Heat resistance differences are common between both vegetative cells and spores of Clostridium perfringens type F isolates carrying a chromosomal vs plasmid-borne enterotoxin gene.

Clostridium perfringens type F isolates utilize C. perfringens enterotoxin (CPE) to cause food poisoning (FP) and nonfoodborne gastrointestinal diseases. The enterotoxin gene (cpe) can be located on either the chromosome or plasmids, but most FP isolates carry a chromosomal cpe (c-cpe) gene. Our 2000 article in Applied and Environmental Microbiology (66:3234-3240, 2000, https://doi.org/10.1128/aem.66.8.3234-3240.2000https://doi.org/10.1128/AEM.66.8.3234-3240.2000) determined that vegetative cells and spores of c-cpe isolates are more heat resistant than those of plasmid cpe (p-cpe) isolates, which is favorable for their survival in improperly cooked or held food. However, that 2000 article was recently retracted (90:e00249-24, 2024, https://doi.org/10.1128/aem.00249-24). To our knowledge, the 2000 article remains the only study reporting that heat resistance differences are common between both vegetative cells and spores of type F c-cpe isolates vs type F p-cpe isolates. To confirm and preserve this information in the literature, the heat resistance portion of the 2000 study has been repeated. The 2024 results reproduced the 2000 results by indicating that, relative to the surveyed type F p-cpe isolates, the vegetative cells of surveyed type F c-cpe isolates are ~2-fold more heat resistant and the spores of most surveyed c-cpe isolates are ~30-fold more heat resistant. However, consistent with several reports since our 2000 paper, one surveyed type F c-cpe isolate (which did not appreciably sporulate in 2000 but sporulated in 2024) produced spores with intermediate heat sensitivity, confirming that spores of some type F c-cpe isolates lack exceptional heat resistance.IMPORTANCEClostridium perfringens type F food poisoning (FP), which is the second most common bacterial cause of FP, involves the production of C. perfringens enterotoxin. While the enterotoxin gene (cpe) can be located on either the chromosome or plasmids in type F isolates, most FP cases are caused by chromosomal cpe isolates. The current results support the conclusion that the vegetative cells and spores of type F chromosomal cpe isolates are often more heat resistant than vegetative cells and spores of type F plasmid cpe isolates. Greater heat resistance should favor the survival of the spores and vegetative cells of those chromosomal cpe isolates in temperature-abused food, which may help explain the strong association of type F chromosomal cpe strains with FP.

The presence of differentiated C2C12 muscle cells enhances toxin production and growth by Clostridium perfringens type A strain ATCC3624.

Clostridium perfringens type A causes gas gangrene, which involves muscle infection. Both alpha toxin (PLC), encoded by the plc gene, and perfringolysin O (PFO), encoded by the pfoA gene, are important when type A strains cause gas gangrene in a mouse model. This study used the differentiated C2C12 muscle cell line to test the hypothesis that one or both of those toxins contributes to gas gangrene pathogenesis by releasing growth nutrients from muscle cells. RT-qPCR analyses showed that the presence of differentiated C2C12 cells induces C. perfringens type A strain ATCC3624 to upregulate plc and pfoA expression, as well as increase expression of several regulatory genes, including virS/R, agrB/D, and eutV/W. The VirS/R two component regulatory system (TCRS) and its coupled Agr-like quorum sensing system, along with the EutV/W TCRS (which regulates expression of genes involved in ethanolamine [EA] utilization), were shown to mediate the C2C12 cell-induced increase in plc and pfoA expression. EA was demonstrated to increase toxin gene expression. ATCC3624 growth increased in the presence of differentiated C2C12 muscle cells and this effect was shown to involve both PFO and PLC. Those membrane-active toxins were each cytotoxic for differentiated C2C12 cells, suggesting they support ATCC3624 growth by releasing nutrients from differentiated C2C12 cells. These findings support a model where, during gas gangrene, increased production of PFO and PLC in the presence of muscle cells causes more damage to those host cells, which release nutrients like EA that are then used to support C. perfringens growth in muscle.

