Kaori Ohtani

Complete genome sequence of Clostridium perfringens, an anaerobic flesh-eater

Clostridium perfringens is a Gram-positive anaerobic spore-forming bacterium that causes life-threatening gas gangrene and mild enterotoxaemia in humans, although it colonizes as normal intestinal flora of humans and animals. The organism is known to produce a variety of toxins and enzymes that are responsible for the severe myonecrotic lesions. Here we report the complete 3,031,430-bp sequence of C. perfringens strain 13 that comprises 2,660 protein coding regions and 10 rRNA genes, showing pronounced low overall G + C content (28.6%). The genome contains typical anaerobic fermentation enzymes leading to gas production but no enzymes for the tricarboxylic acid cycle or respiratory chain. Various saccharolytic enzymes were found, but many enzymes for amino acid biosynthesis were lacking in the genome. Twenty genes were newly identified as putative virulence factors of C. perfringens, and we found a total of five hyaluronidase genes that will also contribute to virulence. The genome analysis also proved an efficient method for finding four members of the two-component VirR/VirS regulon that coordinately regulates the pathogenicity of C. perfringens. Clearly, C. perfringens obtains various essential materials from the host by producing several degradative enzymes and toxins, resulting in massive destruction of the host tissues.

The luxS gene is involved in cell–cell signalling for toxin production in Clostridium perfringens

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. C. perfringens possesses a homologue of the luxS gene that is reported to be responsible for the production of autoinducer 2 (AI‐2), which participates in quorum sensing in bacteria. The luxS mutant was constructed using C. perfringens strain 13, and the role of the luxS gene in toxin production was examined. The cell‐free culture supernatant from wild‐type strain 13 greatly stimulated the luminescence of Vibrio harveyi BB170, whereas that from the luxS mutant caused no significant stimulation, indicating that the luxS gene is necessary for AI‐2 production in C. perfringens. The luxS mutant showed a reduced level of production of alpha‐, kappa‐ and theta‐toxins. In the luxS mutant, the transcription of the theta‐toxin gene (pfoA) was lower at mid‐exponential growth phase, whereas alpha‐ and kappa‐toxin gene transcription was not significantly affected. The production of toxins in the luxS mutant was stimulated by the addition of the culture supernatant from the wild‐type cells, possibly because of the presence of AI‐2. Moreover, the expression of the pfoA gene in the luxS mutant was apparently activated when the mutant cells were cultured in the presence of culture supernatants from the wild‐type C. perfringens, Escherichia coli DH5α carrying the luxS gene of C. perfringens. A deletion analysis of the luxS operon showed that the luxS gene alone is responsible for cell–cell signalling, and that the metB or cysK genes located upstream of luxS are not involved in regulating toxin production. Our results indicate that cell–cell signalling by AI‐2 plays an important role in the regulation of toxin production in C. perfringens.

Clostridial VirR/VirS regulon involves a regulatory RNA molecule for expression of toxins.

We analysed the region encoding VR‐RNA (VirR‐regulated RNA), which has been reported to be positively regulated by the two‐component VirR/VirS system in Clostridium perfringens. The VR‐RNA promoter identified by primer extension analysis was preceded by a probable VirR‐binding site (CCAGTTNNNCAC), which resembled a repeated sequence motif present in the promoter region of the theta‐toxin (pfoA) gene. A VR‐RNA‐null mutant, constructed by a homologous recombination, exhibited a reduced amount of transcription of the alpha‐ (plc) and kappa‐toxin (colA) genes, which was restored by the complementation of intact VR‐RNA, indicating that the VR‐RNA region plays an important role in the regulation of the plc and colA genes in C. perfringens. It was found that the regulatory effect was observed even when the hyp7 gene encoded on VR‐RNA was deleted or a nonsense mutation was introduced in the hyp7‐coding region. We found that the small 3′‐portion of VR‐RNA was sufficient for the activation of toxin genes, which suggested that VR‐RNA itself could act as an RNA regulatory molecule for the plc and colA genes mediating the regulatory information from the VirR/VirS system in C. perfringens.

