Pathogenic Characterization of Clostridium perfringens Strains Isolated From Patients With Massive Intravascular Hemolysis.

Sepsis caused by Clostridium perfringens infection is rare but often fatal. The most serious complication leading to poor prognosis is massive intravascular hemolysis (MIH). However, the molecular mechanism underlying this fulminant form of hemolysis is unclear. In the present study, we employed 11 clinical strains isolated from patients with C. perfringens septicemia and subdivided these isolates into groups H and NH: septicemia with (n = 5) or without (n = 6) MIH, respectively. To elucidate the major pathogenic factors of MIH, biological features were compared between these groups. The isolates of two groups did not differ in growth rate, virulence-related gene expression, or phospholipase C (CPA) production. Erythrocyte hemolysis was predominantly observed in culture supernatants of the strains in group H, and the human erythrocyte hemolysis rate was significantly correlated with perfringolysin O (PFO) production. Correlations were also found among PFO production, human peripheral blood mononuclear cell (PBMC) cytotoxicity, and production of interleukin-6 (IL-6) and interleukin-8 (IL-8) by human PBMCs. Analysis of proinflammatory cytokines showed that PFO induced tumor necrosis factor-α (TNF-α), IL-5, IL-6, and IL-8 production more strongly than did CPA. PFO exerted potent cytotoxic and proinflammatory cytokine induction effects on human blood cells. PFO may be a major virulence factor of sepsis with MIH, and potent proinflammatory cytokine production induced by PFO may influence the rapid progression of this fatal disease caused by C. perfringens.

Gas gangrene-associated gliding motility is regulated by the Clostridium perfringens CpAL/VirSR system.

Clostridium perfringens strains cause a wide variety of human and animal disease, including gas gangrene or myonecrosis. Production of toxins required for myonecrosis, PFO and CPA, is regulated by the C. perfringens Agr-like (CpAL) system via the VirSR two-component system. Myonecrosis begins at the site of infection from where bacteria migrate deep into the host tissue likely using a previously described gliding motility phenotype. We therefore assessed whether gliding motility was under the control of the CpAL/VirSR regulon. The migration rate of myonecrosis-causing C. perfringens strain 13 (S13) was investigated during a 96 h period, including an adaptation phase with bacterial migration (∼1.4 mm/day) followed by a gliding phase allowing bacteria faster migration (∼8.6 mm/day). Gliding required both an intact CpAL system, and signaling through VirSR. Mutants lacking ΔagrB, or ΔvirR, were impaired for onward gliding while a complemented strain S13ΔagrB/pTS1303 had the gliding phenotype restored. Gene expression studies revealed upregulated transcription of pili genes (pilA1, pilA2 and pilT) whose encoded proteins were previously found to be required for gliding motility and CpAL/VirSR-regulated pfoA and cpa toxin genes. Compared to S13, transcription of cpa and pfoA significantly decreased in S13ΔagrB, or S13ΔvirR, strains but not that of pili genes. Further experiments demonstrated that mutants S13ΔpfoA and S13Δcpa migrated at the same rate as S13 wt. We demonstrated that CpAL/VirSR regulates C. perfringens gliding motility and that gliding bacteria have an increased transcription of toxin genes involved in myonecrosis.

Inflammasome Activation Induced by Perfringolysin O of Clostridium perfringens and Its Involvement in the Progression of Gas Gangrene.

Clostridium perfringens (C. perfringens) is Gram-positive anaerobic, spore-forming rod-shaped bacterial pathogen that is widely distributed in nature. This bacterium is known as the causative agent of a foodborne illness and of gas gangrene. While the major virulence factors are the α-toxin and perfringolysin O (PFO) produced by type A strains of C. perfringens, the precise mechanisms of how these toxins induce the development of gas gangrene are still not well understood. In this study, we analyzed the host responses to these toxins, including inflammasome activation, using mouse bone marrow-derived macrophages (BMDMs). Our results demonstrated, for the first time, that C. perfringens triggers the activation of caspase-1 and release of IL-1ß through PFO-mediated inflammasome activation via a receptor of the Nod-like receptor (NLR) family, pyrin-domain containing 3 protein (NLRP3). The PFO-mediated inflammasome activation was not induced in the cultured myocytes. We further analyzed the functional roles of the toxins in inducing myonecrosis in a mouse model of gas gangrene. Although the myonecrosis was found to be largely dependent on the α-toxin, PFO also induced myonecrosis to a lesser extent, again through the mediation of NLRP3. These results suggest that C. perfringens triggers inflammatory responses via PFO-mediated inflammasome activation via NLRP3, and that this axis contributes in part to the progression of gas gangrene. Our findings provide a novel insight into the molecular mechanisms underlying the pathogenesis of gas gangrene caused by C. perfringens.

