) that is a hallmark for the species Salmonella enterica, which is essential for the survival and replication within macrophages. Expression of SPI-2 genes is positively controlled by the two-component system SsrAB. Here, we determined a regulatory mechanism involved in controlling the overgrowth of Salmonella inside macrophages. In this mechanism, CpxRA, a two-component system that is activated by extracytoplasmic stress, directly represses expression of the ssrAB regulatory operon; as a consequence, expression of SsrAB target genes is decreased. Our findings reveal a novel mechanism involved in the intracellular lifestyle of Salmonella, which is expected to sense perturbations in the bacterial envelope that Salmonella faces inside host cells, as the synthesis of the T3SS-2 itself.

An incoherent feedforward loop formed by SirA/BarA, HilE and HilD is involved in controlling the growth cost of virulence factor expression by Salmonella Typhimurium.

An intricate regulatory network controls the expression of Salmonella virulence genes. The transcriptional regulator HilD plays a central role in this network by controlling the expression of tens of genes mainly required for intestinal colonization. Accordingly, the expression/activity of HilD is highly regulated by multiple factors, such as the SirA/BarA two-component system and the Hcp-like protein HilE. SirA/BarA positively regulates translation of hilD mRNA through a regulatory cascade involving the small RNAs CsrB and CsrC, and the RNA-binding protein CsrA, whereas HilE inhibits HilD activity by protein-protein interaction. In this study, we show that SirA/BarA also positively regulates translation of hilE mRNA through the same mentioned regulatory cascade. Thus, our results reveal a paradoxical regulation exerted by SirA/BarA-Csr on HilD, which involves simultaneous opposite effects, direct positive control and indirect negative control through HilE. This kind of regulation is called an incoherent type-1 feedforward loop (I1-FFL), which is a motif present in certain regulatory networks and represents a complex biological problem to decipher. Interestingly, our results, together with those from a previous study, indicate that HilE, the repressor component of the I1-FFL reported here (I1-FFLSirA/BarA-HilE-HilD), is required to reduce the growth cost imposed by the expression of the genes regulated by HilD. Moreover, we and others found that HilE is necessary for successful intestinal colonization by Salmonella. Thus, these findings support that I1-FFLSirA/BarA-HilE-HilD cooperates to control the precise amount and activity of HilD, for an appropriate balance between the growth cost and the virulence benefit generated by the expression of the genes induced by this regulator. I1-FFLSirA/BarA-HilE-HilD represents a complex regulatory I1-FFL that involves multiple regulators acting at distinct levels of gene expression, as well as showing different connections to the rest of the regulatory network governing Salmonella virulence.

Molecular Features and mRNA Expression of the Receptor for Activated C Kinase 1 from Symbiodinium microadriaticum ssp. microadriaticum During Growth and the Light/Dark cycle.

Two genes of the RACK1 homolog from the photosynthetic dinoflagellate Symbiodinium microadriaticum ssp. microadriaticum (SmicRACK1), termed SmicRACK1A and SmicRACK1B, were found tandemly arrayed and displayed a single synonymous substitution (T/C) encoding threonine. They included two exons of 942 bp each, encoding 313 amino acids with seven WD-40 repeats and two PKC-binding motifs. The protein theoretical mass and pI were 34,200 Da and 5.9, respectively. SmicRACK1 showed maximum identities with RACK1 homologs at the amino acid and nucleotide level, respectively, of 92 and 84% with S. minutum, and phylogenetic analysis revealed clustered related RACK1 sequences from the marine dinoflagellates S. minutum, Heterocapsa triquetra, Karenia brevis, and Alexandrium tamarense. Interestingly, light-dependent regulatory elements were found both within the 282 bp SmicRACK1A promotor sequence, and within an intergenic sequence of 359 nucleotides that separated both genes, which strongly suggest light-related functions. This was further supported by mRNA accumulation analysis, which fluctuated along the light and dark phases of the growth cycle showing maximum specific peaks under either condition. Finally, qRT-PCR analysis revealed differential SmicRACK1 mRNA accumulation with maxima at 6 and 20 d of culture. Our SmicRACK1 characterization suggests roles in active growth and proliferation, as well as light/dark cycle regulation in S. microadriaticum.