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Absent in melanoma 2 (AIM2), a key component of the IFI20X/IFI16 (PYHIN) protein family, is characterized as a DNA sensor to detect cytosolic bacteria and DNA viruses. However, little is known about its immunological role during pathogenic Clostridium perfringens ( C . perfringens ) infection, an extracellular bacterial pathogen. In a pathogenic C . perfringens gas gangrene model, Aim2 -/- mice are more susceptible to pathogenic C . perfringens soft tissue infection, revealing the importance of AIM2 in host protection. Notably, Aim2 deficiency leads to a defect in bacterial killing and clearance. Our in vivo and in vitro findings further establish that inflammasome signaling is impaired in the absence of Aim2 in response to pathogenic C . perfringens . Mechanistically, inflammasome signaling downstream of active AIM2 promotes pathogen control. Importantly, pathogenic C . perfringens -derived genomic DNA triggers inflammasome signaling activation in an AIM2-dependent manner. Thus, these observations uncover a central role for AIM2 in host defense and triggering innate immunity to combat pathogenic C . perfringens infections.

Gas gangrene, or clostridial myonecrosis, is usually caused by Clostridium perfringens and may occur spontaneously in association with diabetes mellitus, peripheral vascular disease, or some malignancies but more often after contamination of a deep surgical or traumatic lesion. If not controlled, clostridial myonecrosis results in multiorgan failure, shock, and death, but very little is known about the muscle regeneration process that follows myonecrosis when the infection is controlled. In this study, we characterized the muscle regeneration process after myonecrosis caused in a murine experimental infection with a sublethal inoculum of C. perfringens vegetative cells. The results show that myonecrosis occurs concomitantly with significant vascular injury, which limits the migration of inflammatory cells. A significant increase in cytokines that promote inflammation explains the presence of an inflammatory infiltrate; however, impaired interferon gamma (IFN-γ) expression, a reduced number of M1 macrophages, deficient phagocytic activity, and a prolongation of the permanence of inflammatory cells lead to deficient muscle regeneration. The expression of transforming growth factor β1 (TGF-β1) agrees with the consequent accumulation of collagen in the muscle, i.e., fibrosis observed 30 days after infection. These results provide new information on the pathogenesis of gas gangrene caused by C. perfringens , shed light on the basis of the deficient muscle regenerative activity, and may open new perspectives for the development of novel therapies for patients suffering from this disease., (Copyright © 2019 American Society for Microbiology.)

During bacterial infection, granulocyte colony-stimulating factor (G-CSF) is produced and accelerates neutrophil production from their progenitors. This process, termed granulopoiesis, strengthens host defense, but Clostridium perfringens α-toxin impairs granulopoiesis via an unknown mechanism. Here, we tested whether G-CSF accounts for the α-toxin-mediated impairment of granulopoiesis. We find that α-toxin dramatically accelerates G-CSF production from endothelial cells in response to Toll-like receptor 2 (TLR2) agonists through activation of the c-Jun N-terminal kinase (JNK) signaling pathway. Meanwhile, α-toxin inhibits G-CSF-mediated cell proliferation of Ly-6G + neutrophils by inducing degradation of G-CSF receptor (G-CSFR). During sepsis, administration of α-toxin promotes lethality and tissue injury accompanied by accelerated production of inflammatory cytokines in a TLR4-dependent manner. Together, our results illustrate that α-toxin disturbs G-CSF-mediated granulopoiesis by reducing the expression of G-CSFR on neutrophils while augmenting septic shock due to excess inflammatory cytokine release, which provides a new mechanism to explain how pathogenic bacteria modulate the host immune system., Competing Interests: The authors declare no competing interests.

