Identification and characterization of inosine 5-monophosphate dehydrogenase in Streptococcus suis type 2.
ABSTRACT 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.
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ABSTRACT: Proteomic analysis by two-dimensional electrophoresis (2D)-mass spectrometry was used to identify differentially expressed proteins in the Clostridium sp. native strain (IBUN 158B) in two phases of the 1,3-propanediol (1,3-PD) production (lag phase and exponential growth phase). Intracellular protein fraction extraction conditions were standardised, as well as the 2D electrophoresis. Differences were found between both of the growth phases evaluated here. Thirty-two of the differentially expressed proteins were chosen to be identified by tandem mass spectrometry (MALDI TOF/TOF). The presence of four enzymes implicated in the 1,3-PD metabolic pathway was recorded: one from the reductive route (1,3-propanediol dehydrogenase) and three from the oxidative route (3-hydroxybutyryl CoA dehydrogenase, NADPH-dependent butanol dehydrogenase and phosphate butyryl transferase). The following enzymes which have not been previously reported for Clostridium sp., were also identified: phosphoglycerate kinase, glucose 6-phosphate isomerase, deoxyribose phosphate aldolase, transketolase, cysteine synthetase, O-acetylhomoserine sulphhydrylase, glycyl-tRNA ligase, aspartate β-semialdehyde dehydrogenase, inosine 5-monophosphate dehydrogenase, aconitate hydratase and the PrsA protein. The foregoing provides a novel contribution towards knowledge of the native strain for the purpose to design genetic manipulation strategies, for obtain strains with high producing of 1,3-PD. The article "Proteins identification in two phases of 1,3-propanediol production by proteomic analysis" provides a novel contribution towards knowledge regarding the Colombian Clostridium sp. native strain (IBUN 158B) because is an new approximation in comparative proteomics in two phases of the bacterial growth and 1,3-propanediol (1,3-PD) production conditions. The proteomic studies are very important for identify the enzymes that are expressed at different stages of production and therefore genes of interest in the genetic manipulation strategies, the results can be taken into account in future studies in metabolic engineering in the optimizing of the 1,3-PD production, in a cost-effective process having direct industrial applications.Journal of proteomics 06/2013; 89:255-264. · 5.07 Impact Factor
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ABSTRACT: Streptococcus suis serotype 2 (SS2) is an important zoonotic pathogen that causes severe disease symptoms in pigs and humans. In the present study, we found one isogenic mutant lacking inosine 5-monophosphate dehydrogenase (IMPDH) ΔZY05719 was attenuated in pigs compared with the wild-type SS2 strain ZY05719. Comparative proteome analysis of the secreted proteins expression profiles between ZY05719 and ΔZY05719 allowed us to identify Triosephosphate isomerase (TPI) and glyceraldehyde phosphate dehydrogenase (GAPDH), which were down expressed in the absence of the IMPDH. Both of them are glycolytic enzymes participating in the glycolytic pathway. Compared with ZY05719, ΔZY05719 lost the ability of utilize mannose, which might relate to down expression of TPI and GAPDH. In addition, GAPDH is a well-known factor that involved in adhesion to host cells, and we demonstrated ability of adhesion to HEp-2 and PK15 by ΔZY05719 was significantly weakened, in contrast to ZY05719. The adhesion to host cells is the crucial step to cause infection for pathogen, and the reduction adhesion of ΔZY05719, to some extent illustrates the attenuated virulence of ΔZY05719.Current Microbiology 01/2014; · 1.52 Impact Factor
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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.Journal of Medical Microbiology 07/2013; · 2.30 Impact Factor