Clostridium perfringens virulence factors are nonredundant activators of the NLRP3 inflammasome.

Inflammasome signaling is a central pillar of innate immunity triggering inflammation and cell death in response to microbes and danger signals. Here, we show that two virulence factors from the human bacterial pathogen Clostridium perfringens are nonredundant activators of the NLRP3 inflammasome in mice and humans. C. perfringens lecithinase (also known as phospolipase C) and C. perfringens perfringolysin O induce distinct mechanisms of activation. Lecithinase enters LAMP1+ vesicular structures and induces lysosomal membrane destabilization. Furthermore, lecithinase induces the release of the inflammasome-dependent cytokines IL-1ß and IL-18, and the induction of cell death independently of the pore-forming proteins gasdermin D, MLKL and the cell death effector protein ninjurin-1 or NINJ1. We also show that lecithinase triggers inflammation via the NLRP3 inflammasome in vivo and that pharmacological blockade of NLRP3 using MCC950 partially prevents lecithinase-induced lethality. Together, these findings reveal that lecithinase activates an alternative pathway to induce inflammation during C. perfringens infection and that this mode of action can be similarly exploited for sensing by a single inflammasome.

Clostridium septicum α-toxin activates the NLRP3 inflammasome by engaging GPI-anchored proteins.

Clostridium species are a group of Gram-positive bacteria that cause diseases in humans, such as food poisoning, botulism, and tetanus. Here, we analyzed 10 different Clostridium species and identified that Clostridium septicum, a pathogen that causes sepsis and gas gangrene, activates the mammalian cytosolic inflammasome complex in mice and humans. Mechanistically, we demonstrate that α-toxin secreted by C. septicum binds to glycosylphosphatidylinositol (GPI)-anchored proteins on the host plasma membrane, oligomerizing and forming a membrane pore that is permissive to efflux of magnesium and potassium ions. Efflux of these cytosolic ions triggers the activation of the innate immune sensor NLRP3, inducing activation of caspase-1 and gasdermin D, secretion of the proinflammatory cytokines interleukin-1ß and interleukin-18, pyroptosis, and plasma membrane rupture via ninjurin-1. Furthermore, α-toxin of C. septicum induces rapid inflammasome-mediated lethality in mice and pharmacological inhibition of the NLRP3 inflammasome using MCC950 prevents C. septicum-induced lethality. Overall, our results reveal that cytosolic innate sensing of α-toxin is central to the recognition of C. septicum infection and that therapeutic blockade of the inflammasome pathway may prevent sepsis and death caused by toxin-producing pathogens.

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.

PGFinder, a novel analysis pipeline for the consistent, reproducible, and high-resolution structural analysis of bacterial peptidoglycans.

Many software solutions are available for proteomics and glycomics studies, but none are ideal for the structural analysis of peptidoglycan (PG), the essential and major component of bacterial cell envelopes. It icomprises glycan chains and peptide stems, both containing unusual amino acids and sugars. This has forced the field to rely on manual analysis approaches, which are time-consuming, labour-intensive, and prone to error. The lack of automated tools has hampered the ability to perform high-throughput analyses and prevented the adoption of a standard methodology. Here, we describe a novel tool called PGFinder for the analysis of PG structure and demonstrate that it represents a powerful tool to quantify PG fragments and discover novel structural features. Our analysis workflow, which relies on open-access tools, is a breakthrough towards a consistent and reproducible analysis of bacterial PGs. It represents a significant advance towards peptidoglycomics as a full-fledged discipline.

Chromosome Segregation and Peptidoglycan Remodeling Are Coordinated at a Highly Stabilized Septal Pore to Maintain Bacterial Spore Development.

Asymmetric division, a hallmark of endospore development, generates two cells, a larger mother cell and a smaller forespore. Approximately 75% of the forespore chromosome must be translocated across the division septum into the forespore by the DNA translocase SpoIIIE. Asymmetric division also triggers cell-specific transcription, which initiates septal peptidoglycan remodeling involving synthetic and hydrolytic enzymes. How these processes are coordinated has remained a mystery. Using Bacillus subtilis, we identified factors that revealed the link between chromosome translocation and peptidoglycan remodeling. In cells lacking these factors, the asymmetric septum retracts, resulting in forespore cytoplasmic leakage and loss of DNA translocation. Importantly, these phenotypes depend on septal peptidoglycan hydrolysis. Our data support a model in which SpoIIIE is anchored at the edge of a septal pore, stabilized by newly synthesized peptidoglycan and protein-protein interactions across the septum. Together, these factors ensure coordination between chromosome translocation and septal peptidoglycan remodeling to maintain spore development.

