Clostridium perfringens phospholipase C, an archetypal bacterial virulence factor, induces the formation of extracellular traps by human neutrophils.

Neutrophil extracellular traps (NETs) are networks of DNA and various microbicidal proteins released to kill invading microorganisms and prevent their dissemination. However, a NETs excess is detrimental to the host and involved in the pathogenesis of various inflammatory and immunothrombotic diseases. Clostridium perfringens is a widely distributed pathogen associated with several animal and human diseases, that produces many exotoxins, including the phospholipase C (CpPLC), the main virulence factor in gas gangrene. During this disease, CpPLC generates the formation of neutrophil/platelet aggregates within the vasculature, favoring an anaerobic environment for C. perfringens growth. This work demonstrates that CpPLC induces NETosis in human neutrophils. Antibodies against CpPLC completely abrogate the NETosis-inducing activity of recombinant CpPLC and C. perfringens secretome. CpPLC induces suicidal NETosis through a mechanism that requires calcium release from inositol trisphosphate receptor (IP3) sensitive stores, activation of protein kinase C (PKC), and the mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK/ERK) pathways, as well as the production of reactive oxygen species (ROS) by the metabolism of arachidonic acid. Proteomic analysis of the C. perfringens secretome identified 40 proteins, including a DNAse and two 5´-nucleotidases homologous to virulence factors that could be relevant in evading NETs. We suggested that in gas gangrene this pathogen benefits from having access to the metabolic resources of the tissue injured by a dysregulated intravascular NETosis and then escapes and spreads to deeper tissues. Understanding the role of NETs in gas gangrene could help develop novel therapeutic strategies to reduce mortality, improve muscle regeneration, and prevent deleterious patient outcomes.

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.

Ethanolamine utilization supports Clostridium perfringens growth in infected tissues.

Clostridium perfringens possesses the ethanolamine (EA) utilization (eut) system encoded within the eut operon, which utilizes the EA as a carbon, nitrogen and energy source. To determine the role of the eut system in C. perfringens growth, an in-frame deletion of the eutABC genes was made in strain HN13 to generate the eutABC-deleted mutant strain HY1701. Comparison of HN13 and HY1701 growth in media supplemented with 1.0% glucose and/or 1.0% EA showed that glucose enhanced the growth of both strains, whereas EA enhanced HN13 growth, but not that of HY1701, indicating that the eut system is necessary for C. perfringens to utilize EA. The two-component regulatory system EutVW is needed to induce eut gene expression in response to EA whereas the global virulence regulator VirRS differentially controlled eut gene expression depending on glucose and EA availability. To assess the role of the eut system in vivo, an equal number of HN13 and HY1701 cells were injected into the right thigh muscles of mice. Mice infected with HY1701 showed fewer symptoms than those injected with HN13. The mortality rate of mice infected with HY1701 tended to be lower than for mice infected with HN13. In addition, in infected tissues from mice injected with a mixture of HN13 and HY1701, HN13 outnumbered HY1701. PCR screening demonstrated that C. perfringens isolated from gas gangrene and sporadic diarrhea cases carried both eut genes and the perfringolysin O gene (pfoA) as well as the phospholipase C gene (plc). However, pfoA was not detected in isolates from food poisoning patients and healthy volunteers. Culture supernatants prepared from HN13 grown in media containing 7.5% sheep red blood cells induced significantly higher eutB expression levels compared to those from plc- and/or pfoA-deletion mutants. Together, these results indicate that the eut system plays a nutritional role for C. perfringens during histolytic infection.

Depletion of neurohypophyseal content of vasopressin in septic shock.

OBJECTIVES: To assess the mechanisms underlying the inappropriately low plasma vasopressin levels reported in septic shock. DESIGN: Prospective case series. SETTING: A 26-bed general medical intensive care unit at a university hospital. PATIENTS: Septic shock patients. MEASUREMENTS AND MAIN RESULTS: In three consecutive patients with septic shock, plasma vasopressin levels, circulating vasopressinase activity, baroreflex sensitivity, and neurohypophyseal vasopressin content were assessed. Plasma vasopressin concentration was unexpectedly within normal range in two patients (1.6 pg/mL and 1.8 pg/mL) and increased in one (16 pg/mL). In all cases, vasopressinase activity was undetectable, baroreflex sensitivity was decreased, and the high signal intensity of the posterior lobe of the pituitary gland on T1-weighted magnetic resonance images was absent. Magnetic resonance imaging and plasma vasopressin levels normalized after recovery from shock in the patient who survived. CONCLUSION: These data suggest that in septic shock, inappropriately low plasma levels of vasopressin are at least partly related to a depletion of vasopressin stores in the neurohypophysis.

Clostridial gas gangrene. II. Phospholipase C-induced activation of platelet gpIIbIIIa mediates vascular occlusion and myonecrosis in Clostridium perfringens gas gangrene.

Clostridium perfringens gas gangrene is a fulminant infection, and radical amputation remains the single best treatment. It has been hypothesized that rapid tissue destruction is related to tissue hypoxia secondary to toxin-induced vascular obstruction, and previous studies demonstrated that phospholipase C (PLC) caused a rapid and irreversible decrease in skeletal muscle blood flow that paralleled the formation of intravascular aggregates of activated platelets, fibrin, and leukocytes. In this study, flow cytometry demonstrated that PLC stimulated platelet/neutrophil aggregation in a gpIIbIIIa-dependent fashion. Pretreatment of animals with heparin or depletion of leukocytes reduced blood-flow deficits, and aggregate formation caused by PLC. It is concluded that fulminant tissue destruction in gas gangrene results from profound attenuation of blood flow caused by PLC-induced, gpIIbIIIa-mediated formation of heterotypic platelet/polymorphonuclear leukocyte aggregates. Therapeutic strategies that target gpIIbIIIa may prevent vascular occlusion, maintain tissue viability, and provide an alternative to radical amputation for patients with this infection.

