A gram-positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissue. The agr system is known to be important for the regulation of virulence genes in a quorum-sensing manner in Staphylococcus aureus. A homologue for S. aureus agrBD (agrBD Sa ) was identified in the C. perfringens strain 13 genome, and the role of C. perfringens agrBD (agrBD Cp ) was examined. The agrBD Cp knockout mutant did not express the theta-toxin gene, and transcription of the alpha-and kappa-toxin genes was also significantly decreased in the mutant strain. The mutant strain showed a recovery of toxin production after the addition of the culture supernatant of the wild-type strain, indicating that the agrBD Cp mutant lacks a signal molecule in the culture supernatant. An agr-virR double-knockout mutant was constructed to examine the role of the VirR/VirS two-component regulatory system, a key virulence regulator, in agrBD Cp -mediated regulation of toxin production. The double-mutant strain could not be stimulated for toxin production with the wild-type culture supernatant. These results indicate that the agrBD Cp system plays an important role in virulence regulation and also suggest that VirR/VirS is required for sensing of the extracellular signal and activation of toxin gene transcription in C. perfringens.Clostridium perfringens is a gram-positive, spore-forming, anaerobic bacterium. C. perfringens is the causative agent of several human and animal diseases, including clostridial myonecrosis, or gas gangrene (7). C. perfringens produces various extracellular enzymes and toxins, including alpha-, theta-, and kappa-toxins encoded by plc, pfoA, and colA, respectively (21). These toxin genes are positively regulated by the two-component VirR/VirS system (25) that is a major regulator of virulence in C. perfringens.
Enzymatic disruption of quorum-sensing (QS) pathways in pathogenic organisms is a promising anti-infection therapeutic strategy. AHL-lactonase, a potent tool for biocontrol, can hydrolyze QS signal molecule N-acyl-homoserine lactones (AHLs) into inactive products, thereby blocking the QS systems. A marine bacterial isolate Y2, identified as a Bacillus cereus subsp., was found capable of inactivating AHLs. The aiiA gene encoding the AHL-degrading enzyme from bacterial strain Y2 was cloned and expressed in Escherichia coli. The 28-kDa recombinant Y2-AiiA protein was purified and showed strong AHL-degrading activity. Sequence comparisons of Y2-aiiA with known AHL-lactonases revealed high identities in the deduced amino-acid sequences. Functional determination of a potential catalytic residue Tyr-194 of AHL-lactonases was performed by site-directed mutagenesis. As judged by AHL-degrading bioassay, substitution of Tyr-194 with Ala resulted in a dramatic decrease of activity compared with wild-type (WT) recombinant Y2-AiiA, although the expression level of the mutated Y2-AiiA protein was equivalent to that of WT Y2-AiiA. These results suggested that the conserved residue Tyr-194 is critical for catalytic function of the novel AHL-lactonase.
A Gram-positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissues. The ability of infectious bacteria to acquire sufficient iron during infection is essential for the pathogen to cause disease. In the C. perfringens strain 13 genome, a heme oxygenase gene homologue (CPE0214, hemO) was found and its role was examined. The purified recombinant HemO protein showed heme oxygenase activity that can convert heme to biliverdin. hemO transcription was induced in response to extracellular hemin in a dose-dependent manner. The global two-component VirR/VirS regulatory system and its secondary regulator VR-RNA had positive regulatory effects on the transcription of hemO. These data indicate that heme oxygenase may play important roles in iron acquisition and cellular metabolism, and that the VirR/VirS-VR-RNA system is also involved in the regulation of cellular iron homeostasis, which might be important for the survival of C. perfringens in a human host.
Clostridium perfringens, a Gram-positive anaerobe, is a human pathogen that causes gas gangrene in muscle tissues. Its ability to grow and survive in the host is believed to be due to the production of numerous enzymes that enable the organism to obtain essential nutrients from the host. In this study, CPE0201, a putative acid phosphatase gene deduced by genome analysis, was shown to encode a non-specific acid phosphatase in C. perfringens. Multiple alignments of the amino acid sequence showed that CPE0201 shares two signature motifs that belong to a class C acid phosphatase family. Expression of CPE0201 was shown to be positively regulated by the global VirR/VirS-VR-RNA regulatory cascade at the transcriptional level. To determine the acid phosphatase activity of the CPE0201-encoded protein, cloning, expression, purification and several biochemical characterizations were carried out. The optimum pH for activity of the CPE0201 enzyme was 4.8, and its V max and K m were 3.08 nmol ml−1 min−1 and 2.84 mM, respectively, with p-nitrophenyl phosphate (PNPP) as substrate. A CPE0201 mutant did not grow in a minimal medium containing PNPP, while it showed normal growth when Na2HPO4 was added to the medium. The enzyme appears to be associated with the surface of the cell, where it may function to acquire inorganic phosphate from organic phosphomonoesters in acidic conditions, which could play an important role in the survival and growth of C. perfringens in the host tissue.
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