Genomic epidemiology of Vibrio cholerae reveals the regional and global spread of two epidemic non-toxigenic lineages

Non-toxigenic Vibrio cholerae isolates have been found associated with diarrheal disease globally, however, the global picture of non-toxigenic infections is largely unknown. Among non-toxigenic V. cholerae, ctxAB negative, tcpA positive (CNTP) isolates have the highest risk of disease. From 2001 to 2012, 71 infectious diarrhea cases were reported in Hangzhou, China, caused by CNTP serogroup O1 isolates. We sequenced 119 V. cholerae genomes isolated from patients, carriers and the environment in Hangzhou between 2001 and 2012, and compared them with 850 publicly available global isolates. We found that CNTP isolates from Hangzhou belonged to two distinctive lineages, named L3b and L9. Both lineages caused disease over a long time period with usually mild or moderate clinical symptoms. Within Hangzhou, the spread route of the L3b lineage was apparently from rural to urban areas, with aquatic food products being the most likely medium. Both lineages had been previously reported as causing local endemic disease in Latin America, but here we show that global spread of them has occurred, with the most likely origin of L3b lineage being in Central Asia. The L3b lineage has spread to China on at least three occasions. Other spread events, including from China to Thailand and to Latin America were also observed. We fill the missing links in the global spread of the two non-toxigenic serogroup O1 V. cholerae lineages that can cause human infection. The results are important for the design of future disease control strategies: surveillance of V. cholerae should not be limited to ctxAB positive strains

Transcriptional Regulation of opaR, qrr2–4 and aphA by the Master Quorum-Sensing Regulator OpaR in Vibrio parahaemolyticus

Background: Vibrio parahaemolyticus is a leading cause of infectious diarrhea and enterogastritis via the fecal-oral route. V. harveyi is a pathogen of fishes and invertebrates, and has been used as a model for quorum sensing (QS) studies. LuxR is the master QS regulator (MQSR) of V. harveyi, and LuxR-dependent expression of its own gene, qrr2–4 and aphA have been established in V. harveyi. Molecular regulation of target genes by the V. parahaemolyticus MQSR OpaR is still poorly understood. Methodology/Principal Findings: The bioinformatics analysis indicated that V. parahaemolyticus OpaR, V. harveyi LuxR, V. vulnificu SmcR, and V. alginolyticus ValR were extremely conserved, and that these four MQSRs appeared to recognize the same conserved cis-acting signals, which was represented by the consensus constructs manifesting as a position frequency matrix and as a 20 bp box, within their target promoters. The MQSR box-like sequences were found within the upstream DNA regions of opaR, qrr2–4 and aphA in V. parahaemolyticus, and the direct transcriptional regulation of these target genes by OpaR were further confirmed by multiple biochemical experiments including primer extension assay, gel mobility shift assay, and DNase I footprinting analysis. Translation and transcription starts, core promoter elements for sigma factor recognition, Shine-Dalgarno sequences for ribosome recognition, and OpaR-binding sites were determined for the five target genes of OpaR, which gave a structural map of the OpaR-dependent promoters. Further computational promote

Comparative transcriptomics in Yersinia pestis: a global view of environmental modulation of gene expression

<p>Abstract</p> <p>Background</p> <p>Environmental modulation of gene expression in <it>Yersinia pestis </it>is critical for its life style and pathogenesis. Using cDNA microarray technology, we have analyzed the global gene expression of this deadly pathogen when grown under different stress conditions <it>in vitro</it>.</p> <p>Results</p> <p>To provide us with a comprehensive view of environmental modulation of global gene expression in <it>Y. pestis</it>, we have analyzed the gene expression profiles of 25 different stress conditions. Almost all known virulence genes of <it>Y. pestis </it>were differentially regulated under multiple environmental perturbations. Clustering enabled us to functionally classify co-expressed genes, including some uncharacterized genes. Collections of operons were predicted from the microarray data, and some of these were confirmed by reverse-transcription polymerase chain reaction (RT-PCR). Several regulatory DNA motifs, probably recognized by the regulatory protein Fur, PurR, or Fnr, were predicted from the clustered genes, and a Fur binding site in the corresponding promoter regions was verified by electrophoretic mobility shift assay (EMSA).</p> <p>Conclusion</p> <p>The comparative transcriptomics analysis we present here not only benefits our understanding of the molecular determinants of pathogenesis and cellular regulatory circuits in <it>Y. pestis</it>, it also serves as a basis for integrating increasing volumes of microarray data using existing methods.</p

Characterization of a thermostable β-glucosidase from Aspergillus fumigatus Z5, and its functional expression in Pichia pastoris X33