The alpha-toxin is one of the virulence factors of Clostridium perfringens for gas gangrene in humans and animals or necrotic enteritis in poultry. The C-terminal domain of this toxin ( cpa 247-370 ) was synthesized and cloned into pT1NX vector to construct the pT1NX-alpha plasmid. This surface-expressing plasmid was electroporated into Lactobacillus casei ATCC 393, generating the recombinant L. casei strain expressing alpha-toxoid (LC-α strain). Expression of this modified alpha-toxoid was confirmed by SDS-PAGE, immunoblotting, and direct immunofluorescence microscopy. BALB/c mice, immunized orally by the recombinant LC-α strain, elicited mucosal and significantly humoral immune responses (p < 0.05) and developed a protection against 900 MLD/mL of the standard alpha-toxin. This study showed that this recombinant LC-α strain could be a promising vaccine candidate against gas gangrene and necrotic enteritis.

To obtain an insight into host-pathogen interactions in clostridial myonecrosis, we carried out comparative transcriptome analysis of both the bacterium and the host in a murine Clostridium perfringens infection model, which is the first time that such an investigation has been conducted. Analysis of the host transcriptome from infected muscle tissues indicated that many genes were upregulated compared to the results seen with mock-infected mice. These genes were enriched for host defense pathways, including Toll-like receptor (TLR) and Nod-like receptor (NLR) signaling components. Real-time PCR confirmed that host TLR2 and NLRP3 inflammasome genes were induced in response to C. perfringens infection. Comparison of the transcriptome of C. perfringens cells from the infected tissues with that from broth cultures showed that host selective pressure induced a global change in C. perfringens gene expression. A total of 33% (923) of C. perfringens genes were differentially regulated, including 10 potential virulence genes that were upregulated relative to their expression in vitro These genes encoded putative proteins that may be involved in the synthesis of cell wall-associated macromolecules, in adhesion to host cells, or in protection from host cationic antimicrobial peptides. This report presents the first successful expression profiling of coregulated transcriptomes of bacterial and host genes during a clostridial myonecrosis infection and provides new insights into disease pathogenesis and host-pathogen interactions. IMPORTANCE Clostridium perfringens is the causative agent of traumatic clostridial myonecrosis, or gas gangrene. In this study, we carried out transcriptional analysis of both the host and the bacterial pathogen in a mouse myonecrosis infection. The results showed that in comparison to mock-infected control tissues, muscle tissues from C. perfringens -infected mice had a significantly altered gene expression profile. In particular, the expression of many genes involved in the innate immune system was upregulated. Comparison of the expression profiles of C. perfringens cells isolated from the infected tissues with those from equivalent broth cultures identified many potential virulence genes that were significantly upregulated in vivo These studies have provided a new understanding of the range of factors involved in host-pathogen interactions in a myonecrosis infection., (Copyright © 2018 Low et al.)

Cases of Clostridium perfringens septicemia, such as liver abscess, often develop a rapidly progressive intravascular hemolysis and coagulation; the mortality rate with current standard care including antibiotics and surgery is high. Herein, we firstly investigated the effects of gas gangrene antitoxin (GGA) (antitoxin against C. perfringens) and recombinant human soluble thrombomodulin (rTM) on the hemolysis, coagulation status, inflammatory process, and mortality in α-toxin-treated rats. Male 11-week-old Sprague Dawley rats were randomly divided into five groups: control group, α-toxin group, GGA group, rTM group, and combined GGA and rTM (combination group). After α-toxin injection, mortality and platelet counts, and hemolysis were observed for 6 h. The fibrin/fibrinogen degradation products (FDP), and plasma high-mobility group box 1 (HMGB1) were also measured at 6 h. The combination group demonstrated 100% survival compared with 50% survival in the α-toxin group and demonstrated significantly improved hemolysis, platelet counts, and lactate levels compared with those in the α-toxin group (p < .01). The FDP and HMGB1 levels in the combination therapy group were significantly lower than those in the α-toxin group (p < .05). Combination therapy with GGA and rTM administration is applicable as adjunct therapy for fatal C. perfringens sepsis., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Blood serum from immunized humans or animals (e.g., horses) contains relevant antibodies and has been used as serum therapy to treat many diseases or envenomation events. The effectiveness of blood serum was initially discovered in 1890 when Kitasato and von Behring observed the effectiveness of this type of therapy against diphtheria and tetanus. Serum therapies played an important role in the advancement of modern medicine prior to the development of penicillin and steroids. At present, several types of serum therapy remain in clinical use. However, some physicians have a limited understanding of the nature and the benefits of serum therapy and the factors that require particular attention. In this review, we set out to clarify the benefits, cautions, and potential applications of serum therapy in the context of conditions such as gas gangrene, diphtheria, botulism, and tetanus and bites from three snake species (mamushi, habu, and yamakagashi) and the redback spider. It is hoped that this review will help clinicians to learn about clinical serum therapies and become familiar with their applications.

