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.

Phagocyte extracellular traps formation contributes to host defense against Clostridium perfringens infection.

Clostridium perfringens (C. perfringens) is an important Gram-positive anaerobic spore-forming pathogen that provokes life-threatening gas gangrene and acute enterotoxaemia, although it colonizes as a component of the symbiotic bacteria in humans and animals. However, the mechanisms by which C. perfringens is cleared from the host remains poorly understood, thereby impeding the development of novel strategies for control this infection. Here, we uncover a beneficial effect of extracellular traps (ETs) formation on bacterial killing and clearance by phagocytes. C. perfringens strain ATCC13124, and wild-type isolates CP1 and CP3 markedly trigger ETs formation in macrophages and neutrophils. As expected, visualization of DNA decorated with histone, myeloperoxidase (MPO) and neutrophils elastase (NE) in C. perfringens-triggered classical ETs structures. Notably, the bacteria-induced ETs formation is an ERK1/2-, P38 MAPK-, store-operated calcium entry (SOCE)-, NADPH oxidase-, histone-, NE-, and MPO-dependent process, and is independent of LDH activity. Meanwhile, the defect of bactericidal activity is mediated by impairing ETs formation in phagocytes. Moreover, In vivo studies indicated that degradation of ETs by DNase I administration leads to a defect in the protection against experimental gas gangrene, with higher mortality rates, exacerbated tissue damage, and more bacterial colonization. Together, these results suggest that phagocyte ETs formation is essential for the host defense against C. perfringens infection.

Autolysin as a fibronectin receptor on the cell surface of Clostridium perfringens.

OBJECTIVE: Clostridium perfringens causes food poisoning and gas gangrene, a serious wound-associated infection. C. perfringens cells adhere to collagen via fibronectin (Fn). We investigated whether the peptidoglycan hydrolase of C. perfringens, i.e., autolysin (Acp), is implicated in Fn binding to C. perfringens cells. METHODS: This study used recombinant Acp fragments, human Fn and knockout mutants (C. perfringens 13 acp::erm and HN13 ΔfbpC ΔfbpD). Ligand blotting, Western blotting analysis, and complementation tests were performed. The Fn-binding activity of each mutant was evaluated by ELISA. RESULTS: From an Fn-binding assay using recombinant Acp fragments, Fn was found to bind to the catalytic domain of Acp. In mutant cells lacking Acp, Fn binding was significantly decreased, but was restored by the complementation of the acp gene. There are three known kinds of Fn-binding proteins in C. perfringens: FbpC, FbpD, and glyceraldehyde-3-phosphate dehydrogenase. We found no difference in Fn-binding activity between the mutant cells lacking both FbpC and FbpD (SAK3 cells) and the wild-type cells, indicating that these Fn-binding proteins are not involved in Fn binding to C. perfringens cells. CONCLUSIONS: We found that the Acp is an Fn-binding protein that acts as an Fn receptor on the surface of C. perfringens cells.

Gas-Producing Infections in the Foot at a Large Academic Medical Center: A 10-Year Retrospective Review.

Gas-producing infections, such as clostridial and nonclostridial gas gangrene, crepitant cellulitis, and necrotizing fasciitis, are characterized in the literature by a variety of initial presentations, microbial burdens and surgical outcomes-ranging from debridement to amputation to death. The primary aim of this study was to identify the organisms cultured in gas-producing infections of the foot in patients that presented to a large academic medical center over a 10-year period. Our secondary aims were to report the prevalence of sepsis in this population upon presentation, and patient outcomes upon discharge. After a retrospective chart review of 207,534 procedures, 70 surgical cases met inclusion criteria. The most common organisms that grew in operating room cultures were Staphylococcus aureus, Group B Beta Streptococcus, and Enterococcus species. Just over half of the population presented with sepsis. After an average of 2 or more operations, 64% of patients underwent amputation. One death occurred. Gas-producing infections, or "gas gangrene," are primarily polymicrobial infections, rarely due to Clostridium perfringens, that warrant surgical exploration for optimal outcomes.

Infectious shock after liposuction.

