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 oxidative stress response of pathogenic Leptospira is controlled by two peroxide stress regulators which putatively cooperate in controlling virulence.

Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverished populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans. Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans, the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, a double perRAperRB mutant was avirulent. Interestingly, this correlated with major changes in gene and non-coding RNA expression. Notably, several virulence-associated genes (clpB, ligA/B, and lvrAB) were repressed. By obtaining a double mutant in a pathogenic Leptospira strain, our study has uncovered an interplay of two PerRs in the adaptation of Leptospira to oxidative stress with a putative role in virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network.

The FUR-like regulators PerRA and PerRB integrate a complex regulatory network that promotes mammalian host-adaptation and virulence of Leptospira interrogans.

Leptospira interrogans, the causative agent of most cases of human leptospirosis, must respond to myriad environmental signals during its free-living and pathogenic lifestyles. Previously, we compared L. interrogans cultivated in vitro and in vivo using a dialysis membrane chamber (DMC) peritoneal implant model. From these studies emerged the importance of genes encoding the Peroxide responsive regulators PerRA and PerRB. First described in in Bacillus subtilis, PerRs are widespread in Gram-negative and -positive bacteria, where regulate the expression of gene products involved in detoxification of reactive oxygen species and virulence. Using perRA and perRB single and double mutants, we establish that L. interrogans requires at least one functional PerR for infectivity and renal colonization in a reservoir host. Our finding that the perRA/B double mutant survives at wild-type levels in DMCs is noteworthy as it demonstrates that the loss of virulence is not due to a metabolic lesion (i.e., metal starvation) but instead reflects dysregulation of virulence-related gene products. Comparative RNA-Seq analyses of perRA, perRB and perRA/B mutants cultivated within DMCs identified 106 genes that are dysregulated in the double mutant, including ligA, ligB and lvrA/B sensory histidine kinases. Decreased expression of LigA and LigB in the perRA/B mutant was not due to loss of LvrAB signaling. The majority of genes in the perRA and perRB single and double mutant DMC regulons were differentially expressed only in vivo, highlighting the importance of host signals for regulating gene expression in L. interrogans. Importantly, the PerRA, PerRB and PerRA/B DMC regulons each contain multiple genes related to environmental sensing and/or transcriptional regulation. Collectively, our data suggest that PerRA and PerRB are part of a complex regulatory network that promotes host adaptation by L. interrogans within mammals.

The transcriptional response of pathogenic Leptospira to peroxide reveals new defenses against infection-related oxidative stress.

Pathogenic Leptospira spp. are the causative agents of the waterborne zoonotic disease leptospirosis. Leptospira are challenged by numerous adverse conditions, including deadly reactive oxygen species (ROS), when infecting their hosts. Withstanding ROS produced by the host innate immunity is an important strategy evolved by pathogenic Leptospira for persisting in and colonizing hosts. In L. interrogans, genes encoding defenses against ROS are repressed by the peroxide stress regulator, PerR. In this study, RNA sequencing was performed to characterize both the L. interrogans response to low and high concentrations of hydrogen peroxide and the PerR regulon. We showed that Leptospira solicit three main peroxidase machineries (catalase, cytochrome C peroxidase and peroxiredoxin) and heme to detoxify oxidants produced during peroxide stress. In addition, canonical molecular chaperones of the heat shock response and DNA repair proteins from the SOS response were required for Leptospira recovering from oxidative damage. Identification of the PerR regulon upon exposure to H2O2 allowed to define the contribution of this regulator in the oxidative stress response. This study has revealed a PerR-independent regulatory network involving other transcriptional regulators, two-component systems and sigma factors as well as non-coding RNAs that putatively orchestrate, in concert with PerR, the oxidative stress response. We have shown that PerR-regulated genes encoding a TonB-dependent transporter and a two-component system (VicKR) are involved in Leptospira tolerance to superoxide. This could represent the first defense mechanism against superoxide in L. interrogans, a bacterium lacking canonical superoxide dismutase. Our findings provide an insight into the mechanisms required by pathogenic Leptospira to overcome oxidative damage during infection-related conditions. This will participate in framing future hypothesis-driven studies to identify and decipher novel virulence mechanisms in this life-threatening pathogen.

The Single-Step Method of RNA Purification Applied to Leptospira.

Establishing a rapid method to obtain pure and intact RNA molecules has revolutionized the field of RNA biology, enabling laboratories to routinely perform RNA analysis such as Northern blot, reverse transcriptase quantitative PCR, and RNA sequencing. Here, we describe an application of the effective single-step method of RNA extraction (or guanidinium thiocyanate-phenol-chloroform extraction) applied to Leptospira species. This method is based on the powerful ability of guanidinium thiocyanate to inactivate RNases and on the different solubilities of RNA and DNA in acidic phenol. This method allows one to reproducibly obtain total RNAs with high yield and integrity, as determined by capillary electrophoresis, suitable for the RNA sequencing technology.

SlyA and HilD Counteract H-NS-Mediated Repression on the ssrAB Virulence Operon of Salmonella enterica Serovar Typhimurium and Thus Promote Its Activation by OmpR.