A dynamic, ring-forming MucB / RseB-like protein influences spore shape in Bacillus subtilis.

How organisms develop into specific shapes is a central question in biology. The maintenance of bacterial shape is connected to the assembly and remodelling of the cell envelope. In endospore-forming bacteria, the pre-spore compartment (the forespore) undergoes morphological changes that result in a spore of defined shape, with a complex, multi-layered cell envelope. However, the mechanisms that govern spore shape remain poorly understood. Here, using a combination of fluorescence microscopy, quantitative image analysis, molecular genetics and transmission electron microscopy, we show that SsdC (formerly YdcC), a poorly-characterized new member of the MucB / RseB family of proteins that bind lipopolysaccharide in diderm bacteria, influences spore shape in the monoderm Bacillus subtilis. Sporulating cells lacking SsdC fail to adopt the typical oblong shape of wild-type forespores and are instead rounder. 2D and 3D-fluorescence microscopy suggest that SsdC forms a discontinuous, dynamic ring-like structure in the peripheral membrane of the mother cell, near the mother cell proximal pole of the forespore. A synthetic sporulation screen identified genetic relationships between ssdC and genes involved in the assembly of the spore coat. Phenotypic characterization of these mutants revealed that spore shape, and SsdC localization, depend on the coat basement layer proteins SpoVM and SpoIVA, the encasement protein SpoVID and the inner coat protein SafA. Importantly, we found that the ΔssdC mutant produces spores with an abnormal-looking cortex, and abolishing cortex synthesis in the mutant largely suppresses its shape defects. Thus, SsdC appears to play a role in the proper assembly of the spore cortex, through connections to the spore coat. Collectively, our data suggest functional diversification of the MucB / RseB protein domain between diderm and monoderm bacteria and identify SsdC as an important factor in spore shape development.

Human Plasminogen Exacerbates Clostridioides difficile Enteric Disease and Alters the Spore Surface.

BACKGROUND & AIMS: The protease plasmin is an important wound healing factor, but it is not clear how it affects gastrointestinal infection-mediated damage, such as that resulting from Clostridioides difficile. We investigated the role of plasmin in C difficile-associated disease. This bacterium produces a spore form that is required for infection, so we also investigated the effects of plasmin on spores. METHODS: C57BL/6J mice expressing the precursor to plasmin, the zymogen human plasminogen (hPLG), or infused with hPLG were infected with C difficile, and disease progression was monitored. Gut tissues were collected, and cytokine production and tissue damage were analyzed by using proteomic and cytokine arrays. Antibodies that inhibit either hPLG activation or plasmin activity were developed and structurally characterized, and their effects were tested in mice. Spores were isolated from infected patients or mice and visualized using super-resolution microscopy; the functional consequences of hPLG binding to spores were determined. RESULTS: hPLG localized to the toxin-damaged gut, resulting in immune dysregulation with an increased abundance of cytokines (such as interleukin [IL] 1A, IL1B, IL3, IL10, IL12B, MCP1, MP1A, MP1B, GCSF, GMCSF, KC, TIMP-1), tissue degradation, and reduced survival. Administration of antibodies that inhibit plasminogen activation reduced disease severity in mice. C difficile spores bound specifically to hPLG and active plasmin degraded their surface, facilitating rapid germination. CONCLUSIONS: We found that hPLG is recruited to the damaged gut, exacerbating C difficile disease in mice. hPLG binds to C difficile spores, and, upon activation to plasmin, remodels the spore surface, facilitating rapid spore germination. Inhibitors of plasminogen activation might be developed for treatment of C difficile or other infection-mediated gastrointestinal diseases.

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.

Cephamycins inhibit pathogen sporulation and effectively treat recurrent Clostridioides difficile infection.

Spore-forming bacteria encompass a diverse range of genera and species, including important human and animal pathogens, and food contaminants. Clostridioides difficile is one such bacterium and is a global health threat because it is the leading cause of antibiotic-associated diarrhoea in hospitals. A crucial mediator of C. difficile disease initiation, dissemination and re-infection is the formation of spores that are resistant to current therapeutics, which do not target sporulation. Here, we show that cephamycin antibiotics inhibit C. difficile sporulation by targeting spore-specific penicillin-binding proteins. Using a mouse disease model, we show that combined treatment with the current standard-of-care antibiotic, vancomycin, and a cephamycin prevents disease recurrence. Cephamycins were found to have broad applicability as an anti-sporulation strategy, as they inhibited sporulation in other spore-forming pathogens, including the food contaminant Bacillus cereus. This study could directly and immediately affect treatment of C. difficile infection and advance drug development to control other important spore-forming bacteria that are problematic in the food industry (B. cereus), are potential bioterrorism agents (Bacillus anthracis) and cause other animal and human infections.