The whole genome sequencing and annotation of Clostridium perfringens strains revealed several genes coding for proteins of unknown function with no significant similarities to genes in other organisms. Our previous studies clearly demonstrated that hypothetical proteins CPF&#95;2500, CPF&#95;1441, CPF&#95;0876, CPF&#95;0093, CPF&#95;2002, CPF&#95;2314, CPF&#95;1179, CPF&#95;1132, CPF&#95;2853, CPF&#95;0552, CPF&#95;2032, CPF&#95;0438, CPF&#95;1440, CPF&#95;2918, CPF&#95;0656, and CPF&#95;2364 are genuine proteins of C. perfringens expressed in high abundance. This study explored the putative role of these hypothetical proteins using bioinformatic tools and evaluated their potential as putative candidates for prophylaxis. Apart from a group of eight hypothetical proteins (HPs), a putative function was predicted for the rest of the hypothetical proteins using one or more of the algorithms used. The phylogenetic analysis did not suggest an evidence of a horizontal gene transfer event except for HP CPF&#95;0876. HP CPF&#95;2918 is an abundant extracellular protein, unique to C. perfringens species with maximum strain coverage and did not show any significant match in the database. CPF&#95;2918 was cloned, recombinant protein was purified to near homogeneity, and probing with mouse anti-CPF&#95;2918 serum revealed surface localization of the protein in C. perfringens ATCC13124 cultures. The purified recombinant CPF&#95;2918 protein induced antibody production, a mixed Th1 and Th2 kind of response, and provided partial protection to immunized mice in direct C. perfringens challenge., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Clostridium perfringens type D infects ruminants and causes the enterotoxemia disease by ε-toxin. A mutated ε-toxin gene lacking toxicity was designed, synthesized, and cloned into the pT1NX vector and electroporated into Lactobacillus casei competent cells to yield LC-pT1NX-ε recombinant strain. BALB/c mice, immunized orally with this strain, highly induced mucosal, humoral, and cell-mediated immune responses and developed a protection against 200 MLD/ml of the activated ε-toxin. This study showed that the LC-pT1NX-ε could be a promising vaccine candidate against the enterotoxemia disease., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Clostridium perfringens is an obligately anaerobic rod-shaped bacterium and etiological agent for several diseases in humans and animals. The pathogen has been listed as Validated Biological Agent and warrants development of medical countermeasures. The homologs of some of the lipoproteins identified from various fractions of C. perfringens in our previous studies were observed to be virulence determinants in other pathogenic bacteria. Three putative virulence associated lipoproteins; polysaccharide deacetylase family protein, probable ion-uptake ABC transporter, and a putative lipoprotein of no known function are reported here with respect to their immuno-protective potentials. The three proteins were over expressed and purified to near homogeneity. The lipoproteins were shown to be exposed on the C. perfringens surface and, hence, accessible to antibodies and potentially visible to the host immune system. Immunization of mice with purified recombinant proteins elicited protective immunity against challenge with C. perfringens in mouse gas gangrene model. Distribution and relationship of orthologous proteins across other bacterial select agents especially among the members of Firmicutes, was carried out to look for conserved antigenic determinants., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Clostridium perfringens alpha-toxin is thought to be an important agent in gas gangrene, which is a lifethreatening infection with fever, pain, edema, myonecrosis, and gas production. The toxin (370 residues) is composed of an N-terminal domain (1-250 residues, N-domain) in which the catalytic site is found and a C-terminal domain (251-370 residues, C-domain) responsible for binding to membranes. During the past decade, recombinant DNA technology has been employed to develop second-generation vaccines, including site-directed mutants and the C-domain of the toxin, to prevent gas gangrene. These immunities have led to protection against the lethal effects of wild-type C. perfringens in mice. C-domain vaccines are capable of protecting against heterologous clostridia causing clostridial myonecrosis. This article summarizes the current knowledge on vaccines against alpha-toxin.