The biology and pathogenicity of Clostridium perfringens type F: a common human enteropathogen with a new(ish) name.

SUMMARYIn the 2018-revised Clostridium perfringens typing classification system, isolates carrying the enterotoxin (cpe) and alpha toxin genes but no other typing toxin genes are now designated as type F. Type F isolates cause food poisoning and nonfoodborne human gastrointestinal (GI) diseases, which most commonly involve type F isolates carrying, respectivefooly, a chromosomal or plasmid-borne cpe gene. Compared to spores of other C. perfringens isolates, spores of type F chromosomal cpe isolates often exhibit greater resistance to food environment stresses, likely facilitating their survival in improperly prepared or stored foods. Multiple factors contribute to this spore resistance phenotype, including the production of a variant small acid-soluble protein-4. The pathogenicity of type F isolates involves sporulation-dependent C. perfringens enterotoxin (CPE) production. C. perfringens sporulation is initiated by orphan histidine kinases and sporulation-associated sigma factors that drive cpe transcription. CPE-induced cytotoxicity starts when CPE binds to claudin receptors to form a small complex (which also includes nonreceptor claudins). Approximately six small complexes oligomerize on the host cell plasma membrane surface to form a prepore. CPE molecules in that prepore apparently extend ß-hairpin loops to form a ß-barrel pore, allowing a Ca2+ influx that activates calpain. With low-dose CPE treatment, caspase-3-dependent apoptosis develops, while high-CPE dose treatment induces necroptosis. Those effects cause histologic damage along with fluid and electrolyte losses from the colon and small intestine. Sialidases likely contribute to type F disease by enhancing CPE action and, for NanI-producing nonfoodborne human GI disease isolates, increasing intestinal growth and colonization.

Overexpressing the cpr1953 Orphan Histidine Kinase Gene in the Absence of cpr1954 Orphan Histidine Kinase Gene Expression, or Vice Versa, Is Sufficient to Obtain Significant Sporulation and Strong Production of Clostridium perfringens Enterotoxin or Spo0A by Clostridium perfringens Type F Strain SM101.

The CPR1953 and CPR1954 orphan histidine kinases profoundly affect sporulation initiation and Clostridium perfringens enterotoxin (CPE) production by C. perfringens type F strain SM101, whether cultured in vitro (modified Duncan-Strong sporulation medium (MDS)) or ex vivo (mouse small intestinal contents (MIC)). To help distinguish whether CPR1953 and CPR1954 act independently or in a stepwise manner to initiate sporulation and CPE production, cpr1953 and cpr1954 null mutants of SM101 were transformed with plasmids carrying the cpr1954 or cpr1953 genes, respectively, causing overexpression of cpr1954 in the absence of cpr1953 expression and vice versa. RT-PCR confirmed that, compared to SM101, the cpr1953 mutant transformed with a plasmid encoding cpr1954 expressed cpr1954 at higher levels while the cpr1954 mutant transformed with a plasmid encoding cpr1953 expressed higher levels of cpr1953. Both overexpressing strains showed near wild-type levels of sporulation, CPE toxin production, and Spo0A production in MDS or MIC. These findings suggest that CPR1953 and CPR1954 do not function together in a step-wise manner, e.g., as a novel phosphorelay. Instead, it appears that, at natural expression levels, the independent kinase activities of both CPR1953 and CPR1954 are necessary for obtaining sufficient Spo0A production and phosphorylation to initiate sporulation and CPE production.

The impact of orphan histidine kinases and phosphotransfer proteins on the regulation of clostridial sporulation initiation.