Tranexamic Acid Influences the Immune Response, but not Bacterial Clearance in a Model of Post-Traumatic Brain Injury Pneumonia.

The antifibrinolytic agent, tranexamic acid (TXA), an inhibitor of plasmin formation, currently is evaluated to reduce bleeding in various conditions, including traumatic brain injury (TBI). Because plasmin is implicated in inflammation and immunity, we investigated the effects of plasmin inhibition on the immune response after TBI in the presence or absence of induced pneumonia. Wild-type mice treated with vehicle or TXA or mice deficient in plasminogen (plg-/-) underwent TBI using the controlled cortical impact model. Mice were then subjected to Staphylococcus aureus induced pneumonia and the degree of immune competence determined. Significant baseline changes in the innate immune cell profile were seen in plg-/- mice with increases in spleen weight and white blood cell counts, and elevation in plasma interleukin-6 levels. The plg-/- mice subjected to TBI displayed no additional changes in these parameters at the 72 h or one week time point post-TBI. The plg-/- mice subjected to TBI did not exhibit any further increase in susceptibility to endogenous infection. Pneumonia was induced by intratracheal instillation of S. aureus. The TBI did not worsen pneumonia symptoms or delay recovery in plg-/- mice. Similarly, in wild type mice, treatment with TXA did not impact on the ability of mice to counteract pneumonia after TBI. Administration of TXA after TBI and subsequent pneumonia, however, altered the number and surface marker expression of several myeloid and lymphoid cell populations, consistent with enhanced immune activation at the 72 h time point. This investigation confirms the immune-modulatory properties of TXA, thereby highlighting its effects unrelated to inhibition of fibrinolysis.

Paeniclostridium sordellii and Clostridioides difficile encode similar and clinically relevant tetracycline resistance loci in diverse genomic locations.

BACKGROUND: With the current rise of antibiotic resistance in bacteria, it is important to monitor the efficacy of antimicrobials in clinical use. Paeniclostridium sordellii (previously Clostridium sordellii) is a bacterial pathogen that causes human uterine infection after spontaneous or medically induced abortion, for which mortality rates approach 100%. Prophylactic antibiotics have been recommended for individuals undergoing medically-induced abortion, one of which is doxycycline, a member of the tetracycline antibiotic family. However, tetracycline resistance had not been well characterized in P. sordellii. This study therefore aimed to determine the levels of tetracycline resistance in P. sordellii isolates, and to identify associated loci and their genomic locations. RESULTS: Using a MIC assay, five of 24 P. sordellii isolates were found to be resistant to tetracycline, minocycline, and importantly, doxycycline. Analysis of genome sequence data from 46 isolates found that phenotypically resistant isolates encoded a variant of the Clostridium perfringens tetracycline resistance determinant Tet P. Bioinformatic analysis and comparison of the regions surrounding these determinants found variation in the genomic location of Tet P among P. sordellii isolates. The core genome comparison of the 46 isolates revealed genetic diversity and the absence of dominant genetic types among the isolates. There was no strong association between geographic location of isolation, animal host or Tet P carriage with isolate genetic type. Furthermore, the analysis of the Tet P genotype revealed that Tet P is encoded chromosomally, or on one of two, novel, small plasmids, all consistent with multiple acquisition and recombination events. BLAST analysis of Clostridioides difficile draft genome sequences also identified a Tet P locus, the genomic location of which demonstrated an evolutionary relationship with the P. sordellii locus. CONCLUSIONS: The Tet P determinant is found in variable genomic locations within diverse human and animal isolates of P. sordellii and C. difficile, which suggests that it can undergo horizontal transfer, and may disseminate tetracycline resistance between clostridial species. Doxycycline is a suggested prophylactic treatment for P. sordellii infections, however, a small sub-set of the isolates tested are resistant to this antibiotic. Doxycycline may therefore not be an appropriate prophylactic treatment for P. sordellii infections.

pCP13, a representative of a new family of conjugative toxin plasmids in Clostridium perfringens.