BACKGROUND: Liposuction has become one of the most popular cosmetic surgeries in China. However, few studies have discussed infectious shock caused by C. perfringens as one of the causes of death after liposuction. CASE PRESENTATION: A 24-year-old woman was brought to the emergency department (ED) of Guangzhou Chinese Overseas Hospital for treatment. The patient had undergone liposuction in her bilateral lower limbs two days prior. At the ED, the patient was unconscious, and had bilateral equal-sized (diameter, 6 mm) round pupils, no light reflex, a blood pressure (BP) of 71/33 mmHg, a heart rate of 133 bpm, and an SpO2 of 70%. She had bilateral limb swelling, extensive ecchymoses in her lower abdomen and bilateral thighs, local crepitus, blisters, weak pulses on her femoral artery and dorsalis pedis, high skin tension, and hemoglobin of 32 g/L. The patient was diagnosed with Clostridium perfringens infection, and she underwent debridement surgery and supportive treatment. But the patient's BP could not improve. At 8:28 pm on the day of admission, the patient was declared clinically dead after the electrocardiograph showed a horizontal line and spontaneous respiration ceased. CONCLUSIONS: Failure to meet surgical disinfection and environmental standards may be the cause of infection of C. perfringens through wounds. Therefore, it is necessary to strengthen the environmental disinfection of the operating room, and standardize the sterile conditions of the operation staff and patients before and during operation. Liposuction surgery necrotizing fasciitis is a rare but fatal complications, especially if diagnosis delay, therefore it is critical for early diagnosis and treatment of gas gangrene.

Piceatannol Alleviates Clostridium perfringens Virulence by Inhibiting Perfringolysin O.

Clostridium perfringens (C. perfringens) is an important foodborne pathogen that can cause diseases such as gas gangrene and necrotizing enteritis in a variety of economic animals, seriously affecting public health and the economic benefits and healthy development of the livestock and poultry breeding industry. Perfringolysin O (PFO) is an important virulence factor of C. perfringens and plays critical roles in necrotic enteritis and gas gangrene, rendering it an ideal target for developing new drugs against infections caused by this pathogen. In this study, based on biological activity inhibition assays, oligomerization tests and computational biology assays, we found that the foodborne natural component piceatannol reduced pore-forming activity with an inhibitory ratio of 83.84% in the concentration of 16 µg/mL (IC50 = 7.83 µg/mL) by binding with PFO directly and changing some of its secondary structures, including 3-Helix, A-helix, bend, and in turn, ultimately affecting oligomer formation. Furthermore, we confirmed that piceatannol protected human intestinal epithelial cells from the damage induced by PFO with LDH release reduced by 38.44% at 16 µg/mL, based on a cytotoxicity test. By performing an animal experiment, we found the C. perfringens clones showed an approximate 10-fold reduction in infected mice. These results suggest that piceatannol may be a candidate for anti-C. perfringens drug development.

A rare cause of pneumoperitoneum due to clostridium perfringens in a cirrhotic patient.

INTRODUCTION: Abdominal gas gangrene caused by Clostridium perfringens is a rare differential diagnosis to pneumoperitoneum caused by bowel perforation. There are only a handful of case reports on this topic. PATIENTS AND METHODS: We present the case of a 58 year old cirrhotic patient who represented to our ER after complicated surgery for retroperitoneal liposarcoma. On admission he complained of abdominal pain and mild fever. Due to leukocytosis and CRP a CT scan was performed which showed extensive free air. The patient was taken to the OR for suspected bowel perforation. No perforation could be identified after extensive search and lavage. RESULTS: Twelve hours after surgery microbiology reported extensive growth of clostridium perfringens in the cultures drawn from ascites. The patient was successfully treated with antibiotics and discharged home soon after. CONCLUSION: Gas gangrene is a rare differential diagnosis to bowel perforation. Most reported cases are from cirrhotic patients. If no perforation can be identified in the OR postoperative antibiotics should cover clostridium perfringens.

[Soft tissue necrotizing infection]. / L'image du mois. Infection nécrosante des tissus mous.

