Structural dynamics of the CROPs domain control stability and toxicity of Paeniclostridium sordellii lethal toxin.

Paeniclostridium sordellii lethal toxin (TcsL) is a potent exotoxin that causes lethal toxic shock syndrome associated with fulminant bacterial infections. TcsL belongs to the large clostridial toxin (LCT) family. Here, we report that TcsL with varied lengths of combined repetitive oligopeptides (CROPs) deleted show increased autoproteolysis as well as higher cytotoxicity. We next present cryo-EM structures of full-length TcsL, at neutral (pH 7.4) and acidic (pH 5.0) conditions. The TcsL at neutral pH exhibits in the open conformation, which resembles reported TcdB structures. Low pH induces the conformational change of partial TcsL to the closed form. Two intracellular interfaces are observed in the closed conformation, which possibly locks the cysteine protease domain and hinders the binding of the host receptor. Our findings provide insights into the structure and function of TcsL and reveal mechanisms for CROPs-mediated modulation of autoproteolysis and cytotoxicity, which could be common across the LCT family.

Detection and molecular characterization of Clostridium perfringens, Paeniclostridium sordellii and Clostridium septicum from lambs and goat kids with hemorrhagic abomasitis in Turkey.

BACKGROUND: The pathogenic Clostridia cause neurotoxic, histotoxic and enterotoxic infections in humans and animals. Several Clostridium species have been associated with abomasitis in ruminants. The present study aimed to investigate the frequency, and the presence of virulence genes, of Clostridium perfringens, Paeniclostridium sordellii and Clostridium septicum in lambs and goat kids with hemorrhagic abomasitis. RESULTS: A total of 38 abomasum samples, collected from lambs and goat kids of 1 week to 1 month of age in different farms located in eastern Turkey between 2021 and 2022, were evaluated by histopathology, culture and PCR. At necropsy, the abomasum of the animals was excessively filled with caseinized content and gas, and the abomasum mucosa was hemorrhagic in varying degrees. In histopathological evaluation, acute necrotizing hemorrhagic inflammation was noted in abomasum samples. The examination of swab samples by culture and PCR revealed that C. perfringens type A was the most frequently detected species (86.84%) either alone or in combination with other Clostridium species. P. sordellii, C. perfringens type F and C. septicum were also harboured in the samples, albeit at low rates. Beta2 toxin gene (cpb2) was found in three of C. perfringens type A positive samples. CONCLUSION: It was suggested that vaccination of pregnant animals with toxoid vaccines would be beneficial in terms of protecting newborn animals against Clostridial infections. This study investigated the presence of clostridial toxin genes in abomasal samples for the first time in Turkey.

Infections caused by Clostridium perfringens and Paeniclostridium sordellii after unsafe abortion.

After the legalisation of abortion in the USA in 1973, the risk of infectious morbidity and mortality from this procedure notably decreased. With increasingly restrictive legislation targeting access to safe abortion services, reviewing infectious complications of unsafe pregnancy termination is crucial, particularly the diagnosis and management of life-threatening clostridial (and related anaerobic bacterial) infections that can complicate unsafe abortion. This Review deals with two especially devastating infections that are well-documented causes of septic abortion: the anaerobic, spore-forming pathogens Clostridium perfringens and Paeniclostridium sordellii. We seek to familiarise the reader with these bacteria, the clinical syndromes they can cause (with a focus on toxic shock syndrome), and provide a review of diagnosis and treatment options.

Paeniclostridium sordellii-associated peripartum metritis in goats.

Paeniclostridium sordellii is involved in enteric and histotoxic infections in several animal species. In humans, P. sordellii has been linked to gynecological disease, an association not previously investigated in animals. To unveil a potential association of P. sordellii with veterinary reproductive disease, a retrospective search of the database of the California Animal Health and Food Safety Laboratory System (1990-2020) was conducted and identified 9 cases of goats with P. sordellii-associated metritis or endometritis that were confirmed by immunofluorescence antibody test and/or bacterial isolation, and often co-colonized by Escherichia coli. Six of 9 does were also copper deficient. Polymerase chain reaction (PCR) on formalin-fixed, paraffin-embedded uterine tissue identified the sordellilysin gene in all 9 cases, and the lethal toxin gene in 4. Our findings suggest goats could be predisposed to P. sordellii-associated endometritis/metritis and toxemia when co-infected with E. coli. The role of mineral deficiencies influencing vulnerability to puerperal bacterial infections in goats is possible but remains undetermined. To our knowledge, this is the first report documenting the association of P. sordellii with veterinary gynecological disease.