Clostridium septicum (C. septicum) gas gangrene is well documented in the literature, typically in the setting of trauma or immunosuppression. In this paper, we report a unique case of spontaneous clostridial myonecrosis in a patient with Crohn's disease and sulfasalazine-induced neutropenia. The patient presented with left thigh pain, vomiting and diarrhea. Blood tests demonstrated a profound neutropenia, and magnetic resonance imaging of the thigh confirmed extensive myonecrosis. The patient underwent emergency hip disarticulation, followed by hemicolectomy. C. septicum was cultured from the blood. Following completion of antibiotic therapy, the patient developed myonecrosis of the right pectoral muscle necessitating further debridement, and remains on lifelong prophylactic antibiotic therapy.

Azathioprine and 6-mercaptopurine have been used for many years in the treatment of inflammatory bowel disease. Approximately 0.3% of the population are homozygous for variant alleles associated with extremely low thiopurine S-methyltransferase enzyme activity. We describe the case of a young patient with ulcerative colitis, homozygous for TPMT*3A alleles, who suffered fatal azathioprine-induced myelotoxicity after standard dosing with azathioprine. Screening for decreased activity of TPMT in patients prior to azathioprine treatment is advised to minimize the risk of drug-induced toxicity.

Clostridium perfringens is a common cause of the fatal disease gas gangrene (myonecrosis). Established gas gangrene is notable for a profound absence of neutrophils and monocytic cells (phagocytes), and it has been suggested that the bactericidal activities of these cells play an insignificant role in controlling the progression of the infection. However, large inocula of bacteria are needed to establish an infection in experimental animals, suggesting phagocytes may play a role in inhibiting the initiation of gangrene. Examination of tissue sections of mice infected with a lethal (1 x 10(9)) or sublethal (1 x 10(6)) inoculum of C. perfringens revealed that phagocyte infiltration in the first 3 h postinfection was inhibited with a lethal dose but not with a sublethal dose, indicating that exclusion of phagocytes begins very early in the infection cycle. Experiments in which mice were depleted of either circulating monocytes or neutrophils before infection with C. perfringens showed that monocytes play a role in inhibiting the onset of gas gangrene at intermediate inocula but, although neutrophils can slow the onset of the infection, they are not protective. These results suggest that treatments designed to increase monocyte infiltration and activate macrophages may lead to increased resistance to the initiation of gas gangrene.

We report on a 30-year-old male with ulcerative colitis who developed a spontaneous gas gangrene in the right limb, the gluteal muscles and the retroperitoneal region under immunosuppressive therapy. In spite of immediate aggressive surgical and antibiotic therapy the massive infection led to septicemia and ultimately death. Clostridium septicum was identified with multiple local manifestations in the skeletal muscles. Gas gangrene is extremely rare in patients with ulcerative colitis or Crohn's disease and immunosuppression. The therapeutic options are discussed and the relevant present literature is reviewed.

Various stressors were used to induce stress proteins in Clostridium perfringens. Cultures of C. perfringens FD-1041 were subjected to cold shock (28 degrees C for 1 h), acid shock (pH 4.5 for 30 min), or heat shock (50 degrees C for 30 min). Cells were lysed and protein samples were analyzed by immunoblotting with antiserum derived from rabbits suffering from gas gangrene. Eight cold shock proteins (approximate Mr 101, 82, 70, 37, 22, 12, 10 and 6 kDa) and also eight heat shock proteins (approximate Mr 101, 82, 70, 27, 22, 16, 12 and 10 kDa) were immunoreactive with the serum. No immunoreactive proteins were detected in samples subjected to acid shock proteins and purified DnaK protein was also non-immunoreactive with the serum. These immunogenic stress proteins may be important in regulating diseases caused by C. perfringens. Such proteins could be involved in cell survival mechanisms, serve as targets during infection, or play a role in recognition of the bacteria by the host.