Conjugative transfer is a major contributor to the dissemination of antibiotic resistance and virulence genes in the human and animal pathogen, Clostridium perfringens. The C. perfringens plasmid pCW3 is the archetype of an extensive family of highly related conjugative toxin and antibiotic resistance plasmids found in this bacterium. These plasmids were thought to constitute the only conjugative plasmid family in C. perfringens. Recently, another series of C. perfringens plasmids, the pCP13-like family, have been shown to harbour important toxin genes, including genes that encode the novel binary clostridial enterotoxin, BEC. Based on early bioinformatics analysis this plasmid family was thought to be non-conjugative. Here we demonstrate that pCP13 is in fact conjugative, transfers at high frequency and that the newly defined Pcp conjugation locus encodes putative homologues of a type 4 secretion system (T4SS), one of which, PcpB4, was shown to be essential for transfer. The T4SS of pCP13 also appears to be evolutionarily related to conjugative toxin plasmids from other clostridia-like species, including Paeniclostridium (formerly Clostridium) sordellii, Clostridioides (formerly Clostridium) difficile and Clostridium botulinum. Therefore, it is clear that there are two distinct families of conjugative plasmids in C. perfringens: the pCW3 family and the pCP13 family. This study has significant implications for our understanding of the movement of toxin genes both within C. perfringens, but also potentially to other pathogenic clostridia.

Lectin Activity of the TcdA and TcdB Toxins of Clostridium difficile.

Clostridium difficile is a major cause of hospital-acquired antibiotic-associated diarrhea. C. difficile produces two cytotoxins, TcdA and TcdB; both toxins are multidomain proteins that lead to cytotoxicity through the modification and inactivation of small GTPases of the Rho/Rac family. Previous studies have indicated that host glycans are targets for TcdA and TcdB, with interactions thought to be with both α- and ß-linked galactose. In the current study, screening of glycan arrays with different domains of TcdA and TcdB revealed that the binding regions of both toxins interact with a wider range of host glycoconjugates than just terminal α- and ß-linked galactose, including blood groups, Lewis antigens, N-acetylglucosamine, mannose, and glycosaminoglycans. The interactions of TcdA and TcdB with ABO blood group and Lewis antigens were assessed by surface plasmon resonance (SPR). The blood group A antigen was the highest-affinity ligand for both toxins. Free glycans alone or in combination were unable to abolish Vero cell cytotoxicity by TcdB. SPR competition assays indicate that there is more than one glycan binding site on TcdB. Host glycoconjugates are common targets of bacterial toxins, but typically this binding is to a specific structure or related structures. The binding of TcdA and TcdB is to a wide range of host glycans providing a wide range of target cells and tissues in vivo.

Clostridium sordellii outer spore proteins maintain spore structural integrity and promote bacterial clearance from the gastrointestinal tract.

Bacterial spores play an important role in disease initiation, transmission and persistence. In some species, the exosporium forms the outermost structure of the spore and provides the first point of contact between the spore and the environment. The exosporium may also be involved in spore adherence, protection and germination. Clostridium sordellii is a highly lethal, spore forming pathogen that causes soft-tissue infections, enteritis and toxic-shock syndrome. Despite the importance of C. sordellii spores in disease, spore proteins from this bacterium have not been defined or interrogated functionally. In this study, we identified the C. sordellii outer spore proteome and two of the identified proteins, CsA and CsB, were characterised using a genetic and phenotypic approach. Both proteins were essential for the correct formation and positioning of the C. sordellii spore coat and exosporium. The absence of CsA reduced sporulation levels and increased spore sensitivity to heat, sodium hydroxide and hydrochloric acid. By comparison, CsB was required for normal levels of spore adherence to cervical, but not vaginal, cells, with csB mutant spores having increased adherence properties. The establishment of a mouse infection model of the gastrointestinal tract for C. sordellii allowed the role of CsA and CsB to be interrogated in an infected host. Following the oral administration of spores to mice, the wild-type strain efficiently colonized the gastrointestinal tract, with the peak of bacterial numbers occurring at one day post-infection. Colonization was reduced by two logs at four days post-infection. By comparison, mice infected with the csB mutant did not show a reduction in bacterial numbers. We conclude that C. sordellii outer spore proteins are important for the structural and functional integrity of spores. Furthermore, outer spore proteins are required for wild-type levels of colonization during infection, possibly as a result of the role that the proteins play in spore structure and morphology.

Clostridium sordellii Pathogenicity Locus Plasmid pCS1-1 Encodes a Novel Clostridial Conjugation Locus.