Sporulation is an important feature of the clostridial life cycle, facilitating survival of these bacteria in harsh environments, contributing to disease transmission for pathogenic species, and sharing common early steps that are also involved in regulating industrially important solvent production by some non-pathogenic species. Initial genomics studies suggested that Clostridia lack the classical phosphorelay that phosphorylates Spo0A and initiates sporulation in Bacillus, leading to the hypothesis that sporulation in Clostridia universally begins when Spo0A is phosphorylated by orphan histidine kinases (OHKs). However, components of the classical Bacillus phosphorelay were recently identified in some Clostridia. Similar Bacillus phosphorelay components have not yet been found in the pathogenic Clostridia or the solventogenic Clostridia of industrial importance. For some of those Clostridia lacking a classical phosphorelay, the involvement of OHKs in sporulation initiation has received support from genetic studies demonstrating the involvement of several apparent OHKs in their sporulation. In addition, several clostridial OHKs directly phosphorylate Spo0A in vitro. Interestingly, there is considerable protein domain diversity among the sporulation-associated OHKs in Clostridia. Further adding to the emergent complexity of sporulation initiation in Clostridia, several candidate OHK phosphotransfer proteins that were OHK candidates were shown to function as phosphatases that reduce sporulation in some Clostridia. The mounting evidence indicates that no single pathway explains sporulation initiation in all Clostridia and supports the need for further study to fully understand the unexpected and biologically fascinating mechanistic diversity of this important process among these medically and industrially important bacteria.

Identification of orphan histidine kinases that impact sporulation and enterotoxin production by Clostridium perfringens type F strain SM101 in a pathophysiologically-relevant ex vivo mouse intestinal contents model.

When causing food poisoning or antibiotic-associated diarrhea, Clostridium perfringens type F strains must sporulate to produce C. perfringens enterotoxin (CPE) in the intestines. C. perfringens is thought to use some of its seven annotated orphan histidine kinases to phosphorylate Spo0A and initiate sporulation and CPE production. We previously demonstrated the CPR0195 orphan kinase, but not the putative CPR1055 orphan kinase, is important when type F strain SM101 initiates sporulation and CPE production in modified Duncan-Strong (MDS) sporulation medium. Since there is no small animal model for C. perfringens sporulation, the current study used diluted mouse intestinal contents (MIC) to develop an ex vivo sporulation model and employed this model to test sporulation and CPE production by SM101 CPR0195 and CPR1055 null mutants in a pathophysiologically-relevant context. Surprisingly, both mutants still sporulated and produced CPE at wild-type levels in MIC. Therefore, five single null mutants were constructed that cannot produce one of the previously-unstudied putative orphan kinases of SM101. Those mutants implicated CPR1316, CPR1493, CPR1953 and CPR1954 in sporulation and CPE production by SM101 MDS cultures. Phosphorylation activity was necessary for CPR1316, CPR1493, CPR1953 and CPR1954 to affect sporulation in those MDS cultures, supporting their identity as kinases. Importantly, only the CPR1953 or CPR1954 null mutants exhibited significantly reduced levels of sporulation and CPE production in MIC cultures. These phenotypes were reversible by complementation. Characterization studies suggested that, in MDS or MIC, the CPR1953 and CPR1954 mutants produce less Spo0A than wild-type SM101. In addition, the CPR1954 mutant exhibited little or no Spo0A phosphorylation in MDS cultures. These studies, i) highlight the importance of using pathophysiologically-relevant models to investigate C. perfringens sporulation and CPE production in a disease context and ii) link the CPR1953 and CPR1954 kinases to C. perfringens sporulation and CPE production in disease-relevant conditions.

NanJ Is the Major Sialidase for Clostridium perfringens Type F Food Poisoning Strain 01E809.