The rapid extension of necrosis and an absence of polymorphonuclear leukocytes (PMNL) at the site of infection are two hallmarks of Clostridium perfringens gas gangrene. While both alpha and theta toxins profoundly affect PMNL function and viability in vitro, their roles in muscle destruction and impairment of the inflammatory response in vivo have not been investigated. Comparative histopathologic examinations were performed on animals infected with either wild-type C. perfringens, or isogenic, toxin-deficient mutants of C. perfringens. Tissue destruction was modest in animals infected with the alpha toxin-deficient mutant; destruction was more pronounced in tissues infected with the theta toxin-deficient mutant or the wild-type strain. alpha and theta toxins also displayed differing abilities to modulate the inflammatory response. Histopathologic studies in which recombinant toxins were injected together with killed, washed C. perfringens further substantiated these tissue-destructive and differential antiinflammatory effects.

Within the framework of an extensive research programme, the socio-economic and environmental conditions which influence the emergence of soil-borne diseases in north-eastern Mexico were analysed. Furthermore, specimens collected from carcasses in the field were bacteriologically examined and the causal organisms of soil-borne diseases differentiated by means of gas chromatographic analysis of their metabolic products and the long-chained fatty acids contained in the cell. With experimental clostridial vaccines prepared with the Goettingen Bioreactor Technique, trials to protect cattle and guinea-pigs against gas gangrene were carried out. It was found that the farm structure and the dry climate as well as the specific soil conditions and plant cover favour the emergence of soil-borne diseases. Causal organisms B. anthracis, C. perfringens, C. sordellii, C. haemolyticum, C. chauvoei/septicum, C. novyi A, C. botulinum and site-specific field strains of clostridia were detected. Experimental site-specific vaccines proved to be highly efficient in protecting cattle and guinea pigs.

The effectiveness, both immunological (by an increase in the titers of antitoxins) and protective (by resistance to the inoculation of the absolute lethal dose of infective agents), of the regional (wound) revaccination with tetratoxoid (Clostridium perfringens, C. oedematiens, C. septicum, C. histolyticum) was demonstrated on the experimental model of wound infection (gas gangrene) of guinea pigs. The schedule of rapid immunization with tetratoxoid was developed, which made it possible to create good immunological preparedness (basic immunity) for subsequent revaccination in case of traumas within 6 days. The effectiveness of rapid immunization by the application of tetratoxoid on the wound was shown. This immunization ensured a considered increase in the titers of antitoxins within the first 6 days, which increased the protection of the animals from infection with each of the four causative agents of gas gangrene.

Clostridium septicum bacteremia typically portends a fulminant disease associated with high mortality. We describe the clinical courses of seven survivors of C. septicum infection and their antibody responses to the alpha toxin produced by C. septicum. Three patients had clinical syndromes ranging from uncomplicated bacteremia to early typhlitis, and three patients had syndromes ranging from abscess to myonecrosis and septic shock. In addition, an AIDS patient who developed septic shock and who had extensive gas in the retroperitoneal musculature did not undergo surgery but survived after receiving antimicrobial therapy and intensive supportive care. Both immunocompetent patients with myonecrosis had detectable IgG to alpha toxin by immunoblot analysis. IgG to alpha toxin was not detected in the four immunocompetent patients who had C. septicum bacteremia but who did not have myonecrosis or in the AIDS patient with myonecrosis. Therefore, humoral responses to alpha toxin during C. septicum infection may be related to the host's clinical syndrome and immune status.

A 20 year old leukaemic patient with neutropaenia secondary to chemotherapy, who developed overwhelming sepsis, myonecrosis, vascular occlusion and necrotizing enterocolitis due to Clostridium septicum infection is described. Plain abdominal radiographs and a computed tomography scan of the abdomen and pelvis showed gas in the retroperitoneal soft tissues. Clostridium septicum septicaemia has a recognized association with malignancy and neutropaenia and has a high mortality if not diagnosed and treated early. Computed tomography scanning of the abdomen, pelvis and head is advised in any patient with a positive C. septicum blood culture.