Necrotizing fasciitis and gas gangrene represent a rapidly progressive and fatal tissue infection in the absence of early multidisciplinary treatment. There are multiple risk factors, but diabetes remains the main one. The presence of crackles or extensive lesions are an indication for exploration and surgical treatment. Conservative management is associated with zero survival. Iconography, biology or bacteriology can help in the diagnosis, but the latter is only made during surgery. The associated mortality is high, despite appropriate management. We report here the case of a diabetic patient requiring insulin, having presented this pathology, the starting point of which is a traumatic wound, with an extensive acute course, contra-indicating any surgical procedure.

La fasciite nécrosante et la gangrène gazeuse représentent une infection tissulaire rapidement progressive et létale en l'absence d'un traitement multidisciplinaire précoce. Il existe de multiples facteurs de risques, mais le diabète en reste le principal. La présence de crépitants ou de lésions extensives sont une indication à l'exploration et au traitement chirurgical. Une prise en charge conservatrice est associée à une survie nulle. L'iconographie, la biologie ou la bactériologie permettent d'aider au diagnostic, mais ce dernier est uniquement posé en per-opératoire. La mortalité associée est élevée, malgré une prise en charge adaptée. Nous rapportons ici le cas d'un patient diabétique de type 2 insulino-requérant, ayant présenté cette pathologie dont le point de départ est une plaie traumatique, avec une évolution aiguë extensive, contre-indiquant tout geste chirurgical.

Analyzing Outcomes Among Older Adults With Necrotizing Soft-Tissue Infections in the United States.

BACKGROUND: Necrotizing soft-tissue infections (NSTIs) encompass a group of severe, life-threatening diseases with high morbidity and mortality. Evidence suggests advanced age is associated with worse outcomes. To date, no large data sets exist describing outcomes in older individuals, and risk factor identification is lacking. METHODS: Retrospective data were obtained from the 2015 Medicare 100% sample. Included in the analysis were those aged ≥65 y with a primary diagnosis of an NSTI (gas gangrene, necrotizing fasciitis, cutaneous gangrene, or Fournier's gangrene). Risk factors for in-hospital mortality and discharge disposition were examined. Continuous variables were assessed using central tendency, t-tests, and Wilcoxon rank-sum tests. Categorical variables were assessed using the chi-squared and Fisher's exact tests. Statistical significance was defined as P < 0.05. RESULTS: 1427 patient records were reviewed. 59% of patients were male, and the overall mean age was 75.4±8.6 y. 1385 (97.0%) patients required emergency surgery for their NSTI diagnosis. The overall mortality was 5.3%. Several underlying comorbidities were associated with higher rates of mortality including cancer (OR: 3.50, P = 0.0009), liver disease (OR: 2.97, P = 0.03), and kidney disease (OR: 2.15, P = 0.01). While associated with high in-hospital mortality, these diagnoses were not associated with a difference in the rate of discharge to home compared with skilled nursing or rehab. Overall, patients discharged to skilled nursing facilities or rehab had higher rates of underlying comorbidities than patients who were discharged home (3 or more comorbid illness 84.3% versus 68.6%, P < 0.0001); however, no individual comorbid illness was associated with discharge location. CONCLUSIONS: In our Medicare data set, we identified several medical comorbidities that are associated with increased rates of in-hospital mortality. Patients with underlying cancers had the highest odds of increased mortality. The effect on outcomes of the potentially immunosuppressive cancer treatments in these patients is unknown. These data suggest that patients with underlying illnesses, especially cancer, kidney disease, or liver disease have higher mortalities and are more likely to be discharged to skilled nursing facilities or rehab. It is unclear why these illnesses were associated with these worse outcomes while others including diabetes and heart disease were not. These data suggest that these particular comorbid illnesses may have special prognostic implications, although further analysis is necessary to identify the causative factors.

Gas Where It Shouldn't Be! Imaging Spectrum of Emphysematous Infections in the Abdomen and Pelvis.

OBJECTIVE. The purpose of this article is to review the spectrum, etiopathogenesis, clinical presentation, imaging features, differential diagnoses, and management of emphysematous infections of the abdomen and pelvis. CONCLUSION. Emphysematous infections are associated with high morbidity and mortality and thus need urgent medical and surgical interventions. CT is the most sensitive modality to detect gas; CT provides definitive diagnosis in most cases and can depict the extent of involvement.

Clostridium septicum-induced gangrene in the right lower extremity complicating pneumatosis in the right ventricle and the pulmonary artery and occlusion of right femoral artery: a case report.