Paeniclostridium sordellii uterine infection is dependent on the estrous cycle.

Human infections caused by the toxin-producing, anaerobic and spore-forming bacterium Paeniclostridium sordellii are associated with a treatment-refractory toxic shock syndrome (TSS). Reproductive-age women are at increased risk for P. sordellii infection (PSI) because this organism can cause intrauterine infection following childbirth, stillbirth, or abortion. PSI-induced TSS in this setting is nearly 100% fatal, and there are no effective treatments. TcsL, or lethal toxin, is the primary virulence factor in PSI and shares 70% sequence identity with Clostridioides difficile toxin B (TcdB). We therefore reasoned that a neutralizing monoclonal antibody (mAB) against TcdB might also provide protection against TcsL and PSI. We characterized two anti-TcdB mABs: PA41, which binds and prevents translocation of the TcdB glucosyltransferase domain into the cell, and CDB1, a biosimilar of bezlotoxumab, which prevents TcdB binding to a cell surface receptor. Both mABs could neutralize the cytotoxic activity of recombinant TcsL on Vero cells. To determine the efficacy of PA41 and CDB1 in vivo, we developed a transcervical inoculation method for modeling uterine PSI in mice. In the process, we discovered that the stage of the mouse reproductive cycle was a key variable in establishing symptoms of disease. By synchronizing the mice in diestrus with progesterone prior to transcervical inoculation with TcsL or vegetative P. sordellii, we observed highly reproducible intoxication and infection dynamics. PA41 showed efficacy in protecting against toxin in our transcervical in vivo model, but CDB1 did not. Furthermore, PA41 could provide protection following P. sordellii bacterial and spore infections, suggesting a path for further optimization and clinical translation in the effort to advance treatment options for PSI infection.

Paeniclostridium sordellii hemorrhagic toxin targets TMPRSS2 to induce colonic epithelial lesions.

Hemorrhagic toxin (TcsH) is an important exotoxin produced by Paeniclostridium sordellii, but the exact role of TcsH in the pathogenesis remains unclear, partly due to the lack of knowledge of host receptor(s). Here, we carried out two genome-wide CRISPR/Cas9 screens parallelly with TcsH and identified cell surface fucosylation and TMPRSS2 as host factors contributing to the binding and entry of TcsH. Genetic deletion of either fucosylation biosynthesis enzymes or TMPRSS2 in the cells confers resistance to TcsH intoxication. Interestingly, TMPRSS2 and fucosylated glycans can mediate the binding/entry of TcsH independently, thus serving as redundant receptors. Both TMPRSS2 and fucosylation recognize TcsH through its CROPs domain. By using Tmprss2‒/‒ mice, we show that Tmprss2 is important for TcsH-induced systematic toxicity and colonic epithelial lesions. These findings reveal the importance of TMPRSS2 and surface fucosylation in TcsH actions and further provide insights into host recognition mechanisms for large clostridial toxins.

A Highly Specific Holin-Mediated Mechanism Facilitates the Secretion of Lethal Toxin TcsL in Paeniclostridiumsordellii.

