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: 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.Veterinary Microbiology 05/2013; 165(3-4). DOI:10.1016/j.vetmic.2013.04.025 · 2.73 Impact Factor
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ABSTRACT: Streptococcus suis is recognized as a major swine pathogen and an emerging zoonotic agent. Two large-scale outbreaks of severe S. suis epidemics occurred in China in 1998 and 2005 that posed serious concerns to public health and challenged the conventional conception that opportunistic infections of S. suis serotype 2 (SS2) in humans were only sporadic cases. An extensive, collaborative study on Chinese SS2 variants, which exhibit strong invasiveness and high pathogenicity, has resulted in the description of a new disease form of streptococcal toxic shock syndrome (STSS) and a putative pathogenicity island (termed 89K). The abbreviation of STSS is used for the severe disease caused by both Staphylococci and Streptococci. The main virulence factors involved in STSS caused by either Staphylococcus aureus or Streptococcus pyogenes consist of so-called superantigens or molecules that trigger a nonspecific, uncontrolled activation of T cells and massive cytokine release. However, although a collection of new virulence factors have been described, no superantigen candidates have been found for SS2 strains, implying that a different mechanism could be involved in the STSS form caused by SS2 variants.Trends in Microbiology 03/2010; 18(3):124-31. DOI:10.1016/j.tim.2009.12.003 · 9.81 Impact Factor
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ABSTRACT: In contrast with most bacteria, which harbour a single inosine monophosphate dehydrogenase (IMPDH) gene, the genomic sequence of Mycobacterium tuberculosis H37Rv predicts three genes encoding IMPDH: guaB1, guaB2 and guaB3. These three genes were cloned and expressed in Escherichia coli to evaluate functional IMPDH activity. Purified recombinant Mt-GuaB2, which uses inosine monophosphate as a substrate, was identified as the only active GuaB orthologue in M. tuberculosis and showed optimal activity at pH 8.5 and 37 °C. Mt-GuaB2 was inhibited significantly in vitro by a panel of diphenyl urea-based derivatives, which were also potent anti-mycobacterial agents against M. tuberculosis and Mycobacterium smegmatis, with MICs in the range of 0.2-0.5 μg ml(-1). When Mt-GuaB2 was overexpressed on a plasmid in trans in M. smegmatis, a diphenyl urea analogue showed a 16-fold increase in MIC. Interestingly, when Mt-GuaB orthologues (Mt-GuaB1 and 3) were also overexpressed on a plasmid in trans in M. smegmatis, they also conferred resistance, suggesting that although these Mt-GuaB orthologues were inactive in vitro, they presumably titrate the effect of the inhibitory properties of the active compounds in vivo.Microbiology 11/2010; 157(Pt 1):290-9. DOI:10.1099/mic.0.042549-0 · 2.84 Impact Factor