H-NS-mediated repression of acquired genes and the subsequent adaptation of regulatory mechanisms that counteract this repression have played a central role in the Salmonella pathogenicity evolution. The Salmonella pathogenicity island 2 (SPI-2) is an acquired chromosomal region containing genes necessary for Salmonella enterica to colonize and replicate in different niches of hosts. The ssrAB operon, located in SPI-2, encodes the two-component system SsrA-SsrB, which positively controls the expression of the SPI-2 genes but also other many genes located outside SPI-2. Several regulators have been involved in the expression of ssrAB, such as the ancestral regulators SlyA and OmpR, and the acquired regulator HilD. In this study, we show how SlyA, HilD, and OmpR coordinate to induce the expression of ssrAB under different growth conditions. We found that when Salmonella enterica serovar Typhimurium is grown in nutrient-rich lysogeny broth (LB), SlyA and HilD additively counteract H-NS-mediated repression on ssrAB, whereas in N-minimal medium (N-MM), SlyA antagonizes H-NS-mediated repression on ssrAB independently of HilD. Interestingly, our results indicate that OmpR is required for the expression of ssrAB independently of the growth conditions, even in the absence of repression by H-NS. Therefore, our data support two mechanisms adapted for the expression of ssrAB under different growth conditions. One involves the additive action of SlyA and HilD, whereas the other involves SlyA, but not HilD, to counteract H-NS-mediated repression on ssrAB, thus favoring in both cases the activation of ssrAB by OmpR.IMPORTANCE The global regulator H-NS represses the expression of acquired genes and thus avoids possible detrimental effects on bacterial fitness. Regulatory mechanisms are adapted to induce expression of the acquired genes in particular niches to obtain a benefit from the information encoded in the foreign DNA, as for pathogenesis. Here, we show two mechanisms that were integrated for the expression of virulence genes in Salmonella Typhimurium. One involves the additive action of the regulators SlyA and HilD, whereas the other involves SlyA, but not HilD, to counteract H-NS-mediated repression on the ssrAB operon, thus favoring its activation by the OmpR regulator. To our knowledge, this is the first report involving the coordinated action of two regulators to counteract H-NS-mediated repression.

Self-Conjugation of the Enteropathogenic Escherichia coli Adherence Factor Plasmid of Four Typical EPEC Isolates.

The enteropathogenic Escherichia coli (EPEC) adherence factor plasmid (pEAF) encodes the proteins involved in the biogenesis of the bundle-forming pilus (BFP), a key virulence factor that mediates microcolony formation and the localized adherence phenotype on the surface of the host enterocytes. The presence or absence of this plasmid defines typical EPEC (tEPEC) and atypical EPEC (aEPEC), respectively. Although lateral transfer of pEAF has been evidenced by phylogenetic studies, conjugal transfer ability has been experimentally established only for two pEAF plasmids from strains isolated in the late 60s. In the present work, we tested the self-conjugation ability of four pEAF plasmids from tEPEC strains isolated between 2007 and 2008 from children in Peru and the potential of aEPEC to receive them. A kanamycin resistance cassette was inserted into donor pEAF plasmids in order to provide a selectable marker in the conjugation experiments. Two aEPEC isolated from the same geographic region were used as recipient strains along with the laboratory E. coli DH5α strain. Here we show that the four pEAF plasmids tested are self-conjugative, with transfer frequencies in the range of 10-6 to 10-9. Moreover, the generation of aEPEC strains harboring pEAF plasmids provides valuable specimens to further perform functional studies.

Conjugative transfer of an IncA/C plasmid-borne blaCMY-2 gene through genetic re-arrangements with an IncX1 plasmid.

BACKGROUND: Our observation that in the Mexican Salmonella Typhimurium population none of the ST19 and ST213 strains harbored both the Salmonella virulence plasmid (pSTV) and the prevalent IncA/C plasmid (pA/C) led us to hypothesize that restriction to horizontal transfer of these plasmids existed. We designed a conjugation scheme using ST213 strain YU39 as donor of the blaCMY-2 gene (conferring resistance to ceftriaxone; CRO) carried by pA/C, and two E. coli lab strains (DH5α and HB101) and two Typhimurium ST19 strains (SO1 and LT2) carrying pSTV as recipients. The aim of this study was to determine if the genetic background of the different recipient strains affected the transfer frequencies of pA/C. RESULTS: YU39 was able to transfer CRO resistance, via a novel conjugative mechanism, to all the recipient strains although at low frequencies (10-7 to 10-10). The presence of pSTV in the recipients had little effect on the conjugation frequency. The analysis of the transconjugants showed that three different phenomena were occurring associated to the transfer of blaCMY-2: 1) the co-integration of pA/C and pX1; 2) the transposition of the CMY region from pA/C to pX1; or 3) the rearrangement of pA/C. In addition, the co-lateral mobilization of a small (5 kb) ColE1-like plasmid was observed. The transconjugant plasmids involving pX1 re-arrangements (either via co-integration or ISEcp1-mediated transposition) obtained the capacity to conjugate at very high levels, similar to those found for pX1 (10-1). Two versions of the region containing blaCMY-2 were found to transpose to pX1: the large version was inserted into an intergenic region located where the "genetic load" operons are frequently inserted into pX1, while the short version was inserted into the stbDE operon involved in plasmid addiction system. This is the first study to report the acquisition of an extended spectrum cephalosporin (ESC)-resistance gene by an IncX1 plasmid. CONCLUSIONS: We showed that the transfer of the YU39 blaCMY-2 gene harbored on a non- conjugative pA/C requires the machinery of a highly conjugative pX1 plasmid. Our experiments demonstrate the complex interactions a single strain can exploit to contend with the challenge of horizontal transfer and antibiotic selective pressure.