Protein secretion is generally mediated by a series of distinct pathways in bacteria. Recently, evidence of a novel bacterial secretion pathway involving a bacteriophage-related protein has emerged. TcdE, a holin-like protein encoded by toxigenic isolates of Clostridioides difficile, mediates the release of the large clostridial glucosylating toxins (LCGTs), TcdA and TcdB, and TpeL from C. perfringens uses another holin-like protein, TpeE, for its secretion; however, it is not yet known if TcdE or TpeE secretion is specific to these proteins. It is also unknown if other members of the LCGT-producing clostridia, including Paeniclostridium sordellii (previously Clostridium sordellii), use a similar toxin-release mechanism. Here, we confirm that each of the LCGT-producing clostridia encode functional holin-like proteins in close proximity to the toxin genes. To characterise the respective roles of these holin-like proteins in the release of the LCGTs, P. sordellii and its lethal toxin, TcsL, were used as a model. Construction and analysis of mutants of the P. sordellii tcsE (holin-like) gene demonstrated that TcsE plays a significant role in TcsL release. Proteomic analysis of the secretome from the tcsE mutant confirmed that TcsE is required for efficient TcsL secretion. Unexpectedly, comparative sample analysis showed that TcsL was the only protein significantly altered in its release, suggesting that this holin-like protein has specifically evolved to function in the release of this important virulence factor. This specificity has, to our knowledge, not been previously shown and suggests that this protein may function as part of a specific mechanism for the release of all LCGTs.

Persistent bacteremia and psoas abscess caused by a lethal toxin-deficient Paeniclostridiumsordellii.

We present a case of persistent bacteremia and psoas abscess from Paeniclostridium sordellii without severe symptoms or the classically associated toxic shock syndrome. Further laboratory evaluation demonstrated that the Paeniclostridium sordellii isolate lacked the lethal toxin gene and there was no cytotoxicity to exposed Vero cells.

Characterization of Paeniclostridium sordellii Metalloproteinase-1 in vitro and in an experimental model of infection.

OBJECTIVE: Paeniclostridium sordellii is a pathogen that causes rapidly fatal infections characterized by severe edema, extreme leukemoid reaction and lack of an innate immune response. We recently identified a metalloproteinase of P. sordellii-1 (Mcs1) that cleaves human vascular cell adhesion molecule 1, an adhesion molecule important to hematopoietic precursor retention and leukocyte diapedesis. In the current study, we further characterize Mcs1 activity and investigate its role in pathogenesis. METHODS: Mcs1 peptide cleavage sequence and activity conditions were identified using a semi-quantitative fluorescence-based reporter assay. Additional host targets for Mcs1 protease activity were tested and confirmed by gel electrophoreses and western blots. Finally, Mcs1 knock out (ΔMcs1) and complemented (cMcs1) strains were developed for assessment in our animal model of myonecrosis. RESULTS: Data show that Mcs1 prefers aliphatic amino acid residues, I or L, especially when adjacent to negatively charged or noncharged-polar residues. In vitro, Mcs1 cleaved or partially cleaved human cell adhesion molecules, E-selectin and intracellular adhesion molecule-1 (ICAM-1), and mediators of innate immune infection defense, complement protein-3 and antimicrobial peptide LL-37. In vivo, infection with the ΔMcs1 P. sordellii strain had little effect on animal survival, tissue destruction or circulating white blood cell counts compared to wild type and cMcs1 strains. CONCLUSIONS: Similar to proteolytic virulence factors from other pathogens, Mcs1 is a promiscuous protease that cleaves multiple human-host factors. Despite minimal impact of Mcs1 on the murine model of P. sordellii infection, it is worth considering its role in humans and other animal models.

The comparative pathology of enterocolitis caused by Clostridium perfringens type C, Clostridioides difficile, Paeniclostridium sordellii, Salmonella enterica subspecies enterica serovar Typhimurium, and nonsteroidal anti-inflammatory drugs in horses.

To determine if there were significant differences produced by 5 of the most prevalent causes of equine enterocolitis, we studied retrospectively the gross and microscopic pathology of 90 cases of enterocolitis submitted to the San Bernardino laboratory of the California Animal Health and Food Safety Laboratory. Included were cases caused by Clostridium perfringens type C (CP; n = 20), Clostridioides difficile (CD; n = 20), Paeniclostridium sordellii (PS; n = 15), Salmonella enterica subspecies enterica serovar Typhimurium (ST; n = 20), and NSAID intoxication (NS; n = 15). Grossly, necrotizing hemorrhagic typhlocolitis was seen most frequently in cases of CD, ST, and NS disease. Cases of CP and PS had enteritis or colitis in similar percentages. Congestion, hemorrhage, and pleocellular inflammatory infiltrates followed by mucosal and submucosal necrosis were the main lesions found in horses with enteritis or colitis produced by any of the etiologic agents investigated. Severe lesions were more frequent in cases of CD and CP than in cases associated with any of the other 3 etiologies. Pseudomembranes were observed with similar prevalence in the small intestine and colon affected by all agents studied. Thrombosis of the lamina propria and/or submucosa was observed in ~50% of the cases of enteritis and colitis by all etiologies, except for PS, in which the majority of the cases had thrombosis. Gross and microscopic lesions of enterocolitis were not sufficiently specific for any of these etiologic agents to enable these enteritides to be distinguished by gross and/or histologic examination.