Clostridium perfringens type F strains cause food poisoning (FP) when they sporulate and produce C. perfringens enterotoxin (CPE) in the intestines. Most type F FP strains carry a chromosomal cpe gene (c-cpe strains). C. perfringens produces up to three different sialidases, named NanH, NanI, and NanJ, but some c-cpe FP strains carry only nanJ and nanH genes. This study surveyed a collection of such strains and showed that they produce sialidase activity when cultured in Todd-Hewitt broth (TH) (vegetative cultures) or modified Duncan-Strong (MDS) medium (sporulating cultures). Sialidase null mutants were constructed in 01E809, a type F c-cpe FP strain carrying the nanJ and nanH genes. Characterization of those mutants identified NanJ as the major sialidase of 01E809 and showed that, in vegetative and sporulating cultures, nanH expression affects nanJ expression and vice versa; those regulatory effects may involve media-dependent changes in transcription of the codY or ccpA genes but not nanR. Additional characterization of these mutants demonstrated the following: (i) NanJ contributions to growth and vegetative cell survival are media dependent, with this sialidase increasing 01E809 growth in MDS but not TH; (ii) NanJ enhances 24-h vegetative cell viability in both TH and MDS cultures; and (iii) NanJ is important for 01E809 sporulation and, together with NanH, CPE production in MDS cultures. Lastly, NanJ was shown to increase CPE-induced cytotoxicity and CH-1 pore formation in Caco-2 cells. Collectively, these results suggest that NanJ may have a contributory role in FP caused by type F c-cpe strains that carry the nanH and nanJ genes.

Epsilon toxin-producing Clostridium perfringens colonize the multiple sclerosis gut microbiome overcoming CNS immune privilege.

Multiple sclerosis (MS) is a complex disease of the CNS thought to require an environmental trigger. Gut dysbiosis is common in MS, but specific causative species are unknown. To address this knowledge gap, we used sensitive and quantitative PCR detection to show that people with MS were more likely to harbor and show a greater abundance of epsilon toxin-producing (ETX-producing) strains of C. perfringens within their gut microbiomes compared with individuals who are healthy controls (HCs). Isolates derived from patients with MS produced functional ETX and had a genetic architecture typical of highly conjugative plasmids. In the active immunization model of experimental autoimmune encephalomyelitis (EAE), where pertussis toxin (PTX) is used to overcome CNS immune privilege, ETX can substitute for PTX. In contrast to PTX-induced EAE, where inflammatory demyelination is largely restricted to the spinal cord, ETX-induced EAE caused demyelination in the corpus callosum, thalamus, cerebellum, brainstem, and spinal cord, more akin to the neuroanatomical lesion distribution seen in MS. CNS endothelial cell transcriptional profiles revealed ETX-induced genes that are known to play a role in overcoming CNS immune privilege. Together, these findings suggest that ETX-producing C. perfringens strains are biologically plausible pathogens in MS that trigger inflammatory demyelination in the context of circulating myelin autoreactive lymphocytes.

Characterization of NanR Regulation of Sialidase Production, Sporulation and Enterotoxin Production by Clostridium perfringens Type F Strains Carrying a Chromosomal Enterotoxin Gene.

Clostridium perfringens type F food poisoning (FP) strains produce C. perfringens enterotoxin (CPE) to cause a common bacterial food-borne illness in the United States. During FP, CPE is synthesized in the intestines when C. perfringens sporulates. Besides CPE, FP strains also produce sialidases. Most FP strains carry their cpe gene on the chromosome and all surveyed chromosomal cpe (c-cpe) FP strains produce NanH sialidase or both NanJ and NanH sialidases. NanR has been shown previously to regulate sialidase activity in non-FP strains. The current study investigated whether NanR also regulates sialidase activity or influences sporulation and CPE production for c-cpe FP strains SM101 and 01E809. In sporulation medium, the SM101 nanR null mutant showed lower sialidase activity, sporulation, and CPE production than its wild-type parent, while the 01E809 nanR null mutant showed roughly similar sialidase activity, sporulation, and CPE production as its parent. In vegetative medium, the nanR null mutants of both strains produced more spores than their parents while NanR repressed sialidase activity in SM101 but positively regulated sialidase activity in 01E809. These results demonstrate that NanR regulates important virulence functions of c-cpe strains, with this control varying depending on strain and culture conditions.

Characterizing the Contributions of Various Clostridium perfringens Enterotoxin Properties to In Vivo and In Vitro Permeability Effects.