Metabolic dependent and independent pH-drop shuts down VirSR quorum sensing in Clostridium perfringens.

Clostridium perfringens produces various exotoxins and enzymes that cause food poisoning and gas gangrene. The genes involved in virulence are regulated by the agr-like quorum sensing (QS) system, which consists of a QS signal synthesis system and a VirSR two-component regulatory system (VirSR TCS) which is a global regulatory system composed of signal sensor kinase (VirS) and response regulator (VirR). We found that the perfringolysin O gene (pfoA) was transiently expressed during mid-log phase of bacterial growth; its expression was rapidly shut down thereafter, suggesting the existence of a self-quorum quenching (sQQ) system. The sQQ system was induced by the addition of stationary phase culture supernatant (SPCS). Activity of the sQQ system was heat stable, and was present following filtration through the ultrafiltration membrane, suggesting that small molecules acted as sQQ agents. In addition, sQQ was also induced by pure acetic and butyric acids at concentrations equivalent to those in the stationary phase culture, suggesting that organic acids produced by C. perfringens were involved in sQQ. In pH-controlled batch culture, sQQ was greatly diminished; expression level of pfoA extended to late-log growth phase, and was eventually increased by one order of magnitude. Furthermore, hydrochloric acid induced sQQ at the same pH as was used in organic acids. SPCS also suppressed the expression of genes regulated by VirSR TCS. Overall, the expression of virulence factors of C. perfringens was downregulated by the sQQ system, which was mediated by primary acidic metabolites and acidic environments. This suggested the possibility of pH-controlled anti-virulence strategies.

Inhibition of biofilm formation on iodine-supported titanium implants.

PURPOSE: We have developed iodine-supported titanium implants that suppress microbial activities and conducted in vivo and in vitro studies to determine their antimicrobial properties. METHODS: The implants were Ti-6Al-4 V titanium implants either untreated (Ti), treated with oxide film on the Ti surface by anodization (Ti-O), or treated with an iodine coating on oxidation film (Ti-I). The strain of bacteria used in this study was Gram-positive Staphylococcus aureus strain ATCC 25923. We analyzed the antibacterial attachment effects in vivo by using rats. The attachment bacteria on the implant surface were evaluated using a spread-plate method assay. A biofilm study was performed in vitro. The biofilm formed after bacterial attachment was qualitatively studied with fluorescence microscopy (FM) and scanning electron microscopy (SEM). Also, the formed biofilm was quantitatively studied with a spread-plate method assay. RESULTS: In vivo analysis of antimicrobial attachment effects showed that the mean viable bacterial number was significantly lower on Ti-I than Ti or Ti-O surfaces. In the in vitro biofilm study, FM and SEM images showed thick and mature biofilm formation on Ti and Ti-O and thin, small biofilm formation on Ti-I. A quantitative biofilm analysis found a significant difference in the number of viable bacteria between Ti-I and Ti or Ti-O. CONCLUSIONS: This study showed that iodine-supported implants have a good antibacterial attachment effect and inhibit biofilm formation and growth. Iodine-supported implants may have great potential as innovative antibacterial implants that can prevent implant related infection in orthopaedic surgery.

Regulation of Toxin Production in Clostridium perfringens.

The Gram-positive anaerobic bacterium Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tracts of humans and animals. C. perfringens causes gas gangrene and food poisoning, and it produces extracellular enzymes and toxins that are thought to act synergistically and contribute to its pathogenesis. A complicated regulatory network of toxin genes has been reported that includes a two-component system for regulatory RNA and cell-cell communication. It is necessary to clarify the global regulatory system of these genes in order to understand and treat the virulence of C. perfringens. We summarize the existing knowledge about the regulatory mechanisms here.