Rapidly fatal necrotizing soft tissue infection due to Clostridium sordellii in an injection drug user.

Clostridium sordellii infections are known to be associated with high morbidity and mortality. To date, only a small number of cases with necrotizing soft tissue infection due to C. Sordellii have been reported. We report a case presented with necrotizing soft tissue infection of the right upper extremity caused by C. sordellii in a patient with known use of injected heroin. Despite broad spectrum antibiotics and surgical debridement, the patient's clinical course became rapidly fatal, within 24 h of admission. C. sordellii necrotizing soft tissue infections are particularly virulent. Even in the context of appropriate surgical debridement, these infections can be rapidly fatal. This case highlights the importance of high suspicion for C. sordellii as potential pathogen of necrotizing soft tissue infection in injection drug users.

Skin and Soft Tissue Infections in Persons Who Inject Drugs.

Persons who inject drugs are at high risk for skin and soft tissue infections. Infections range from simple abscesses and uncomplicated cellulitis to life-threatening and limb-threatening infections. These infections are predominantly caused by gram-positive organisms with Staphylococcus aureus, Streptococcus pyogenes, and other streptococcal species being most common. Although antimicrobial therapy has an important role in treatment of these infections, surgical incision, drainage, and debridement of devitalized tissue are primary. Strategies that decrease the frequency of injection drug use, needle sharing, use of contaminated equipment, and other risk behaviors may be effective in preventing these infections in persons who inject drugs.

Recognition of Semaphorin Proteins by P. sordellii Lethal Toxin Reveals Principles of Receptor Specificity in Clostridial Toxins.

Pathogenic clostridial species secrete potent toxins that induce severe host tissue damage. Paeniclostridium sordellii lethal toxin (TcsL) causes an almost invariably lethal toxic shock syndrome associated with gynecological infections. TcsL is 87% similar to C. difficile TcdB, which enters host cells via Frizzled receptors in colon epithelium. However, P. sordellii infections target vascular endothelium, suggesting that TcsL exploits another receptor. Here, using CRISPR/Cas9 screening, we establish semaphorins SEMA6A and SEMA6B as TcsL receptors. We demonstrate that recombinant SEMA6A can protect mice from TcsL-induced edema. A 3.3 Å cryo-EM structure shows that TcsL binds SEMA6A with the same region that in TcdB binds structurally unrelated Frizzled. Remarkably, 15 mutations in this evolutionarily divergent surface are sufficient to switch binding specificity of TcsL to that of TcdB. Our findings establish semaphorins as physiologically relevant receptors for TcsL and reveal the molecular basis for the difference in tissue targeting and disease pathogenesis between highly related toxins.

Bacteriemia por Clostridium sordellii en paciente con neoplasia gastrointestinal. Reporte de caso y revisión de literatura / Clostridium sordellii bacteremia in a patient with gastrointestinal neoplasia. Case report and review of the literatura

La bacteriemia por Clostridium sordellii es infrecuente y usualmente se origina a partir de infecciones de etiología generalmente ginecológica y puerperal, con una mortalidad de aproximadamente el 70%. Existen pocas herramientas para el diagnóstico rápido y oportuno, siendo así la experiencia de tratamiento para este germen muy limitada en otros escenarios, lo que probablemente sea la causa de su alta mortalidad. Presentamos una paciente con antecedente de masa abdominal expansiva de larga data, con diagnóstico por histopatología e inmunohistoquimica compatibles con tumor del estroma gastrointestinal (GIST por sus siglas en inglés) y estudios de extensión que confirman compromiso metastásico hepático, en quien se documenta bacteriemia por Clostridium sordellii.

