Edmundo Calva

Transcriptional regulation of type III secretion genes in enteropathogenic Escherichia coli: Ler antagonizes H-NS-dependent repression.

Secretion of effector proteins in enteropathogenic Escherichia coli (EPEC) is mediated by a specialized type III secretion system whose components are encoded in the LEE1, LEE2 and LEE3 operons. Using cat transcriptional fusions and primer extension analysis, we determined that the LEE2 and LEE3 operons are expressed from two overlapping divergent promoters, whose expression is negatively regulated by flanking common upstream and downstream silencing regulatory sequences (SRS1 and SRS2). In the absence of either SRS1 or SRS2, expression of the LEE2 and LEE3 operons became independent of Ler, a positive regulatory protein encoded by the first gene of the LEE1 operon. Similarly, in the absence of the histone‐like protein H‐NS, expression from both promoters became Ler independent even if both SRSs were present. In addition, the efficient expression of both the LEE2 and the LEE3 promoters required PerC (BfpW), a protein coded by the third gene of the per (bfpTVW) locus, but only in the presence of the EAF plasmid. Our deletion analysis also showed that the negative regulation observed in the presence of ammonium or at temperatures above 37°C (e.g. 40°C) required the SRSs or elements located therein. In contrast, the negative regulation observed in LB or at temperatures below 37°C (e.g. 25°C) was still observed even in the absence of both SRSs and seems to act only on the promoters. Together, these results suggest that Ler acts as an antirepressor protein that overcomes the H‐NS‐mediated silencing on the LEE2/LEE3 divergent promoter region, which is probably caused by the formation of a repressing H‐NS–nucleoprotein complex.

Transcriptional Regulation of the orf19 Gene and the tir-cesT-eae Operon of Enteropathogenic Escherichia coli

To establish an intimate interaction with the host epithelial cell surface, enteropathogenic Escherichia coli (EPEC) produces Tir, a bacterial protein that upon translocation and insertion into the epithelial cell membrane constitutes the receptor for intimin. The tir gene is encoded by the locus for enterocyte effacement (LEE), where it is flanked upstream by orf19 and downstream by the cesT and eae genes. With the use of a series of cat transcriptional fusions and primer extension analysis, we confirmed that tir, cesT, and eae form the LEE5 operon, which is under the control of a promoter located upstream from tir, and found that the orf19 gene is transcribed as a monocistronic unit. We also demonstrated that the LEE-encoded regulator Ler was required for efficient activation of both the tir and the orf19 promoters and that a sequence motif located between positions -204 and -157 was needed for the Ler-dependent activation of the tir operon. Sequence elements located between positions -204 and -97 were determined to be required for the differential negative modulatory effects exerted by unknown regulatory factors under specific growth conditions. Upon deletion of the upstream sequences, the tir promoter was fully active even in the absence of Ler, indicating that tir expression is subject to a repression mechanism that is counteracted by this regulatory protein. However, its full activation was still repressed by growth in rich medium or at 25 degrees C, suggesting that negative regulation also occurs at or downstream of the promoter. Expression of orf19, but not of the tir operon, became Ler independent in an hns mutant strain, suggesting that Ler overcomes the repression exerted by H-NS (histone-like nucleoid structuring protein) on this gene.

LeuO antagonizes H-NS and StpA-dependent repression in Salmonella enterica ompS1

The ompS1 gene encodes a quiescent porin in Salmonella enterica. We analysed the effects of H‐NS and StpA, a paralogue of H‐NS, on ompS1 expression. In an hns single mutant expression was derepressed but did not reach the maximum level. Expression in an stpA single mutant showed the same low repressed level as the wild type. In contrast, in an hns stpA background, OmpS1 became abundant in the outer membrane. The expression of ompS1 was positively regulated by LeuO, a LysR‐type quiescent regulator that has been involved in pathogenesis. Upon induction of the cloned leuO gene into the wild type, ompS1 was completely derepressed and the OmpS1 porin was detected in the outer membrane. LeuO activated the P1 promoter in an OmpR‐dependent manner and P2 in the absence of OmpR. LeuO bound upstream of the regulatory region of ompS1 overlapping with one nucleation site of H‐NS and StpA. Our results are thus consistent with a model where H‐NS binds at a nucleation site and LeuO displaces H‐NS and StpA.