A major virulence factor in Clostridium sordellii-mediated infection is the toxin TcsL, which is encoded within a region of the genome called the pathogenicity locus (PaLoc). C. sordellii isolates carry the PaLoc on the pCS1 family of plasmids, of which there are four characterized members. Here, we determined the potential mobility of pCS1 plasmids and characterized a fifth unique pCS1 member. Using a derivative of the pCS1-1 plasmid from strain ATCC 9714 which had been marked with the ermB erythromycin resistance gene, conjugative transfer into a recipient C. sordellii isolate, R28058, was demonstrated. Bioinformatic analysis of pCS1-1 identified a novel conjugation gene cluster defined as the C. sordellii transfer (cst) locus. Interruption of genes within the cst locus resulted in loss of pCS1-1 transfer, which was restored upon complementation in trans These studies provided clear evidence that genes within the cst locus are essential for the conjugative transfer of pCS1-1. The cst locus is present on all pCS1 subtypes, and homologous loci were identified on toxin-encoding plasmids from Clostridium perfringens and Clostridium botulinum and also carried within genomes of Clostridium difficile isolates, indicating that it is a widespread clostridial conjugation locus. The results of this study have broad implications for the dissemination of toxin genes and, potentially, antibiotic resistance genes among members of a diverse range of clostridial pathogens, providing these microorganisms with a survival advantage within the infected host.IMPORTANCEC. sordellii is a bacterial pathogen that causes severe infections in humans and animals, with high mortality rates. While the pathogenesis of C. sordellii infections is not well understood, it is known that the toxin TcsL is an important virulence factor. Here, we have shown the ability of a plasmid carrying the tcsL gene to undergo conjugative transfer between distantly related strains of C. sordellii, which has far-reaching implications for the ability of C. sordellii to acquire the capacity to cause disease. Plasmids that carry tcsL encode a previously uncharacterized conjugation locus, and individual genes within this locus were shown to be required for conjugative transfer. Furthermore, homologues on toxin plasmids from other clostridial species were identified, indicating that this region represents a novel clostridial conjugation locus. The results of this study have broad implications for the dissemination of virulence genes among members of a diverse range of clostridial pathogens.

Structural Characterization of Clostridium sordellii Spores of Diverse Human, Animal, and Environmental Origin and Comparison to Clostridium difficile Spores.

Clostridium sordellii is an often-lethal bacterium causing human and animal disease. Crucial to the infectious cycle of C. sordellii is its ability to produce spores, which can germinate into toxin-producing vegetative bacteria under favorable conditions. However, structural details of the C. sordellii spore are lacking. Here, we used a range of electron microscopy techniques together with superresolution optical microscopy to characterize the C. sordellii spore morphology with an emphasis on the exosporium. The C. sordellii spore is made up of multiple layers with the exosporium presenting as a smooth balloon-like structure that is open at the spore poles. Focusing on the outer spore layers, we compared the morphologies of C. sordellii spores derived from different strains and determined that there is some variation between the spores, most notably with spores of some strains having tubular appendages. Since Clostridium difficile is a close relative of C. sordellii, their spores were compared by electron microscopy and their exosporia were found to be distinctly different from each other. This study therefore provides new structural details of the C. sordellii spore and offers insights into the physical structure of the exosporium across clostridial species. IMPORTANCEClostridium sordellii is a significant pathogen with mortality rates approaching 100%. It is the bacterial spore that is critical in initiating infection and disease. An understanding of spore structures as well as spore morphology across a range of strains may lead to a better understanding of C. sordellii infection and disease. However, the structural characteristics of the C. sordellii spores are limited. In this work, we have addressed this lack of detail and characterized the C. sordellii spore morphology. The use of traditional and advanced microscopy techniques has provided detailed new observations of C. sordellii spore structural features, which serve as a reference point for structural studies of spores from other bacterial species.

The NEAT Domain-Containing Proteins of Clostridium perfringens Bind Heme.

The ability of a pathogenic bacterium to scavenge iron from its host is important for its growth and survival during an infection. Our studies on C. perfringens gas gangrene strain JIR325, a derivative of strain 13, showed that it is capable of utilizing both human hemoglobin and ferric chloride, but not human holo-transferrin, as an iron source for in vitro growth. Analysis of the C. perfringens strain 13 genome sequence identified a putative heme acquisition system encoded by an iron-regulated surface gene region that we have named the Cht (Clostridium perfringens heme transport) locus. This locus comprises eight genes that are co-transcribed and includes genes that encode NEAT domain-containing proteins (ChtD and ChtE) and a putative sortase (Srt). The ChtD, ChtE and Srt proteins were shown to be expressed in JIR325 cells grown under iron-limited conditions and were localized to the cell envelope. Moreover, the NEAT proteins, ChtD and ChtE, were found to bind heme. Both chtDE and srt mutants were constructed, but these mutants were not defective in hemoglobin or ferric chloride utilization. They were, however, attenuated for virulence when tested in a mouse myonecrosis model, although the virulence phenotype could not be restored via complementation and, as is common with such systems, secondary mutations were identified in these strains. In summary, this study provides evidence for the functional redundancies that occur in the heme transport pathways of this life threatening pathogen.