Clostridium sordellii bacteriemia is infrequent and usually comes from infections of gynecological and puerperal etiology, with mortality near 70%. There are few tools for rapid and timely diagnosis. Thus, treatment experience for this pathogen is very limited in other scenarios, which is probably the cause of high mortality rates. We describe a patient with a history of expansive abdominal mass, diagnosed with metastasic Gastrointestinal Stromal Tumor (GIST), with Clostridium sordellii bacteremia.

Fatal Clostridium sordellii-mediated hemorrhagic and necrotizing gastroenteropathy in a dog: case report.

BACKGROUND: Canine hemorrhagic gastroenteritis (also canine gastrointestinal hemorrhagic syndrome) is commonly associated with Clostridium perfringens, although in some cases the etiology remains unclear. This report describes a fatal acute hemorrhagic and necrotizing gastroenteropathy in a dog associated with Clostridium sordellii, a bacterial species never before identified as the etiological agent of hemorrhagic and necrotizing gastroenteropathy in dogs. CASE PRESENTATION: A fully vaccinated, eight-year-old, female neutered Labrador presented with a history of vomiting without diarrhea. Clinical examination revealed pink mucous membranes, adequate hydration, normothermia, and normocardia. The dog was discovered deceased the following day. Post-mortem examination showed moderate amounts of dark red, non-clotted fluid within the stomach that extended into the jejunum. Discoloration was noted in the gastric mucosa, liver, lungs, and kidneys, with small petechial hemorrhages present in the endocardium over the right heart base and thymic remnants. Histological analysis demonstrated that the gastric fundic mucosa, the pyloric region, small intestine, and large intestine exhibited superficial coagulative necrosis and were lined with a layer of short Gram-positive rods. Anaerobic culture of the gastric content revealed C. sordellii as the dominant bacterial species and neither Salmonella spp., Campylobacter spp., C. perfringens, nor C. difficile were isolated. Unexpectedly, whole genome sequencing of the C. sordellii isolate showed that it lacked the main plasmid-encoded virulence factors typical of the species, indicating that the genetic determinants of pathogenicity of this strain must be chromosomally encoded. Further phylogenetic analysis revealed it to be genetically similar to C. sordellii isolates associated with gastroenteric disease in livestock, indicating that the infection may have been acquired from the environment. CONCLUSIONS: This case demonstrates that C. sordellii can associate with a canine hemorrhagic and necrotizing gastroenteropathy in the absence of C. perfringens and illustrates the benefits of using bacterial whole genome sequencing to support pathological investigations in veterinary diagnostics. These data also update the molecular phylogeny of C. sordellii, indicating a possible pathogenic clade in the environment that is distinct from currently identified clades.

Genome-Wide CRISPR Screen Identifies Semaphorin 6A and 6B as Receptors for Paeniclostridium sordellii Toxin TcsL.

The exotoxin TcsL is a major virulence factor in Paeniclostridium (Clostridium) sordellii and responsible for the high lethality rate associated with P. sordellii infection. Here, we present a genome-wide CRISPR-Cas9-mediated screen using a human lung carcinoma cell line and identify semaphorin (SEMA) 6A and 6B as receptors for TcsL. Disrupting SEMA6A/6B expression in several distinct human cell lines and primary human endothelial cells results in reduced TcsL sensitivity, while SEMA6A/6B over-expression increases their sensitivity. TcsL recognizes the extracellular domain (ECD) of SEMA6A/6B via a region homologous to the receptor-binding site in Clostridioides difficile toxin B (TcdB), which binds the human receptor Frizzled. Exchanging the receptor-binding interfaces between TcsL and TcdB switches their receptor-binding specificity. Finally, administration of SEMA6A-ECD proteins protects human cells from TcsL toxicity and reduces TcsL-induced damage to lung tissues and the lethality rate in mice. These findings establish SEMA6A and 6B as pathophysiologically relevant receptors for TcsL.