Clostridium perfringens α-Toxin Impairs Innate Immunity via Inhibition of Neutrophil Differentiation.

Although granulopoiesis is accelerated to suppress bacteria during infection, some bacteria can still cause life-threatening infections, but the mechanism behind this remains unclear. In this study, we found that mature neutrophils in bone marrow cells (BMCs) were decreased in C. perfringens-infected mice and also after injection of virulence factor α-toxin. C. perfringens infection interfered with the replenishment of mature neutrophils in the peripheral circulation and the accumulation of neutrophils at C. perfringens-infected sites in an α-toxin-dependent manner. Measurements of bacterial colony-forming units in C. perfringens-infected muscle revealed that α-toxin inhibited a reduction in the load of C. perfringens. In vitro treatment of isolated BMCs with α-toxin (phospholipase C) revealed that α-toxin directly decreased mature neutrophils. α-Toxin did not influence the viability of isolated mature neutrophils, while simultaneous treatment of BMCs with granulocyte colony-stimulating factor attenuated the reduction of mature neutrophils by α-toxin. Together, our results illustrate that impairment of the innate immune system by the inhibition of neutrophil differentiation is crucial for the pathogenesis of C. perfringens to promote disease to a life-threatening infection, which provides new insight to understand how pathogenic bacteria evade the host immune system.

Gene regulation by the VirS/VirR system in Clostridium perfringens.

The Gram-positive anaerobic spore-forming rod, Clostridium perfringens, is widely distributed in nature, especially in soil and the gastrointestinal tract of humans and animals. C. perfringens produces many secreted toxins and enzymes that are involved in the pathogenesis of gas gangrane and gastrointestinal disease. One of the most important systems regulating the production of these proteins in C. perfringens is the VirS/VirR-VR-RNA signal transduction cascade. The Agr system also important for the regulation of toxin genes. VirS appears to sense the peptide produced by the Agr (accessory gene regulator) system. The VirS/VirR-VR-RNA cascade controls the pathogenesis of C. perfringens infections by regulating virulence related genes and genes for energy metabolism. These systems are important for the host cell-induced upregulation of toxin production.

Rationale design of quorum-quenching peptides that target the VirSR system of Clostridium perfringens.

In Clostridium perfringens, a 5-membered thiolactone peptide acts as an autoinducing peptide (AIPCp) to activate the VirSR two-component signal transduction system, which in turn controls the expression of genes encoding multiple toxins, including α, θ and κ. To develop anti-pathogenic agents against virulent C. perfringens, quorum-quenching peptides were rationally designed based on the structure-activity relationship (SAR) data on AIPCp. Alanine scanning study of AIPCp suggested that Trp(3) and Phe(4) are involved in receptor binding and activation, respectively. On the basis of the SAR, we designed two quorum-quenching peptides with different modes of action: Z-AIPCp-L2A/T5A (partial agonist) and Z-AIPCp-F4A/T5S (partial antagonist). Both peptides significantly attenuated transcription of θ toxin gene (pfoA) in a virulent strain of C. perfringens with IC50 = 0.32 and 0.72 µM, respectively.

Cyclodepsipeptides produced by actinomycetes inhibit cyclic-peptide-mediated quorum sensing in Gram-positive bacteria.

Cyclic peptides are commonly used as quorum-sensing autoinducers in Gram-positive Firmicutes bacteria. Well-studied examples of such molecules are thiolactone and lactone, used to regulate the expression of a series of virulence genes in the agr system of Staphylococcus aureus and the fsr system of Enterococcus faecalis, respectively. Three cyclodepsipeptides WS9326A, WS9326B and cochinmicin II/III were identified as a result of screening actinomycetes culture extracts for activity against the agr/fsr system. These molecules are already known as receptor antagonists, the first two for tachykinin and the last one for endothelin. WS9326A also inhibited the transcription of pfoA regulated by the VirSR two-component system in Clostridium perfringens. Receptor-binding assays using a fluorescence-labeled autoinducer (FITC-GBAP) showed that WS9326A and WS9326B act as receptor antagonists in this system. In addition, an ex vivo assay showed that WS9326B substantially attenuated the toxicity of S. aureus for human corneal epithelial cells. These results suggest that these three natural cyclodepsipeptides have therapeutic potential for targeting the cyclic peptide-mediated quorum sensing of Gram-positive pathogens.