<p>Abstract</p> <p>Background</p> <p>Recently, the increased demand of energy has strongly stimulated the research on the conversion of lignocellulosic biomass into reducing sugars for the subsequent production, and β-glucosidases have been the focus because of their important roles in a variety fundamental biological processes and the synthesis of useful β-glucosides. Although the β-glucosidases of different sources have been investigated, the amount of β-glucosidases are insufficient for effective conversion of cellulose. The goal of this work was to search for new resources of β-glucosidases, which was thermostable and with high catalytic efficiency.</p> <p>Results</p> <p>In this study, a thermostable native β-glucosidase (nBgl3), which is secreted by the lignocellulose-decomposing fungus <it>Aspergillus fumigatus </it>Z5, was purified to electrophoretic homogeneity. Internal sequences of nBgl3 were obtained by LC-MS/MS, and its encoding gene, <it>bgl3</it>, was cloned based on the peptide sequences obtained from the LC-MS/MS results. <it>bgl</it>3 contains an open reading frame (ORF) of 2622 bp and encodes a protein with a predicted molecular weight of 91.47 kDa; amino acid sequence analysis of the deduced protein indicated that nBgl3 is a member of the glycoside hydrolase family 3. A recombinant β-glucosidase (rBgl3) was obtained by the functional expression of <it>bgl</it>3 in <it>Pichia pastoris </it>X33. Several biochemical properties of purified nBgl3 and rBgl3 were determined - both enzymes showed optimal activity at pH 6.0 and 60°C, and they were stable for a pH range of 4-7 and a temperature range of 50 to 70°C. Of the substrates tested, nBgl3 and rBgl3 displayed the highest activity toward 4-Nitrophenyl-β-D-glucopyranoside (pNPG), with specific activities of 103.5 ± 7.1 and 101.7 ± 5.2 U mg^-1, respectively. However, these enzymes were inactive toward carboxymethyl cellulose, lactose and xylan.</p> <p>Conclusions</p> <p>An native β-glucosidase nBgl3 was purified to electrophoretic homogeneity from the crude extract of <it>A. fumigatus </it>Z5. The gene <it>bgl</it>3 was cloned based on the internal sequences of nBgl3 obtained from the LC-MS/MS results, and the gene <it>bgl3 </it>was expressed in <it>Pichia pastoris </it>X33. The results of various biochemical properties of two enzymes including specific activity, pH stability, thermostability, and kinetic properties (Km and Vmax) indicated that they had no significant differences.</p

Direct and negative regulation of the sycO-ypkA-ypoJ operon by cyclic AMP receptor protein (CRP) in Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>Pathogenic yersiniae, including <it>Y. pestis</it>, share a type III secretion system (T3SS) that is composed of a secretion machinery, a set of translocation proteins, a control system, and six Yop effector proteins including YpkA and YopJ. The cyclic AMP receptor protein (CRP), a global regulator, was recently found to regulate the laterally acquired genes (<it>pla </it>and <it>pst</it>) in <it>Y. pestis</it>. The regulation of T3SS components by CRP is unknown.</p> <p>Results</p> <p>The <it>sycO</it>, <it>ypkA </it>and <it>yopJ </it>genes constitute a single operon in <it>Y. pestis</it>. CRP specifically binds to the promoter-proximate region of <it>sycO</it>, and represses the expression of the <it>sycO-ypkA-yopJ </it>operon. A single CRP-dependent promoter is employed for the <it>sycO-ypkA-yopJ </it>operon, but two CRP binding sites (site 1 and site 2) are detected within the promoter region. A CRP box homologue is found in site 1 other than site 2. The determination of CRP-binding sites, transcription start site and core promoter element (-10 and -35 regions) promotes us to depict the structural organization of CRP-dependent promoter, giving a map of CRP-promoter DNA interaction for <it>sycO-ypkA-yopJ</it>.</p> <p>Conclusion</p> <p>The <it>sycO-ypkA-yopJ </it>operon is under the direct and negative regulation of CRP in <it>Y. pestis</it>. The <it>sycO-ypkA-yopJ </it>promoter-proximate regions are extremely conserved in <it>Y. pestis</it>, <it>Y. pseudotuberculosis </it>and <it>Y. enterocolitica</it>. Therefore, data presented here can be generally applied to the above three pathogenic yersiniae.</p