Autoactivation and environmental regulation of bfpT expression, the gene coding for the transcriptional activator of bfpA in enteropathogenic Escherichia coli.

Expression of bfpA, the gene coding for the structural subunit of the bundle‐forming pili (BFP) in enteropathogenic Escherichia coli (EPEC), requires the product of bfpT (also called perA), a member of the AraC family of transcriptional regulators. Here, we show that bfpT–cat fusions were not expressed in a bfpT − or in a non‐EPEC strain, unless a functional bfpT was present, indicating that an autoregulatory mechanism is involved in expression. Further experiments with bfpT–cat fusions and primer extension analysis showed that bfpT is transcribed from a conventional sigma‐70 promoter and that it is expressed throughout the growth curve. It is regulated in response to the ammonium concentration, temperature and growth media, in the same proportions as those described previously for bfpA. In addition, bfpT and bfpA expression was also modulated by osmolarity, but was not affected by pH, iron excess or limitation. Deletion analysis of the bfpT upstream region revealed that a DNA segment of 81 bp, extending upstream from the transcriptional start site, contained all the sequence elements required for maximal expression of bfpT. Furthermore, it shares significant homology with a bfpA upstream AT‐rich region, which has been shown to be involved in the BfpT‐dependent regulation of bfpA. Interestingly, ammonium repression was observed only when bfpT–cat or bfpA–cat expression was complemented in an EPEC background, whereas low‐temperature regulation was observed in both EPEC and non‐EPEC strains. This suggests that specific regulatory elements are present in EPEC, while others are shared with non‐pathogenic E. coli.

Salmonella porins induce a sustained, lifelong specific bactericidal antibody memory response

We examined the ability of porins from Salmonella enterica serovar typhi to induce a long‐term antibody response in BALB/c mice. These porins triggered a strong lifelong production of immunoglobulin G (IgG) antibody in the absence of exogenous adjuvant. Analysis of the IgG subclasses produced during this antibody response revealed the presence of the subclasses IgG2b, IgG1, IgG2a and weak IgG3. Despite the high homology of porins, the long‐lasting anti‐S. typhi porin sera did not cross‐react with S. typhimurium. Notably, the antiporin sera showed a sustained lifelong bactericidal‐binding activity to the wild‐type S. typhi strain, whereas porin‐specific antibody titres measured by enzyme‐linked immunosorbent assay (ELISA) decreased with time. Because our porin preparations contained the outer membrane proteins C and F (OmpC and OmpF), we evaluated the individual contribution of each porin to the long‐lasting antibody response. OmpC and OmpF induced long‐lasting antibody titres, measured by ELISA, which were sustained for 300 days. In contrast, although OmpC induced sustained high bactericidal antibody titres for 300 days, postimmunization, the bactericidal antibody titre induced by OmpF was not detected at day 180. These results indicate that OmpC is the main protein responsible for the antibody‐mediated memory bactericidal response induced by porins. Taken together, our results show that porins are strong immunogens that confer lifelong specific bactericidal antibody responses in the absence of added adjuvant.

The CRISPR/Cas Immune System Is an Operon Regulated by LeuO, H-NS, and Leucine-Responsive Regulatory Protein in Salmonella enterica Serovar Typhi

Prokaryotes have developed multiple strategies to survive phage attack and invasive DNA. Recently, a novel genetic program denominated the CRISPR/Cas system was demonstrated to have a role in these biological processes providing genetic immunity. This defense mechanism is widespread in the Archaea and Bacteria, suggesting an ancient origin. In the last few years, progress has been made regarding the functionality of the CRISPR/Cas system; however, many basic aspects of the system remain unknown. For instance, there are few studies about the conditions and regulators involved in its transcriptional control. In this work, we analyzed the transcriptional organization of the CRISPR/Cas system as well as the positive and negative regulators involved in its genetic expression in Salmonella enterica serovar Typhi. The results obtained show that in S. Typhi the CRISPR/Cas system is a LeuO-dependent operon silenced by the global regulator LRP, in addition to the previously known nucleoid-associated protein H-NS; both LRP and H-NS bind upstream and downstream of the transcriptional start site of casA. In this study, relevant nucleotides of the casA regulatory region that mediate its LeuO transcriptional activation were identified. Interestingly, specific growth conditions (N-minimal medium) were found for the LeuO-independent expression of the CRISPR/Cas system in S. Typhi. Thus, our work provides evidence that there are multiple modulators involved in the genetic expression of this immune system in S. Typhi IMSS-1.