Regulation of toxin gene expression in Clostridium perfringens.

The Gram-positive, anaerobic, spore-forming, rod-shaped Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tract of humans and animals. C. perfringens causes clostridial myonecrosis (or gas gangrene), enteritis and enterotoxemia in humans and livestock by producing numerous extracellular toxins and enzymes. The toxin gene expression is regulated by a two-component regulatory system and regulatory RNA VirR/VirS-VR-RNA cascade. The VirR/VirS system was originally found in a type A strain, but a recent report showed that it is also important for the toxin gene regulation in other types of strains. Two types of cell-cell signaling, i.e., agr-system and AI-2 signaling, are also important for the regulation of toxin genes. Several regulatory systems independent from the VirR/VirS system, including virX, the orphan histidine kinase ReeS and orphan response regulator RevR, are also involved in the regulation of toxin genes. In addition, the expression of toxin genes is upregulated after contact with Caco-2 cells. C. perfringens has a complex regulatory network for toxin gene expression and thus the coordination of toxin gene expression is important for the process of infection.

Identification of a two-component signal transduction system that regulates maltose genes in Clostridium perfringens.

Clostridium perfringens is a Gram-positive rod that is widely distributed in nature and is the etiological agent of several human and animal diseases. The complete genome sequence of C. perfringens strain 13 has been determined and multiple two-component signal transduction systems identified. One of these systems, designated here as the MalNO system, was analyzed in this study. Microarray analysis was used to carry out functional analysis of a malO mutant. The results, which were confirmed by quantitative reverse-transcriptase PCR, indicated that genes putatively involved in the uptake and metabolism of maltose were up-regulated in the malO mutant. These effects were reversed by complementation with the wild-type malO gene. Growth of these isogenic strains in medium with and without maltose showed that the malO mutant recovered more quickly from maltose deprivation when compared to the wild-type and complemented strains, leading to the conclusion that the MalNO system regulates maltose utilization in C. perfringens. It is postulated that this regulatory network may allow this soil bacterium and opportunistic pathogen to respond to environmental conditions where there are higher concentrations of maltose or maltodextrins, such as in the presence of decaying plant material in rich soil.

Regulation of sialidase production in Clostridium perfringens by the orphan sensor histidine kinase ReeS.

Clostridium perfringens is ubiquitous in nature and is often found as a commensal of the human and animal gastrointestinal tract. It is the primary etiological agent of clostridial myonecrosis, or gas gangrene, a serious infection that results in extensive tissue necrosis due to the action of one or more potent extracellular toxins. α-toxin and perfringolysin O are the major extracellular toxins involved in the pathogenesis of gas gangrene, but histotoxic strains of C. perfringens, such as strain 13, also produce many degradative enzymes such as collagenases, hyaluronidases, sialidases and the cysteine protease, α-clostripain. The production of many of these toxins is regulated either directly or indirectly by the global VirSR two-component signal transduction system. By isolating a chromosomal mutant and carrying out microarray analysis we have identified an orphan sensor histidine kinase, which we have named ReeS (regulator of extracellular enzymes sensor). Expression of the sialidase genes nanI and nanJ was down-regulated in a reeS mutant. Since complementation with the wild-type reeS gene restored nanI and nanJ expression to wild-type levels, as shown by quantitative reverse transcription-PCR and sialidase assays we concluded that ReeS positively regulates the expression of these sialidase genes. However, mutation of the reeS gene had no significant effect on virulence in the mouse myonecrosis model. Sialidase production in C. perfringens has been previously shown to be regulated by both the VirSR system and RevR. In this report, we have analyzed a previously unknown sensor histidine kinase, ReeS, and have shown that it also is involved in controlling the expression of sialidase genes, adding further complexity to the regulatory network that controls sialidase production in C. perfringens.

Unique regulatory mechanism of sporulation and enterotoxin production in Clostridium perfringens.