Regulatory effects of cAMP receptor protein (CRP) on porin genes and its own gene in Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>The cAMP receptor protein (CRP) is a global bacterial regulator that controls many target genes. The CRP-cAMP complex regulates the <it>ompR-envZ </it>operon in <it>E. coli </it>directly, involving both positive and negative regulations of multiple target promoters; further, it controls the production of porins indirectly through its direct action on <it>ompR-envZ</it>. Auto-regulation of CRP has also been established in <it>E. coli</it>. However, the regulation of porin genes and its own gene by CRP remains unclear in <it>Y. pestis</it>.</p> <p>Results</p> <p><it>Y. pestis </it>employs a distinct mechanism indicating that CRP has no regulatory effect on the <it>ompR-envZ </it>operon; however, it stimulates <it>ompC </it>and <it>ompF </it>directly, while repressing <it>ompX</it>. No transcriptional regulatory association between CRP and its own gene can be detected in <it>Y. pestis</it>, which is also in contrast to the fact that CRP acts as both repressor and activator for its own gene in <it>E. coli</it>. It is likely that <it>Y. pestis </it>OmpR and CRP respectively sense different signals (medium osmolarity, and cellular cAMP levels) to regulate porin genes independently.</p> <p>Conclusion</p> <p>Although the CRP of <it>Y. pestis </it>shows a very high homology to that of <it>E. coli</it>, and the consensus DNA sequence recognized by CRP is shared by the two bacteria, the <it>Y. pestis </it>CRP can recognize the promoters of <it>ompC</it>, <it>F</it>, and <it>X </it>directly rather than that of its own gene, which is different from the relevant regulatory circuit of <it>E. coli</it>. Data presented here indicate a remarkable remodeling of the CRP-mediated regulation of porin genes and of its own one between these two bacteria.</p

Phenotypic and transcriptional analysis of the osmotic regulator OmpR in Yersinia pestis

<p>Abstract</p> <p>Background</p> <p>The osmotic regulator OmpR in <it>Escherichia coli </it>regulates differentially the expression of major porin proteins OmpF and OmpC. In <it>Yersinia enterocolitica </it>and <it>Y. pseudotuberculosis</it>, OmpR is required for both virulence and survival within macrophages. However, the phenotypic and regulatory roles of OmpR in <it>Y. pestis </it>are not yet fully understood.</p> <p>Results</p> <p><it>Y. pestis </it>OmpR is involved in building resistance against phagocytosis and controls the adaptation to various stressful conditions met in macrophages. The <it>ompR </it>mutation likely did not affect the virulence of <it>Y. pestis </it>strain 201 that was a human-avirulent enzootic strain. The microarray-based comparative transcriptome analysis disclosed a set of 224 genes whose expressions were affected by the <it>ompR </it>mutation, indicating the global regulatory role of OmpR in <it>Y. pestis</it>. Real-time RT-PCR or <it>lacZ </it>fusion reporter assay further validated 16 OmpR-dependent genes, for which OmpR consensus-like sequences were found within their upstream DNA regions. <it>ompC</it>, <it>F</it>, <it>X</it>, and <it>R </it>were up-regulated dramatically with the increase of medium osmolarity, which was mediated by OmpR occupying the target promoter regions in a tandem manner.</p> <p>Conclusion</p> <p>OmpR contributes to the resistance against phagocytosis or survival within macrophages, which is conserved in the pathogenic yersiniae. <it>Y. pestis </it>OmpR regulates <it>ompC</it>, <it>F</it>, <it>X</it>, and <it>R </it>directly through OmpR-promoter DNA association. There is an inducible expressions of the pore-forming proteins OmpF, C, and × at high osmolarity in <it>Y. pestis</it>, in contrast to the reciprocal regulation of them in <it>E. coli</it>. The main difference is that <it>ompF </it>expression is not repressed at high osmolarity in <it>Y. pestis</it>, which is likely due to the absence of a promoter-distal OmpR-binding site for <it>ompF</it>.</p

Lack of association between polymorphisms of MASP2 and susceptibility to SARS coronavirus infection

<p>Abstract</p> <p>Background</p> <p>The pathogenesis of severe acute respiratory disease syndrome (SARS) is not fully understood. One case-control study has reported an association between susceptibility to SARS and <it>mannan-binding lectin </it>(<it>MBL</it>) in China. As the downstream protein of <it>MBL</it>, variants of the <it>MBL</it>-associated serine protease-2 (<it>MASP2</it>) gene may be associated with SARS coronavirus (SARS-CoV) infection in the same population.</p> <p>Methods</p> <p>Thirty individuals with SARS were chosen for analysis of <it>MASP2 </it>polymorphisms by means of PCR direct sequencing. Tag single nucleotide polymorphisms (tagSNPs) were chosen using pairwise tagging algorithms. The frequencies of four tag SNPs (rs12711521, rs2261695, rs2273346 and rs7548659) were ascertained in 376 SARS patients and 523 control subjects, using the Beckman SNPstream Ultra High Throughput genotyping platform.</p> <p>Results</p> <p>There is no significant association between alleles or genotypes of the <it>MASP2 </it>tagSNP and susceptibility to SARS-CoV in both Beijing and Guangzhou populations. Diplotype (rs2273346 and rs12711521)were analyzed for association with susceptibility to SARS, no statistically significant evidence of association was observed. The Beijing and Guangzhou sample groups were homogeneous regarding demographic and genetic parameters, a joined analysis also showed no statistically significant evidence of association.</p> <p>Conclusion</p> <p>Our data do not suggest a role for <it>MASP2 </it>polymorphisms in SARS susceptibility in northern and southern China.</p