Clostridium perfringens enterotoxin (CPE) is thought to cause lethal enterotoxemia when absorbed from the intestinal lumen into the circulation. CPE action sequentially involves receptor-binding, oligomerization into a prepore, and pore formation. To explore the mechanistic basis by which CPE alters permeability, this study tested the permeability effects of several recombinant CPE (rCPE) species: rCPE and rCPEC186A (which form pores), rC-CPE and rCPED48A (which bind to receptors but cannot oligomerize), rCPEC186A/F91C (which binds and oligomerizes without pore formation), and rCPEY306A/L315A (which has poor receptor-binding ability). On Caco-2 cells, i) only rCPE and rCPEC186A were cytotoxic; ii) rCPE and rCPEC186A affected transepithelial resistance (TEER) and 4 kDa fluorescent dextran (FD4) transit more quickly than binding-capable, but noncytotoxic, rCPE variants; whereas iii) rCPEY306A/L315A did not affect TEER or FD4 transit. Using mouse intestinal loops, rCPE (but not noncytotoxic rC-CPE, rCPED48A or rCPEY306A/L315A) was lethal and caused intestinal histologic damage within 4 h. After 2 h of treatment, rCPE was more strongly absorbed into the serum than those noncytotoxic rCPE species but by 4 h rC-CPE and rCPED48A became absorbed similarly as rCPE, while rCPEY306A/L315A absorption remained low. This increased rC-CPE and rCPED48A absorption from 2 to 4 h did not involve a general intestinal permeability increase because Evans Blue absorption from the intestines did not increase between 2 and 4 h of treatment with rC-CPE or rCPED48A. Collectively, these results indicate that CPE receptor binding is sufficient to slowly affect permeability, but CPE-induced cytotoxicity is necessary for rapid permeability changes and lethality. IMPORTANCE Clostridium perfringens enterotoxin (CPE) causes lethal enterotoxemia when absorbed from the intestines into the bloodstream. Testing recombinant CPE (rCPE) or rCPE variants impaired for various specific steps in CPE action showed that full CPE-induced cytotoxicity causes rapid Caco-2 monolayer permeability alterations, as well as enterotoxemic lethality and rapid CPE absorption in mouse small intestinal loops. However, receptor binding-capable, but noncytotoxic, rCPE variants did cause slow-developing in vitro and in vivo permeability effects. Absorption of binding-capable, noncytotoxic rCPE variants from the intestines did not correlate with general intestinal permeability alterations, suggesting that CPE binding can induce its own uptake. These findings highlight the importance of binding and, especially, cytotoxicity for CPE absorption during enterotoxemia and may assist development of permeability-altering rCPE variants for translational purposes.

Reevaluation of whether a Functional Agr-like Quorum-Sensing System Is Necessary for Production of Wild-Type Levels of Epsilon-Toxin by Clostridium perfringens Type D Strains.

Clostridium perfringens type B and D strains produce epsilon-toxin (ETX). Our 2011 mBio study (mBio 2:e00275-11, 2011, https://doi.org/10.1128/mBio.00275-11) reported that the Agr quorum-sensing (QS) system regulates ETX production by type D strain CN3718. However, subsequent studies have brought that conclusion into question. For example, we reported in 2012 (Infect Immun 80:3008-3017, 2012, https://doi.org/10.1128/IAI.00438-12) that the Agr-like QS system is not required for wild-type ETX production levels by two type B strains. Consequently, we reexamined whether the Agr-like QS system regulates ETX production in type D strains by using Targetron insertional mutagenesis to construct new agrB null mutants of two type D strains, CN3718 and CN2068. Western blotting showed that both agrB mutants still produce wild-type ETX levels. However, the newly constructed agrB mutants of both type D strains produced reduced amounts of alpha-toxin, and this effect was reversible by complementation, which confirms loss of functional AgrB production by these mutants since alpha-toxin production is known to be regulated by AgrB. Coupled with the previously published results for type B strains, these new findings indicate the Agr-like QS system is not usually necessary for C. perfringens to produce wild-type ETX levels. IMPORTANCE Since epsilon-toxin (ETX) is necessary for the virulence of C. perfringens type D and, likely, type B strains, understanding the regulation of ETX production is important. In 2011, we reported that an agrB null mutant of type D strain CN3718 produces less ETX than its wild-type parent. However, when new agrB mutants were constructed in type D strains CN3718 and C2068, ETX production was unaffected. Those newly constructed agrB mutants produced less alpha-toxin, and this phenotype was reversible by complementation, confirming construction of agrB null mutants since alpha-toxin production is regulated by AgrB. Coupled with previous results for type B strains, these new type D results support the conclusion that the Agr QS is not usually necessary for wild-type ETX production levels.

