Identification and characterization of inosine 5-monophosphate dehydrogenase in Streptococcus suis type 2
Institute of Veterinary Medicine, Jiangsu Academy of Agricultural Sciences, National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014 China. Microbial Pathogenesis
(Impact Factor: 1.79).
10/2009; 47(5):267-73. DOI: 10.1016/j.micpath.2009.09.001
Streptococcus suis type 2 is a swine pathogen responsible for diverse diseases. Although many virulent factors have been identified and studied, relatively little is known about the pathogenic mechanisms of type 2. The aim of the study was to identify and understand the characterization of Inosine 5-monophosphate dehydrogenase (IMPDH). A 957-bp gene, impdh, was identified in the virulent S. suis serotype 2 (SS2), and analysis of the predicted IMPDH sequence revealed IMP dehydrogenase/GMP reductase domain. The gene encoding for the IMPDH of S. suis was cloned and sequenced. The DNA sequence contained an open reading frame encoding for a 318 amino acid polypeptide exhibiting 23% sequence identity with the IMPDH from Streptococcus pyogenes (YP281355) and Streptococcus pneumoniae (ZP00404150). Using the pET(32) expression plasmid, the impdh gene was inducibly overexpressed in Escherichia coli to produce IMPDH with a hexahistidyl N-terminus to permit its purification. The (His)6 IMPDH protein was found to possess functional IMPDH enzymatic activity after the purification. The impdh-knockout SS2 mutant ( Delta IMPDH) constructed in this study was slower in growth and one pH unit higher than SS2-H after 6 h of culturing, and found to be attenuated in mouse models of infection for 2.5 times and not be capable of causing death in porcine models of infection in contrast with the parent SS2-H.
- "The SS2 strain was recovered from 280 uC freezer stocks and grown overnight (for 18–20 h) at 37 uC in Todd–Hewitt broth (THB) medium supplemented with 2 % Minima Essential Medium (MEM) (Zhang et al., 2009). Other bacterial strains were also prepared by transferring 50 ml of a thawed, previously frozen, culture into 5 ml of Luria–Bertani (LB) broth and incubated overnight at 37 uC. "
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ABSTRACT: Intimin harbored by pathogenic Escherichia coli (E. coli) strains is a key virulence factor involved in host cell adherence and colonization. Twenty-seven Intimin-encoding E. coli attaching and effacing (eae) gene variants have been reported according to their 3' binding domain sequences. In our study, we developed a specific and sensitive loop-mediated isothermal amplification (LAMP) assay to detect all known Intimin variants. Four primers specific for six regions of eae genes were designed using online software. The eae-LAMP assay was highly specific and detected all 27 tested eae variants; no cross-reactions were observed with genes from enterotoxigenic E. coli (ETEC), E. coli BL21, Salmonella, Shigella, Listeria monocytogenes, or Streptococcus suis type 2 (SS2). With the lowest detection limit of approximately 10 copies per reaction the eae-LAMP assay was 100 times more sensitive than conventional PCR.These results and the results of tests involving food and fecal samples artificially contaminated with E. coli O157:H7 (eaeγ+) show that the eae-LAMP assay is a simple, rapid, sensitive, and specific tool for detecting Intimin variants from pathogenic strains of E. coli. The eae-LAMP assay has great potential for wider applications not only in the laboratory but also in the field setting as it does not require specialized equipment.