BACKGROUND: Gas gangrene is usually manifested as myonecrosis and subcutaneous gas accumulation, but rarely manifested as arterial occlusion or pneumatosis in the right ventricle and the pulmonary artery. CASE PRESENTATION: We report a case of gas gangrene caused by Clostridium septicum. The patient developed gas gangrene after being pecked by a chicken but turned for the better following antibiotic treatment and debriment. Imaging test revealed a rare occlusion of the right femoral artery and pneumatosis in the right ventricle and the main pulmonary artery. CONCLUSIONS: In the presence of gas gangrene, special care must be taken to prevent against the formation of circulatory air embolism. The gas gangrene-induced gangrene in the limb of this patient might be attributed to the combined action of infection and arterial occlusion. MDT (Multidisciplinary team)-Green Channel mode is conductive to treatment success of gas gangrene.

Essential oil combinations against Clostridium perfringens and Clostridium septicum - the causative agents of gas gangrene.

AIMS: The inhibitory and bactericidal effect of a wide range of essential oils, and their selected combinations against two pathogens (Clostridium perfringens and Clostridium septicum), the causative pathogens of gas gangrenous infections were investigated. Fractional inhibitory indices were also calculated to determine the interactions. METHODS AND RESULTS: The Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC) assays were used to determine the efficacy of the essential oils. Santalum austrocaledonicum demonstrated the highest activity inhibiting both Clostridial pathogens at the lowest concentration of 0·02 mg ml-1 . Santalum austrocaledonicum combined with Cymbopogon martinii had the strongest inhibition against C. perfringens (MIC 0·02 mg ml-1 ) and C. septicum (MIC 0·01 mg ml-1 ). Selected combinations demonstrated synergy (ΣFIC ≤ 0·50) in combination against both pathogens tested. Antagonism was also observed in many combinations. CONCLUSIONS: Selected essential oils, when studied either individually or in combination, have high inhibitory and bactericidal effects against both Clostridial strains. Nine combinations have proven to be synergistic with 23 combinations additive; 96 indifferent and 77 having an antagonistic effect against the pathogenic strains. Some combinations demonstrated extreme antagonism and as such, careful consideration needs to be given to essential oil selection against these pathogens. SIGNIFICANT IMPACT OF THE STUDY: Very few essential oils have been antimicrobially screened (MIC and MBC) against Clostridial strains and furthermore, the efficacies in combination are not known.

Fatal Clostridium septicum myonecrosis from gastric perforation: A case report.

Clostridial myonecrosis is a medical and surgical emergency which requires early and aggressive intervention to reduce mortality. We report a rare case of Clostridium septicum myonecrosis that disseminated hematogenously from a gastric perforation. The patient was afebrile and hemodynamically stable upon admission. He rapidly developed spontaneous clostridial myonecrosis and succumbed to septic shock 36 hours after presentation. In our extensive literature review this is the only case with blood cultures confirming Clostridium septicum bacteremia with a surgically confirmed gastric perforation source in the setting of spontaneous clostridial myonecrosis.

Gas Gangrene Following Posterior Tibial Tendon Transfer of a 34-Year-Old Patient - a Case Report.

Gas gangrene is a rare and potentially fatal surgical complication, most often associated with contaminated traumatic wounds or more rarely following cases of abdominal surgery. The following article describes a case of gas gangrene in the right lower limb of a 34-year-old patient following an elective posterior tibial tendon transfer. The surgery was performed in bloodless fashion, utilizing a surgical tourniquet. Fifteen months prior to the procedure, the patient was attacked by a wild boar, resulting in a tear of the upper calf with complete transection of the right common fibular nerve. The patient underwent a total of three acute surgical procedures, of which the third resulted in below knee amputation. The patient then underwent a final corrective procedure and was fitted with a suitable leg prosthesis. In the discussion, three possible pathophysiological mechanisms of gas gangrene development are described - iatrogenic inoculation of bacteria during injection of local anesthetics for conduction anesthesia, iatrogenic inoculation of bacteria during the posterior tibial tendon transfer and activation of latent clostridial spores within the original wound caused by the wild boar. We consider the third mechanism most likely, as boar tusks contaminated with soil and debris are a more likely source of clostridial spores, than sterile surgical instruments. Furthermore, it is likely the surgical tourniquet played a key role in activating latent spores within the patient wound, as changes in tissue oxygen levels are a common cause of spore activation. Thus, we suggest caution in utilizing bloodless operating fields in elective cases with a history of open contaminated wounds, as the iatrogenic hypoxia can potentially activate sporulent bacteria within the patients wound.

