Absent in Melanoma 2 Mediates Inflammasome Signaling Activation against Clostridium perfringens Infection.

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.

Deficient Skeletal Muscle Regeneration after Injury Induced by a Clostridium perfringens Strain Associated with Gas Gangrene.

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 ß1 (TGF-ß1) 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.

Clostridium perfringens α-toxin impairs granulocyte colony-stimulating factor receptor-mediated granulocyte production while triggering septic shock.

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.

Concurrent Host-Pathogen Transcriptional Responses in a Clostridium perfringens Murine Myonecrosis Infection.

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.IMPORTANCEClostridium 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.

A Recombinant Probiotic, Lactobacillus casei, Expressing the Clostridium perfringens α-toxoid, as an Orally Vaccine Candidate Against Gas Gangrene and Necrotic Enteritis.

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.

Combined therapy with gas gangrene antitoxin and recombinant human soluble thrombomodulin for Clostridium perfringens sepsis in a rat model.

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.

Clinical Serum Therapy: Benefits, Cautions, and Potential Applications.

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.

Putative function of hypothetical proteins expressed by Clostridium perfringens type A strains and their protective efficacy in mouse model.

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_2500, CPF_1441, CPF_0876, CPF_0093, CPF_2002, CPF_2314, CPF_1179, CPF_1132, CPF_2853, CPF_0552, CPF_2032, CPF_0438, CPF_1440, CPF_2918, CPF_0656, and CPF_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_0876. HP CPF_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_2918 was cloned, recombinant protein was purified to near homogeneity, and probing with mouse anti-CPF_2918 serum revealed surface localization of the protein in C. perfringens ATCC13124 cultures. The purified recombinant CPF_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.

Oral immunization of mice against Clostridium perfringens epsilon toxin with a Lactobacillus casei vector vaccine expressing epsilon toxoid.

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.

Lipoproteins from Clostridium perfringens and their protective efficacy in mouse model.

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.

Vaccines against Clostridium perfringens alpha-toxin.

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.

A case of gas gangrene in an immunosuppressed Crohn's patient.

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.

Fatal myelotoxicity after azathioprine treatment.

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.

The role of neutrophils and monocytic cells in controlling the initiation of Clostridium perfringens gas gangrene.

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.

[Gas gangrene with ulcerative colitis under immunosuppressive therapy: report of a case]. / Gasbrandinfektion unter Immunsuppression bei Colitis ulcerosa: Ein Fallbericht.

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.

Stress proteins of Clostridium perfringens type A immunoreact with antiserum from rabbits infected with gas gangrene.

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.

Clostridial gas gangrene: evidence that alpha and theta toxins differentially modulate the immune response and induce acute tissue necrosis.

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.

Environment, incidence, aetiology, epizootiology and immunoprophylaxis of soil-borne diseases in north-east Mexico.

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 experimental validation of methods for the emergency immunoprophylaxis of gas gangrene]. / Eksperimental'noe obosnovanie metodov ékstrennoi immunoprofilaktiki gazovoi gangreny.

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.