Clostridium perfringens causes gas gangrene and gastrointestinal (GI) diseases in humans. The most common cause of C. perfringens-associated food poisoning is the consumption of C. perfringens vegetative cells followed by sporulation and production of enterotoxin in the gut. Despite the importance of spore formation in C. perfringens pathogenesis, the details of the regulation of sporulation have not yet been defined fully. In this study, microarray and bioinformatic analyses identified a candidate gene (the RNA regulator virX) for the repression of genes encoding positive regulators (Spo0A and sigma factors) of C. perfringens sporulation. A virX mutant constructed in the food poisoning strain SM101 had a much higher sporulation efficiency than that of the wild type. The transcription of sigE, sigF, and sigK was strongly induced at 2.5 h of culture of the virX mutant. Moreover, the transcription of the enterotoxin gene was also strongly induced in the virX mutant. Western blotting confirmed that the levels of enterotoxin production were higher in the virX mutant than in the wild type. These observations indicated that the higher levels of sporulation and enterotoxin production in the virX mutant were specifically due to inactivation of the virX gene. Since virX homologues were not found in any Bacillus species but were present in other clostridial species, our findings identify further differences in the regulation of sporulation between Bacillus and certain Clostridium species. The virX RNA regulator plays a key role in the drastic shift in lifestyle of the anaerobic flesh eater C. perfringens between the vegetative state (for gas gangrene) and the sporulating state (for food poisoning).

Phosphodiesterase inhibitors suppress Lactobacillus casei cell-wall-induced NF-κB and MAPK activations and cell proliferation through protein kinase A--or exchange protein activated by cAMP-dependent signal pathway.

Specific strains of Lactobacillus have been found to be beneficial in treating some types of diarrhea and vaginosis. However, a high mortality rate results from underlying immunosuppressive conditions in patients with Lactobacillus casei bacteremia. Cyclic AMP (cAMP) is a small second messenger molecule that mediates signal transduction. The onset and progression of inflammatory responses are sensitive to changes in steady-state cAMP levels. L. casei cell wall extract (LCWE) develops arteritis in mice through Toll-like receptor-2 signaling. The purpose of this study was to investigate whether intracellular cAMP affects LCWE-induced pathological signaling. LCWE was shown to induce phosphorylation of the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways and cell proliferation in mice fibroblast cells. Theophylline and phosphodiesterase inhibitor increased intracellular cAMP and inhibited LCWE-induced cell proliferation as well as phosphorylation of NF-κB and MAPK. Protein kinase A inhibitor H89 prevented cAMP-induced MAPK inhibition, but not cAMP-induced NF-κB inhibition. An exchange protein activated by cAMP (Epac) agonist inhibited NF-κB activation but not MAPK activation. These results indicate that an increase in intracellular cAMP prevents LCWE induction of pathological signaling pathways dependent on PKA and Epac signaling.

Ezrin overexpression by transformed human ovarian surface epithelial cells, ovarian cleft cells, and serous ovarian adenocarcinoma cells.

OBJECTIVES: We have shown that ezrin expression correlates with ovarian epithelial cancer (OVCA) cell proliferation and metastatic behavior. In this study, we evaluated ezrin expression in transformed ovarian superficial epithelial cells (OSE) in ovarian clefts and in culture. STUDY DESIGN: Immunohistochemistry and Western blotting for immunoreactive ezrin (ir-ezrin) in normal ovarian tissue, cultured OSE, and ovarian epithelial cancer cells. RESULTS: While ir-ezrin was not demonstrable in normal cuboidal surface cells or interior ovarian organelles, cells lining the ovarian clefts strongly expressed ir-ezrin. Long-term culture of OSE increased ezrin expression and cytological abnormalities. Administration of estradiol and insulin at levels reported in inclusions dramatically induced OSE ir-ezrin expression to OVCA levels and membrane specializations; ruffling, pseudopodia and filopodia. Moreover epidermal growth factor (EGF) drastically increased ezrin translocation in OSE cells in a time-dependent manner. CONCLUSIONS: Ezrin expression by OSE increases during transformation. Ezrin expression is responsive to estradiol and growth factors previously shown to be present in ovarian inclusions. These findings suggest that the microenvironment in ovarian inclusions and clefts contributes to the development of OVCA. Our findings elaborate on the mechanism of the ovarian origin of OVCA.

Complex transcriptional regulation of citrate metabolism in Clostridium perfringens.