The EngCP endo α-N-acetylgalactosaminidase is a virulence factor involved in Clostridium perfringens gas gangrene infections.

Clostridium perfringens is the causative agent of human clostridial myonecrosis; the major toxins involved in this disease are α-toxin and perfringolysin O. The RevSR two-component regulatory system has been shown to be involved in regulating virulence in a mouse myonecrosis model. Previous microarray and RNAseq analysis of a revR mutant implied that factors other than the major toxins may play a role in virulence. The RNAseq data showed that the expression of the gene encoding the EngCP endo α-N-acetylgalactosaminidase (CPE0693) was significantly down-regulated in a revR mutant. Enzymes from this family have been identified in several Gram-positive pathogens and have been postulated to contribute to their virulence. In this study, we constructed an engCP mutant of C. perfringens and showed that it was significantly less virulent than its wild-type parent strain. Virulence was restored by complementation in trans with the wild-type engCP gene. We also demonstrated that purified EngCP was able to hydrolyse α-dystroglycan derived from C2C12 mouse myotubes. However, EngCP had little effect on membrane permeability in mice, suggesting that EngCP may play a role other than the disruption of the structural integrity of myofibres. Glycan array analysis indicated that EngCP could recognise structures containing the monosaccharide N-acetlygalactosamine at 4C, but could recognise structures terminating in galactose, glucose and N-acetylglucosamine under conditions where EngCP was enzymatically active. In conclusion, we have obtained evidence that EngCP is required for virulence in C. perfringens and, although classical exotoxins are important for disease, we have now shown that an O-glycosidase also plays an important role in the disease process.

Clostridium septicum-infected aortic aneurysm or graft is a deadly diagnosis.

BACKGROUND: Clostridium septicum is an anaerobic, motile, spore-forming, toxin-producing gram-positive bacillus that can lead to rapidly progressive gas gangrene due to the release of alpha toxin. Aortic aneurysm secondary to C. septicum infection is a rare condition with 60 cases reported in the literature; however, we have recently treated several patients with the condition in our large tertiary care and aortic center. METHODS: Blood and tissue culture results collected between January 2005 and January 2018 and maintained in the microbiology laboratory database at the Cleveland Clinic were reviewed to identify those with C. septicum reported. Each was reviewed to determine radiographic or histopathologic correlation with aortic disease. RESULTS: Seven cases of C. septicum aortitis were reviewed. Underlying malignant disease was found in four cases and a history of remote malignant disease in one case. The most common location for infection was the infrarenal abdominal aorta. Vascular surgery had previously been performed in three of the cases. Five of the seven patients underwent operative repair. All patients were treated with ß-lactam antibiotics. The two patients who did not undergo an operation died, which is consistent with the 100% mortality described in the literature. Of the five patients who underwent an operation, there was only one documented survivor and one was lost to follow-up. CONCLUSIONS: In the largest reported case series, only a small percentage of patients with C. septicum-infected aortic aneurysms survived >1 year. In the patients described, those who did not receive an operation had 100% mortality. Earlier recognition and prompt operation with appropriate antimicrobial therapy are needed to improve the outcome of patients diagnosed with this rare infection.

Health-Related Quality of Life Is Decreased After Necrotizing Soft-Tissue Infections.