Paeniclostridium (Clostridium) sordellii-associated enterocolitis in 7 horses.

Enteric disease in horses may be caused by a variety of microorganisms, including several clostridial species. Paeniclostridium sordellii (previously Clostridium sordellii) has been frequently associated with gas gangrene in humans and several animal species, including horses. However, its role in enteric diseases of animals has not been fully determined. We describe herein 7 cases of enteric disease in horses associated with P. sordellii infection. Grossly, the small and/or large intestines were necrotic, hemorrhagic, and edematous. Microscopically, there was severe mucosal necrosis and hemorrhage of the small and/or large intestine of all horses. P. sordellii was isolated and/or demonstrated by immunohistochemistry and/or PCR in the intestine of all horses. All other known causes of enteric disease in horses were ruled out in these 7 cases. P. sordellii should be considered among the differential diagnoses in cases of enteric disease in horses.

Clostridium sordellii-associated gas gangrene in 8 horses, 1998-2019.

Gas gangrene occurs in several animal species and is caused by one or more clostridial species. In horses, the disease is most often caused by Clostridium perfringens type A. Although Clostridium sordellii has been associated with gas gangrene in ruminants and humans, cases of the disease associated with this microorganism have not been described in horses, to our knowledge. We report herein 8 cases of gas gangrene caused by C. sordellii in horses. These cases were characterized by myonecrosis and cellulitis, associated with systemic changes suggestive of toxic shock. The diagnosis was confirmed by gross and microscopic changes combined with anaerobic culture, fluorescent antibody test, immunohistochemistry, and/or PCR. The predisposing factor in these cases was an injection or a traumatic skin injury. C. sordellii should be considered as a possible etiologic agent in cases of gas gangrene in horses.

The antibiotic susceptibility pattern of gas gangrene-forming Clostridium spp. clinical isolates from South-Eastern Hungary.

Introduction:Clostridium perfringens and other gas gangrene-forming clostridia are commensals of the human gut and vaginal microbiota, but can cause serious or even fatal infections. As there are relatively few published studies on antibiotic susceptibility of these bacteria, we decided to perform a 10-year retrospective study in a South-Eastern Hungarian clinical centre.Methods: A total of 372 gas gangrene-forming Clostridium spp. were isolated from clinically relevant samples and identified with rapid ID 32A (bioMérieux, France) and MALDI-TOF MS (Bruker Daltinics, Germany) methods. Antibiotic susceptibility was determined with E-tests.Results: We identified 313 C. perfringens, 20 C. septicum, 10 C. sordellii, 10 C. sporogenes, 9 C. tertium, 6 C. bifermentans, 4 C. histolyticum isolates. In C. perfringens isolates, the rate of penicillin resistance was 2.6% and the rate of clindamycin resistance 3.8%. Penicillin resistance was found in 6.8% and clindamycin resistance in 8.5% of the non-perfringens Clostridium spp. isolates.Conclusion: The antibiotic susceptibility of C. perfringens isolates was in good agreement with previous publications. The rates of resistance to penicillin and clindamycin were very low. The resistance rates of non-perfringens Clostridium spp. isolates were higher than those of C. perfringens strains, but lower than those published in the literature.

Virulence Plasmids of the Pathogenic Clostridia.

The clostridia cause a spectrum of diseases in humans and animals ranging from life-threatening tetanus and botulism, uterine infections, histotoxic infections and enteric diseases, including antibiotic-associated diarrhea, and food poisoning. The symptoms of all these diseases are the result of potent protein toxins produced by these organisms. These toxins are diverse, ranging from a multitude of pore-forming toxins to phospholipases, metalloproteases, ADP-ribosyltransferases and large glycosyltransferases. The location of the toxin genes is the unifying theme of this review because with one or two exceptions they are all located on plasmids or on bacteriophage that replicate using a plasmid-like intermediate. Some of these plasmids are distantly related whilst others share little or no similarity. Many of these toxin plasmids have been shown to be conjugative. The mobile nature of these toxin genes gives a ready explanation of how clostridial toxin genes have been so widely disseminated both within the clostridial genera as well as in the wider bacterial community.