Prevention of pin tract infection with titanium‐copper alloys

The most frequent complication in external fixation is pin tract infection. To reduce the incidence of implant-associated infection, many published reports have looked at preventing bacterial adhesion by treating the pin surface. This study aimed to evaluate the antibacterial activity of a Titanium-Copper (Ti-Cu) alloy on implant infection, and to determine the potential use of the Ti-Cu alloy as a biomaterial. Two forms of Ti-Cu alloys were synthesized: one with 1% Cu and the other with 5% Cu. For analyzing infectious behavior, the implants were exposed to Staphylococcus aureus and Escherichia coli. The reaction of pathogens to the Ti-Cu alloys was compared with their reaction to stainless steel and pure titanium as controls. Both Ti-Cu alloys evidently inhibited colonization by both bacteria. Conversely, cytocompatibility studies were performed using fibroblasts and colony formation on the metals was assessed by counting the number of colonies. Ti-1% Cu alloy showed no difference in the number of colonies compared with the control. External fixator pins made of Ti-Cu alloys were evaluated in a rabbit model. The tissue-implant interactions were analyzed for the presence of infection, inflammatory changes and osteoid-formation. Ti-1% Cu alloy significantly inhibited inflammation and infection, and had excellent osteoid-formation. Copper blood levels were measured before surgery and at 14 days postoperatively. Preoperative and postoperative blood copper values were not statistically different. Overall, it was concluded that Ti-Cu alloys have antimicrobial activity and substantially reduce the incidence of pin tract infection. Ti-1% Cu alloy shows particular promise as a biomaterial.

Virulence Gene Regulation by the agr System in Clostridium perfringens

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.

Antibacterial iodine-supported titanium implants

Deep infection remains a serious complication in orthopedic implant surgery. In order to reduce the incidence of implant-associated infections, several biomaterial surface treatments have been proposed. This study focused on evaluating the antibacterial activity of iodine-supported titanium (Ti-I(2)) and its impact on post-implant infection, as well as determining the potential suitability of Ti-I(2) as a biomaterial. External fixation pins were used in this experiment as trial implants because of the ease of making the septic models. The antibacterial activity of the metal was measured using a modification of the Japanese Industrial Standards method. Activity was evaluated by exposing the implants to Staphylococcus aureus or Escherichia coli and comparing reaction of pathogens to Ti-I(2) vs. stainless steel and titanium controls. Ti-I(2) clearly inhibited bacterial colonization more than the control metals. In addition, cytocompatibility was assessed by counting the number of colonies that formed on the metals. The three metals showed the same amount of fibroblast colony formation. Japanese white rabbits were used as an in vivo model. Three pins were inserted into both femora of six rabbits for histological analysis. Pin sites were inspected and graded for infection and inflammation. Fewer signs of infection and inflammatory changes were observed in conjunction with the Ti-I(2) pins. Furthermore, osteoconductivity of the implant was evaluated with osteoid formation surface of the pin. Consecutive bone formation was observed around the Ti-I(2) and titanium pins, while little osteoid formation was found around the stainless steel pins. These findings suggest that Ti-I(2) has antimicrobial activity and exhibits cytocompatibility. Therefore, Ti-I(2) substantially reduces the incidence of implant infection and shows particular promise as a biomaterial.

Identification of novel VirR/VirS-regulated genes in Clostridium perfringens.