BACKGROUND: A necrotizing soft-tissue infection (NSTI) is a rare but severe infection with a high mortality rate of 12%-20%. Diagnosing is challenging and often delayed. Treatment consists of surgical debridement of all necrotic tissue and administration of antibiotics. Despite adequate treatment, survivors are often left with extensive wounds, resulting in mutilating scars and functional deficits. Both the disease and the subsequent scars can negatively influence the health-related quality of life (HRQoL). The present study was performed to contribute to the knowledge about HRQoL in patients after NSTI. METHODS: We retrospectively identified patients treated for NSTI in a tertiary center in the Netherlands. Patient and treatment characteristics were collected and patients were asked to fill in a Short Form 36 questionnaire. RESULTS: Forty-six patients with a diagnosis of NSTI were identified. Twenty-eight (61%) were male and mean age was 57 y. Thirty-nine patients (80%) survived. Thirty-one (84%) of the survivors returned the questionnaire after a median follow-up of 4.1 y (interquartile range [IQR], 2.4-5.9 y). Statistically significantly decreased scores when compared to the Dutch reference values were observed for the Short Form 36 domains, physical functioning, role-physical functioning, general health, and the combined Physical Component Score. No differences were observed for the other five domains or for the Mental Component Score. CONCLUSIONS: This study confirms that NSTI negatively affects HRQoL as reported by the patient, especially on the physical domains. To learn more about HRQoL in patients after NSTI, studies in larger groups with a more disease-specific questionnaire should be performed. LEVEL OF EVIDENCE: Level 3, prognostic and epidemiological.

A case report of iatrogenic gas gangrene post colonoscopy successfully treated with conservative management- is surgery always necessary?

BACKGROUND: Colonoscopy is a routine procedure in diagnosis and treatment of colonic disease. While generally regarded as a safe procedure, potentially fatal complications can occur. Gas gangrene is one such complication, with very high mortality. There are few cases of gas gangrene occurring after colonoscopy, making it one of the rarer complications of this procedure. There have been no previously reported cases of a patient surviving such an infection and the optimal treatment strategy is contentious. This report describes a case of intramural gas gangrene of the colon, treated conservatively with antibiotic therapy in which the patient survived with full recovery. CASE PRESENTATION: A 71-year-old, previously healthy male presented 6 h post apparently uncomplicated colonoscopic polypectomy with rigors, nausea, vomiting and right upper quadrant pain. At presentation he was febrile at 40.1 °C but hemodynamically stable. Abdominal computed tomography revealed substantial colonic thickening and several focal intramural gas bubbles (pneumatosis intestinalis) surrounding the polypectomy site. Within 24 h post procedure he became hypotensive and was admitted to ICU in frank septic shock requiring inotropes, and with demonstrable septic myocardial depression. Bloods showed multi-organ derangement with leukocytosis, lactic acidosis, haemolytic anaemia and hyperbilirubinemia. A diagnosis of presumed Clostridial gas gangrene was made, and treatment was initiated with benzylpenicillin, clindamycin, metronidazole and vancomycin. After 4 days in ICU he was stepped down, and discharged after a further 10 days with no surgical or endoscopic interventions. At three-month review he reported being back to full health. CONCLUSIONS: This case demonstrates that gas gangrene infection is a possible complication of colonoscopic polypectomy. This is a cause of rapid deterioration in post-colonoscopy patients and has been misdiagnosed as colonic perforation in previously reported cases of retroperitoneal gas gangrene. Such misdiagnosis delays antibiotic therapy, which likely plays a role in the high mortality of this condition. Early diagnosis and initiation of antibiotic therapy with benzylpenicillin and clindamycin as seen in this case is essential for patient survival. While surgery is typically performed, non-operative management of pneumatosis intestinalis, and potentially gas gangrene is becoming more common and was utilized effectively in this patient.

Clostridium perfringens α-toxin specifically induces endothelial cell death by promoting ceramide-mediated apoptosis.

Clostridium perfringens type A-induced gas gangrene is characterized by severe myonecrosis, and α-toxin has been revealed to be a major virulence factor involved in the pathogenesis. However, the detailed mechanism is unclear. Here, we show that CD31+ endothelial cell counts decrease in muscles infected with C. perfringens in an α-toxin-dependent manner. In vitro experiments revealed that α-toxin preferentially and rapidly induces the death of human umbilical vein endothelial cells (HUVECs) compared with C2C12 murine muscle cells. The toxin induces apoptosis of HUVECs by increasing ceramide. Furthermore, the specificity might be dependent on differences in the sensitivity to ceramide between these cell lines. Together, our results suggest that α-toxin-induced endothelial cell death promotes severe myonecrosis and is involved in the pathogenesis of 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.