Available from: José Francisco Fernández-Garayzábal
- ". 2.3. Virulence factors Detection of the virulence associated genes for capsular polysaccharide (cps), muramidase released protein (mrp), extracellular protein factor (epf), suilysin (sly), surface antigen protein (sao), enolase (eno), orphan response regulator (CovR), di-peptidyl peptidase IV (dpp), inosine 5-monophosphate dehydrogenase (impdh), transpeptidase mediating covalent linkage of surface proteins to peptidoglycan (sortA) and D-alanine-D-alanyl ligase (dltA) was performed by PCR as described previously (Silva et al., 2006; Feng et al., 2007, 2009; Fittipaldi et al., 2008; Ge et al., 2009; Pan et al., 2009; Wang et al., 2009; Zhang et al., 2009 "
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ABSTRACT: This work aims to investigate the presence of Streptococcus suis in wild rabbits. A total of 65 S. suis isolates were recovered from 33.3% of the wild rabbits examined. Most isolates (86.2%) belong to genotype cps9. These isolates were further characterized by pulsed field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and virulence genotyping. Overall, S. suis exhibited a low genetic diversity. Only 5 genetic profiles were obtained by PFGE and most isolates (71.4%) were included in two pulsotypes that were also widely distributed among the wild rabbit population. MLST analysis assigned all cps9 isolates into three new singlestones (ST216, ST217 and ST284), which were not genetically related to the European ST87 and Spanish ST61 widespread swine clones, indicating a different genetic background for the S. suis isolates from wild rabbits and pigs. Wild rabbit isolates exhibited the genotype mrp-/epf-/sly-, different from those showed by most of the swine S. suis isolates of the ST87 and ST61 clones. None of the S. suis isolated from wild rabbits exhibited the genotype cps2/mrp+/epf+/sly+ associated with human infections. These results indicate that S. suis isolates from wild rabbits are not genetically related with prevalent clones usually associated with infections in pigs or humans in Europe and do not exhibit either their virulence genotypes. Therefore, although wild rabbits could represent an unknown reservoir of this pathogen, they could not represent a potential risk for pigs or humans.
Available from: Astrid De Greeff
- "Naming (SSU numbering) is derived from the annotated genome sequence of P1/7 . Presence of 25 described putative virulence factors was studied: muramidase released protein (mrp), and extracullar factor (epf) , suilysin (sly) , sortases (srtA, srtBCD, srtF) , surface antigen one (sao) , hyaluronidase (hylA) [17,43], opacity factor (ofs) , fibronectin binding protein (fbps) , arginin deiminase (arcA) , glyceraldehyde-3-phosphate dehydrogenase (gapdh) , regulator of virulence (revS) [35,47], enolase (eno) , glutamine synthetase (glnA) , igA1 protease , inosine 5-monophosphate dehydrogenase (impdh) , dipeptidyl peptidase IV (dppIV) , ferrous iron transporter (feoB) , subtilisin like serine protease (sspA) , amylopullulanase (apuA) , ferric uptake regulator (fur), and adhesion competence repressor (adcR) . * hylA is present as pseudogene in P1/7 and does not have a SSU-number. "
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ABSTRACT: Streptococcus suis is a zoonotic pathogen that causes infections in young piglets. S. suis is a heterogeneous species. Thirty-three different capsular serotypes have been described, that differ in virulence between as well as within serotypes.
In this study, the correlation between gene content, serotype, phenotype and virulence among 55 S. suis strains was studied using Comparative Genome Hybridization (CGH). Clustering of CGH data divided S. suis isolates into two clusters, A and B. Cluster A isolates could be discriminated from cluster B isolates based on the protein expression of extracellular factor (EF). Cluster A contained serotype 1 and 2 isolates that were correlated with virulence. Cluster B mainly contained serotype 7 and 9 isolates. Genetic similarity was observed between serotype 7 and serotype 2 isolates that do not express muramidase released protein (MRP) and EF (MRP⁻EF⁻), suggesting these isolates originated from a common founder. Profiles of 25 putative virulence-associated genes of S. suis were determined among the 55 isolates. Presence of all 25 genes was shown for cluster A isolates, whereas cluster B isolates lacked one or more putative virulence genes. Divergence of S. suis isolates was further studied based on the presence of 39 regions of difference. Conservation of genes was evaluated by the definition of a core genome that contained 78% of all ORFs in P1/7.
In conclusion, we show that CGH is a valuable method to study distribution of genes or gene clusters among isolates in detail, yielding information on genetic similarity, and virulence traits of S. suis isolates.
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