NanI Sialidase Enhances the Action of Clostridium perfringens Enterotoxin in the Presence of Mucus.

Clostridium perfringens enterotoxin (CPE) is the main virulence factor for C. perfringens type F strains to cause human gastrointestinal diseases, which can involve lethal enterotoxemia. During type F disease, CPE encounters an adherent mucus layer overlying the intestines, so the current study evaluated if NanI potentiates CPE activity in the presence of adherent mucus. CPE alone caused more cytotoxicity transepithelial electrical resistance (TEER) and permeability to fluorescent dextran (FD) for minimal mucus-producing HT29 cells versus that in their derivative HT29-MTX-E12 cells, which produce abundant adherent mucus. However, for HT29-MTX-E12 cells, the presence of NanI significantly increased CPE binding and pore formation, which enhanced their sensitivity to CPE effects on cytotoxicity, TEER, and FD permeability. When the ability of NanI to potentiate CPE-induced enterotoxemia was then tested in a mouse small intestinal loop enterotoxemia model, a pathophysiologically relevant 50 µg/mL dose of CPE did not kill mice. However, the copresence of purified NanI resulted in significant CPE-induced lethality. More CPE was detected in the sera of mice challenged with 50 µg/mL of CPE when NanI was copresent during challenge. The copresence of NanI and CPE during challenge also significantly increased intestinal histologic damage compared to that after challenge with CPE alone, suggesting that NanI enhancement of CPE-induced intestinal damage may increase CPE absorption into blood. Overall, these results indicate that (i) mucus inhibits CPE action and (ii) NanI can potentiate CPE action in the presence of mucus, which may help explain why type F strains that produce relatively low levels of CPE are still pathogenic. IMPORTANCE NanI is a sialidase produced by some Clostridium perfringens type F strains. Here, we found that NanI can significantly increase the action of C. perfringens enterotoxin (CPE), which is the main toxin responsible for severe human enteric disease caused by type F strains. This effect likely helps to explain why even some type F strains that produce small amounts of CPE are pathogenic.

NanI Sialidase Contributes to the Growth and Adherence of Clostridium perfringens Type F Strain F4969 in the Presence of Adherent Mucus.

Clostridium perfringens type F strains causing nonfoodborne human gastrointestinal diseases (NFD) typically produce NanI sialidase as their major secreted sialidase. Type F NFDs can persist for several weeks, indicating their pathogenesis involves intestinal colonization, including vegetative cell growth and adherence, with subsequent sporulation that fosters enterotoxin production and release. We previously reported that NanI contributes to type F NFD strain adherence and growth using Caco-2 cells. However, Caco-2 cells make minimal amounts of mucus, which is significant because the intestines are coated with adherent mucus. Therefore, it was important to assess if NanI contributes to the growth and adherence of type F NFD strains in the presence of adherent mucus. Consequently, the current study first demonstrated greater growth of nanI-carrying versus non-nanI-carrying type F strains in the presence of HT29-MTX-E12 cells, which produce an adherent mucus layer, versus their parental HT29 cells, which make minimal mucus. Demonstrating the specific importance of NanI for this effect, type F NFD strain F4969 or a complementing strain grew and adhered better than an isogenic nanI null mutant in the presence of HT29-MTX-E12 cells versus HT29 cells. Those effects involved mucus production by HT29-MTX-E12 cells since mucus reduction using N-acetyl cysteine reduced F4969 growth and adherence. Consistent with those in vitro results, NanI contributed to growth of F4969 in the mouse small intestine. By demonstrating a growth and adherence role for NanI in the presence of adherent mucus, these results further support NanI as a potential virulence factor during type F NFDs.