Novel genes that are regulated in Clostridium perfringens by the two‐component regulatory system, VirR/VirS, were identified using a differential display method. A plasmid library was constructed from C. perfringens chromosomal DNA, and the plasmids were hybridized with cDNA probes prepared from total RNA of wild‐type strain 13 and its virR mutant derivative TS133. Three clones were identified that carry newly identified VirR/VirS‐regulated genes, two of which were positively regulated and one of which was negatively regulated. Genes located on the identified clones were deduced by nucleotide sequencing, and the target genes of the VirR/VirS system were identified with a set of Northern hybridizations. A 4.9 kb mRNA transcribing the metB (cystathionine gamma‐synthase), cysK (cysteine synthase) and ygaG (hypothetical protein) genes was negatively regulated, whereas 1.6 and 6.0 kb transcripts encoding ptp (protein tyrosine phosphatase) and cpd (2′,3′‐cyclic nucleotide 2′‐phosphodiesterase) respectively, were shown to be positively regulated by the VirR/VirS system. The other gene, hyp7, whose transcript was positively regulated by the VirR/VirS system, was shown to activate the transcription of the colA (kappa‐toxin) and plc (alpha‐toxin) genes, but not the pfoA (theta‐toxin) gene in C. perfringens. These results suggested that the global regulatory system VirR/VirS could regulate various genes, other than toxin genes, both positively and negatively and that the hyp7 gene might encode a novel regulatory factor for toxin production in C. perfringens.

The VirR/VirS regulatory cascade affects transcription of plasmid-encoded putative virulence genes in Clostridium perfringens strain 13

We analyzed the transcriptional regulation of the putative virulence genes encoded on the plasmid pCP13 from Clostridium perfringens strain 13. The transcription of the beta2-toxin (cpb2) and possible collagen adhesin (cna) genes were regulated in both a positive and negative manner, respectively, by the two-component VirR/VirS system. The secondary regulator of the VirR/VirS system, VR-RNA, also affects the expression of both of these genes in the same fashion as the VirR/VirS system. This indicates that the global regulatory cascade of the VirR/VirS system controls the expression of virulence genes located on the plasmid, as well as those found chromosomally in C. perfringens strain 13.

Identification of a two-component VirR/VirS regulon in Clostridium perfringens

Clostridium perfringens, a Gram-positive anaerobic pathogen, is a causative agent of human gas gangrene that leads to severe rapid tissue destruction and can cause death within hours unless treated immediately. Production of several toxins is known to be controlled by the two-component VirR/VirS system involving a regulatory RNA (VR-RNA) in C. perfringens. To elucidate the precise regulatory network governed by VirR/VirS and VR-RNA, a series of microarray screening using VirR/VirS and VR-RNA-deficient mutants was performed. Finally, by qRT-PCR analysis, 147 genes (30 single genes and 21 putative operons) were confirmed to be under the control of the VirR/VirS-VR-RNA regulatory cascade. Several virulence-related genes for alpha-toxin, kappa-toxin, hyaluronidases, sialidase, and capsular polysaccharide synthesis were found. Furthermore, some genes for catalytic enzymes, various genes for transporters, and many genes for energy metabolism were also found to be controlled by the cascade. Our data indicate that the VirR/VirS-VR-RNA system is a global gene regulator that might control multiple cellular functions to survive and multiply in the host, which would turn out to be a lethal flesh-eating infection.

Contact with enterocyte-like Caco-2 cells induces rapid upregulation of toxin production by Clostridium perfringens type C isolates.

Clostridium perfringens type C isolates cause necrotizing enteritis in humans and domestic animals. In vitro, type C isolates often produce β toxin (CPB), β2 toxin (CPB2), α toxin (CPA), perfringolysin O (PFO) and TpeL during (or after) late log‐phase growth. In contrast, the current study found that many type C isolates respond to close contact with enterocyte‐like Caco‐2 cells by producing all toxins, except TpeL, much more rapidly than occurs during in vitro growth. This in vivo effect involves rapid transcriptional upregulation of the cpb, cpb2, pfoA and plc toxin genes. Rapid Caco‐2 cell‐induced upregulation of CPB and PFO production involves the VirS/VirR two‐component system, since upregulated in vivo transcription of the pfoA and cpb genes was blocked by inactivating the virR gene and was reversible by complementation to restore VirR expression. However, the luxS quorum‐sensing system is not required for the rapid upregulation of type C toxin production induced by contact with Caco‐2 cells. These results provide the first indication of host cell:pathogen cross‐talk affecting toxin production kinetics by any pathogenic Clostridium spp., identify in vivo versus in vitro differences in C. perfringens toxin expression, and implicate VirS/VirR as a possible contributor to some C. perfringens enteric diseases.