Hyperbaric oxygen therapy in acute necrotizing infections with a special reference to the effects on tissue gas tensions.

Clostridial gas gangrene and perineal necrotizing fasciitis or Fournier's gangrene are rare but serious infections with an acute onset, rapid progression, systemic toxemia and a high mortality rate. The aim of this study was to investigate the efficacy of surgery, antibiotic treatment, surgical intensive care and in particular the role of hyperbaric oxygen (HBO) in the management of these infections. An experimental rat model was used to investigate the possibilities for measuring tissue oxygen and carbon dioxide tensions during hyperbaric oxygen treatment. In addition to this preliminary experimental study, Silastic tube tonometer and capillary sampling techniques were tested to measure the effect of hyperbaric oxygen treatment on subcutaneous oxygen and carbon dioxide tensions in patients with necrotizing fasciitis and healthy controls. Between January 1971 and April 1997, 53 patients with Clostridial gas gangrene were treated in the Department of Surgery, University of Turku. The patients underwent surgical debridement, broad spectrum antibiotic therapy and a series of hyperbaric oxygen treatments at 2.5 atmospheres absolute pressure (ATA). Twelve patients died (22.6%). Hyperbaric oxygen therapy in gas gangrene seems to be life-, limb- and tissue saving. Early diagnosis remains essential. Patient survival can be improved if the disease is recognized early and appropriate therapy instituted promptly. Between February 1971 and September 1996, 33 patients with perineal necrotizing fasciitis were treated in the Department of Surgery, University of Turku. The management included surgical debridement of the necrotic tissue with incisions and drainage of the involved areas, antibiotic therapy, hyperbaric oxygen treatment at 2.5 ATA pressure and surgical intensive care. Three patients died giving a mortality rate of 9.1%. The survivors received hyperbaric oxygen therapy for 2-12 times. Our results indicate that hyperbaric oxygenation is an important therapeutic adjunct in the treatment of Fournier's gangrene. Electrical equipment should not be used unsheltered in a hyperbaric chamber due to the increased risk of fire. The subcutaneous tissue gas tensions of rats were therefore measured using a subcutaneously implanted Silastic tube tonometer and a capillary sampling technique. The method was successfully adapted to hyperbaric conditions. The subcutaneous oxygen tension levels increased five fold and the carbon dioxide tension levels two fold compared to initial levels. The PO2 and PCO2 of subcutaneous tissue and arterial blood were measured directly in six patients with necrotizing fasciitis and three healthy volunteers in normobaric conditions and during hyperbaric oxygen exposure at 2.5 ATA pressure. The measurements were carried out in healthy tissue and at the same time in the vicinity of the infected area of the patients. During HBO at 2.5 ATA subcutaneous oxygen tensions increased several fold from baseline values and carbon dioxide tensions also increased, but to a lesser degree in both healthy and infected tissues. When examining the subcutaneous PO2 levels measured from patients with necrotizing fasciitis, the PO2 was regularly higher in the vicinity of the infected area than in healthy tissue. In general, HBO treatment resulted in a marked increase in tissue oxygenation in both healthy tissue and in the vicinity of infected tissue. The hyper-oxygenated tissue zone surrounding the infected area may be of significance in preventing the extension of invading microorganisms.

Gas composition in Clostridium septicum gas gangrene.

Clostridial gas gangrene (myonecrosis) is a rare but catastrophic condition that usually occurs in patients with underlying diseases. This paper reports a fatal case of spontaneous clostridial gas gangrene in a 60-year-old female diabetic patient. The composition of gas samples from the patient's damaged muscle was analyzed. The results showed 5.9% hydrogen, 3.4% carbon dioxide, 74.5% nitrogen and 16.1% oxygen. This gas composition supports the belief that such gas production occurs via glucose fermentation. This is the first time such an analysis has been performed in a clinical case of spontaneous clostridial gas gangrene.

Skeletal muscle PO2 in anaerobic soft tissue infections during hyperbaric oxygen therapy.

In the present study skeletal muscle PO2 measurements were performed in patients with gas gangrene and anaerobic soft tissue infections before, during and after hyperbaric oxygen therapy. Polarographic PO2 needle electrodes appeared to be suitable for application during different ambient pressures. We found that patients with gas gangrene revealed higher skeletal muscle PO2 values than patients with an anaerobic soft tissue infection. This may be explained by a higher metabolic rate within the anaerobically infected soft tissues. The higher PO2 values in gas gangrene may be caused by alpha toxins, affecting cellular and intracellular membranes thus destroying PO2 diffusion barriers.

[Metabolic effects of anaerobic gas infection toxins]. / Metabolicheskie éffekty toksinov anaérobnoi gazovoi infektsii.

Activity of blood serum transaminases correlated distinctly with severity of clinical symptoms of gas-gangrenous intoxication, studied in rats in dynamics of the impairment. Activity of the tissue enzymes did not exhibit this correlation. Evaluation of transaminases activity may be used as an objective criterion of the toxic infection development as well as of complex therapy efficiency.