A Gram-positive, spore-forming bacterium, Clostridium perfringens, possesses genes for citrate metabolism, which might play an important role in the utilization of citrate as a sole carbon source. In this study, we identified a chromosomal citCDEFX-mae-citS operon in C. perfringens strain 13, which is transcribed on three mRNAs of different sizes. Expression of the cit operon was significantly induced when 5 mM extracellular citrate was added to the growth medium. Most interestingly, three regulatory systems were found to be involved in the regulation of the expression of cit genes: 1) the two upstream divergent genes citG and citI; 2) two different two-component regulatory systems, CitA/CitB (TCS6 consisted of CPE0531/CPE0532) and TCS5 (CPE0518/CPE0519); and 3) the global two-component VirR/VirS-VR-RNA regulatory system known to regulate various genes for toxins and degradative enzymes. Our results suggest that in C. perfringens the citrate metabolism might be strictly controlled by a complex regulatory system.

Regulation of virulence by the RevR response regulator in Clostridium perfringens.

Clostridium perfringens causes clostridial myonecrosis or gas gangrene and produces several extracellular hydrolytic enzymes and toxins, many of which are regulated by the VirSR signal transduction system. The revR gene encodes a putative orphan response regulator that has similarity to the YycF (WalR), VicR, PhoB, and PhoP proteins from other Gram-positive bacteria. RevR appears to be a classical response regulator, with an N-terminal receiver domain and a C-terminal domain with a putative winged helix-turn-helix DNA binding region. To determine its functional role, a revR mutant was constructed by allelic exchange and compared to the wild type by microarray analysis. The results showed that more than 100 genes were differentially expressed in the mutant, including several genes involved in cell wall metabolism. The revR mutant had an altered cellular morphology; unlike the short rods observed with the wild type, the mutant cells formed long filaments. These changes were reversed upon complementation with a plasmid that carried the wild-type revR gene. Several genes encoding extracellular hydrolytic enzymes (sialidase, hyaluronidase, and α-clostripain) were differentially expressed in the revR mutant. Quantitative enzyme assays confirmed that these changes led to altered enzyme activity and that complementation restored the wild-type phenotype. Most importantly, the revR mutant was attenuated for virulence in the mouse myonecrosis model compared to the wild type and the complemented strains. These results provide evidence that RevR regulates virulence in C. perfringens; it is the first response regulator other than VirR to be shown to regulate virulence in this important pathogen.

Global regulation of gene expression in response to cysteine availability in Clostridium perfringens.

BACKGROUND: Cysteine has a crucial role in cellular physiology and its synthesis is tightly controlled due to its reactivity. However, little is known about the sulfur metabolism and its regulation in clostridia compared with other firmicutes. In Clostridium perfringens, the two-component system, VirR/VirS, controls the expression of the ubiG operon involved in methionine to cysteine conversion in addition to the expression of several toxin genes. The existence of links between the C. perfringens virulence regulon and sulfur metabolism prompted us to analyze this metabolism in more detail. RESULTS: We first performed a tentative reconstruction of sulfur metabolism in C. perfringens and correlated these data with the growth of strain 13 in the presence of various sulfur sources. Surprisingly, C. perfringens can convert cysteine to methionine by an atypical still uncharacterized pathway. We further compared the expression profiles of strain 13 after growth in the presence of cystine or homocysteine that corresponds to conditions of cysteine depletion. Among the 177 genes differentially expressed, we found genes involved in sulfur metabolism and controlled by premature termination of transcription via a cysteine specific T-box system (cysK-cysE, cysP1 and cysP2) or an S-box riboswitch (metK and metT). We also showed that the ubiG operon was submitted to a triple regulation by cysteine availability via a T-box system, by the VirR/VirS system via the VR-RNA and by the VirX regulatory RNA.In addition, we found that expression of pfoA (theta-toxin), nagL (one of the five genes encoding hyaluronidases) and genes involved in the maintenance of cell redox status was differentially expressed in response to cysteine availability. Finally, we showed that the expression of genes involved in [Fe-S] clusters biogenesis and of the ldh gene encoding the lactate dehydrogenase was induced during cysteine limitation. CONCLUSION: Several key functions for the cellular physiology of this anaerobic bacterium were controlled in response to cysteine availability. While most of the genes involved in sulfur metabolism are regulated by premature termination of transcription, other still uncharacterized mechanisms of regulation participated in the induction of gene expression during cysteine starvation.