The Sialidase NanS Enhances Non-TcsL Mediated Cytotoxicity of Clostridium sordellii.

The clostridia produce an arsenal of toxins to facilitate their survival within the host environment. TcsL is one of two major toxins produced by Clostridium sordellii, a human and animal pathogen, and is essential for disease pathogenesis of this bacterium. C. sordellii produces many other toxins, but the role that they play in disease is not known, although previous work has suggested that the sialidase enzyme NanS may be involved in the characteristic leukemoid reaction that occurs during severe disease. In this study we investigated the role of NanS in C. sordellii disease pathogenesis. We constructed a nanS mutant and showed that NanS is the only sialidase produced from C. sordellii strain ATCC9714 since sialidase activity could not be detected from the nanS mutant. Complementation with the wild-type gene restored sialidase production to the nanS mutant strain. Cytotoxicity assays using sialidase-enriched culture supernatants applied to gut (Caco2), vaginal (VK2), and cervical cell lines (End1/E6E7 and Ect1/E6E7) showed that NanS was not cytotoxic to these cells. However, the cytotoxic capacity of a toxin-enriched supernatant to the vaginal and cervical cell lines was substantially enhanced in the presence of NanS. TcsL was not the mediator of the observed cytotoxicity since supernatants harvested from a TcsL-deficient strain displayed similar cytotoxicity levels to TcsL-containing supernatants. This study suggests that NanS works synergistically with an unknown toxin or toxins to exacerbate C. sordellii-mediated tissue damage in the host.

Functional analysis of an feoB mutant in Clostridium perfringens strain 13.

Bacterial pathogens have adopted numerous mechanisms for acquiring iron from host proteins during an infection, including the direct acquisition of ferric iron from heme-associated proteins or from iron-scavenging siderophores. Ferric iron then is transported into the cytosol, where it can be utilized by the bacterial pathogen. Under anaerobic conditions bacteria can also transport ferrous iron using the transmembrane complex FeoAB, but little is known about iron transport systems in anaerobic bacteria such as the pathogenic clostridia. In this study we sought to characterize the iron acquisition process in Clostridium perfringens. Bioinformatic analysis of the Clostridium perfringens strain 13 genome sequence revealed that it has seven potential iron acquisition systems: three siderophore-mediated systems, one ferric citrate uptake system, two heme-associated acquisition systems and one ferrous iron uptake system (FeoAB). The relative level of expression of these systems was determined using quantitative real-time RT-PCR assays that were specific for one gene from each system. Each of these genes was expressed, with the feoAB genes generating the most abundant iron-uptake related transcripts. To further examine the role of this system in the growth of C. perfringens, insertional inactivation was used to isolate a chromosomal feoB mutant. Growth of this mutant in the presence and absence of iron revealed that it had altered growth properties and a markedly reduced total iron and manganese content compared to the wild type; effects that were reversed upon complementation with the wild-type feoB gene. These studies suggest that under anaerobic conditions FeoB is the major protein required for the uptake of iron into the cell and that it may play an important role in the pathogenesis of C. perfringens infections.

Disruption of the Gut Microbiome: Clostridium difficile Infection and the Threat of Antibiotic Resistance.

Clostridium difficile is well recognized as the leading cause of antibiotic-associated diarrhea, having a significant impact in both health-care and community settings. Central to predisposition to C. difficile infection is disruption of the gut microbiome by antibiotics. Being a Gram-positive anaerobe, C. difficile is intrinsically resistant to a number of antibiotics. Mobile elements encoding antibiotic resistance determinants have also been characterized in this pathogen. While resistance to antibiotics currently used to treat C. difficile infection has not yet been detected, it may be only a matter of time before this occurs, as has been seen with other bacterial pathogens. This review will discuss C. difficile disease pathogenesis, the impact of antibiotic use on inducing disease susceptibility, and the role of antibiotic resistance and mobile elements in C. difficile epidemiology.