Two-Component Signal Transduction Systems, Environmental Signals, and Virulence

The relevance toward virulence of a variety of two-component signal transduction systems is reviewed for 16 pathogenic bacteria, together with the wide array of environmental signals or conditions that have been implicated in their regulation. A series of issues is raised, concerning the need to understand the environmental cues that determine their regulation in the infected host and in the environment outside the laboratory, which shall contribute toward the bridging of bacterial pathogenesis and microbial ecology.

The Complexities of Porin Genetic Regulation

Our understanding of porin regulation has revealed not only increasing complexity in the regulatory mechanisms, but also that the porin repertoire is more extensive than previously conceived. Initially, the OmpR response regulator was described as the master regulator of porin genes, but many more regulators are involved such as CpxR, PhoB, Lrp, Rob, MarA, SoxS, CadC, CRP, Fnr, ToxR, H-NS, StpA, IHF, HU and LeuO. In addition to MicF, the first small RNA (sRNA) that was proposed to regulate porin expression, porins are post-transcriptionally regulated by a variety of other sRNAs, namely, MicC, MicA, IpeX, RseX, InvR, CyaR and RybB. Future challenges include the full integration of all the regulatory circuitries. Whether quiescent porins are merely replacements of the major porins or are part of novel metabolic programs has yet to be elucidated. The comprehensive exploration of the environmental determinants that affect porin gene expression should yield valuable new information about the functions of these important proteins.

Expression of Salmonella typhi and Escherichia coli OmpC is influenced differently by medium osmolarity; dependence on Escherichia coli OmpR.

OmpC, a major outer‐membrane protein, is highly expressed when Salmonella typhi is grown in nutrient broth (NB) of either low (NB + 0% sucrose) or high (NB + 20% sucrose) osmolarity. This contrasts with the expression of Escherichia coli OmpC, which is inhibited in low osmolarity and enhanced in high osmolarity, as has been described previously (van Alphen and Lugtenberg, 1977; Verhoef et al., 1979; Kawaji et al., 1979). Nevertheless, expression of S. typhi OmpC is dependent on the E. coli OmpR transcriptional activator. These findings suggest differences between the mechanisms of osmoregulation of gene expression in both bacteria, although common effectors appear to be shared.

Association of virulence plasmid and antibiotic resistance determinants with chromosomal multilocus genotypes in Mexican Salmonella enterica serovar Typhimurium strains.

BackgroundBacterial genomes are mosaic structures composed of genes present in every strain of the same species (core genome), and genes present in some but not all strains of a species (accessory genome). The aim of this study was to compare the genetic diversity of core and accessory genes of a Salmonella enterica subspecies enterica serovar Typhimurium (Typhimurium) population isolated from food-animal and human sources in four regions of Mexico. Multilocus sequence typing (MLST) and macrorestriction fingerprints by pulsed-field gel electrophoresis (PFGE) were used to address the core genetic variation, and genes involved in pathogenesis and antibiotic resistance were selected to evaluate the accessory genome.ResultsWe found a low genetic diversity for both housekeeping and accessory genes. Sequence type 19 (ST19) was supported as the founder genotype of STs 213, 302 and 429. We found a temporal pattern in which the derived ST213 is replacing the founder ST19 in the four geographic regions analyzed and a geographic trend in the number of resistance determinants. The distribution of the accessory genes was not random among chromosomal genotypes. We detected strong associations among the different accessory genes and the multilocus chromosomal genotypes (STs). First, the Salmonella virulence plasmid (pSTV) was found mostly in ST19 isolates. Second, the plasmid-borne betalactamase cmy-2 was found only in ST213 isolates. Third, the most abundant integron, IP-1 (dfrA12, orfF and aadA2), was found only in ST213 isolates. Fourth, the Salmonella genomic island (SGI1) was found mainly in a subgroup of ST19 isolates carrying pSTV. The mapping of accessory genes and multilocus genotypes on the dendrogram derived from macrorestiction fingerprints allowed the establishment of genetic subgroups within the population.ConclusionDespite the low levels of genetic diversity of core and accessory genes, the non-random distribution of the accessory genes across chromosomal backgrounds allowed us to discover genetic subgroups within the population. This study provides information about the importance of the accessory genome in generating genetic variability within a bacterial population.