Possible prolongation of the biological effect of some available immunostimulators such as prodigiozan, salmozan, polyribonate and thymalin by their sorption on aluminium hydrate was studied. It was shown that in comparison to the native immunostimulators the sorbed ones had a more pronounced biological action and provided a more prolonged increase in the host resistance to the causative agents of gas gangrene and typhoid fever. Using prodigiozan as an example it was demonstrated that the observed increase in the anti-infective activity of the sorbed drugs was associated with more intensive stimulation of some immunological factors involved in regulation of host nonspecific resistance. The results of the study are likely to indicate that it was experiment to further investigate the drugs to reveal their efficacy in other infection models and to optimize the schemes of their use.

A method for isolation of the ribosomal fraction (RF) from the cytoplasm of type-A C. perfringens strain BP6K was developed and its chemical and antigenic properties characterized. RF has been found to possess protective properties: two subcutaneous immunizations of mice with RF preparations adsorbed on Al(OH)3 in doses of 250 and 500 micrograms (dry weight) has ensured, on the average, the protection of 41.9% of the immunized animals from 1 DCL of type-A C. perfringens strain BP6K culture.

The data on the study of the protective activity of theta hemolysin and Cl. perfringens lecithinase preparations and the corresponding antitoxic sera obtained by indirect immune affinity chromatography are presented. Experiments in mice and guinea pigs indicate that the injection of antihemolytic serum and immunization with anatheta hemolysin ensures the protection of the animals from theta toxin. The enrichment of analecithinase preparation with anatheta hemolysin has been found to increase its protective properties against Cl. perfringens culture and toxin.

Nineteen patients have been treated with gas gangrene infection proved on cultures to be caused by Clostridium septicum. Infection occurred after trauma in three patients and occurred spontaneously in 16, including six patients with peritonitis. None of the instances occurred as a postoperative infection. Ten of the patients had an associated malignant disease including carcinoma of the colon and rectum in six, hematologic malignant conditions in three and carcinoma of the gallbladder in one. Of the remaining six patients, four had evidence of immune suppression (two had leukopenia, one was preleukemic and one had undergone a postcadaver renal transplant), one had diabetes and one had overwhelming sepsis due to a perforated gallbladder with peritonitis. Only three of the 19 patients were otherwise healthy, and infection occurred after trauma in all of these patients. Four patients were moribund upon admission and died before initial therapy was completed. In the other 15 patients, treatment consisted of administration of penicillins and broad spectrum antibiotics and débridement, as well as hyperbaric oxygen in 13 patients. Thirteen of 19 patients died and eight of ten with malignant disease died. The data show a striking relationship between Clostridium septicum and both malignant disease and immune suppression.

Experiments on 575 noninbred white mice have revealed that the nonspecific resistance of the animals to type A C. perfringens toxin can be enhanced by the administration of Prodigiosan, a commercial immunostimulating agent. Prodigiosan, introduced in 3-4 injections (the last one made 24 hours before intoxication) has been found to enhance the resistance of the animals to the subcutaneous injection of type A C. perfringens toxin by 40-60% and to its intraperitoneal injection by 60-97%.

Polymeric alpha-toxoid with a molecular weight of 450 000--600 000 was obtained by condensation of alpha-toxoid of Cl. perfringens, type A, with glutaric aldehyde. Experiments on guinea pigs showed that in the adsorbed preparations the immunogenic properties of both monomeric and polymeric alpha-toxoids are practically identical. The primary immune response after the immunization with nonadsorbed antigens was 3 times greater than that induced by the monomer. Polymerization of alpha-toxoid failed to change its thymus dependency.

Comparative experiments were conducted on rabbits and guinea pigs; a study was made of the immunological efficacy of the toxoids with a different content of the immunizing units (IU/ml). There was revealed a regular quantitative interrelationship between the content of the IU/ml in the toxoids, the level of the antitoxin formation and the extent of protection against the experimental gas gangrene in the immunized animals.