Identifying the Basis for VirS/VirR Two-Component Regulatory System Control of Clostridium perfringens Beta-Toxin Production.

Clostridium perfringens toxin production is often regulated by the Agr-like quorum sensing (QS) system signaling the VirS/VirR two-component regulatory system (TCRS), which consists of the VirS membrane sensor histidine kinase and the VirR response regulator. VirS/VirR is known to directly control expression of some genes by binding to a DNA binding motif consisting of two VirR boxes located within 500 bp of the target gene start codon. Alternatively, the VirS/VirR system can indirectly regulate production levels of other proteins by increasing expression of a small regulatory RNA, VR-RNA. Previous studies demonstrated that C. perfringens beta-toxin (CPB) production by C. perfringens type B and C strains is positively regulated by both the Agr-like QS and the VirS/VirR TCRS, but the mechanism has been unclear. The current study first inactivated the vrr gene encoding VR-RNA to show that VirS/VirR regulation of cpb expression does not involve VR-RNA. Subsequently, bioinformatic analyses identified a potential VirR binding motif, along with a predicted strong promoter, ∼1.4 kb upstream of the cpb open reading frame (ORF). Two insertion sequences were present between this VirR binding motif/promoter region and the cpb ORF. PCR screening of a collection of strains carrying cpb showed that the presence and sequence of this VirR binding motif/promoter is highly conserved among CPB-producing strains. Reverse transcription-PCR (RT-PCR) and a GusA reporter assay showed this VirR binding motif is important for regulating CPB production. These findings indicate that VirS/VirR directly regulates cpb expression via VirS binding to a VirR binding motif located unusually distant from the cpb start codon. IMPORTANCE Clostridium perfringens beta-toxin (CPB) is only produced by type B and C strains. Production of CPB is essential for the pathogenesis of type C-associated infections, which include hemorrhagic necrotizing enteritis and enterotoxemia in both humans and animals. In addition, CPB can synergize with other toxins during C. perfringens gastrointestinal diseases. CPB toxin production is cooperatively regulated by the Agr-like quorum sensing (QS) system and the VirS/VirR two-component regulatory system. This study now reports that the VirS/VirR regulatory cascade directly controls expression of the cpb gene via a process involving a VirR box binding motif located unusually far (∼1.4 kb) upstream of the cpb ORF. This study provides a better understanding of the regulatory mechanisms for CPB production by the VirS/VirR regulatory cascade.

NanH Is Produced by Sporulating Cultures of Clostridium perfringens Type F Food Poisoning Strains and Enhances the Cytotoxicity of C. perfringens Enterotoxin.

Clostridium perfringens type F food poisoning (FP) strains cause one of the most common foodborne illnesses. This FP develops when type F FP strains sporulate in the intestines and produce C. perfringens enterotoxin (CPE), which is responsible for the diarrhea and abdominal cramps of this disease. While C. perfringens can produce up to three different sialidases, the current study surveyed FP strains, which confirmed the results of a previous study that they consistently carry the nanH sialidase gene, often as their only sialidase gene. NanH production was found to be associated with sporulating cultures of the surveyed type F FP strains, including SM101 (a transformable derivative of a FP strain). The sporulation-associated regulation of NanH production by strain SM101 growing in modified Duncan-Strong medium (MDS) was shown to involve Spo0A, but it did not require the completion of sporulation. NanH production was not necessary for either the growth or sporulation of SM101 when cultured in MDS. In those MDS cultures, NanH accumulated in the sporulating mother cell until it was released coincidently with CPE. Since CPE becomes extracellular when mother cells lyse to release their mature spores, this indicates that mother cell lysis is also important for NanH release. The copresence of NanH and CPE in supernatants from lysed sporulating cultures was shown to enhance CPE cytotoxicity for Caco-2 cells. This enhancement was attributable to NanH increasing CPE binding and could be replicated with purified recombinant NanH. These in vitro findings suggest that NanH may be an accessory virulence factor during type F FP.IMPORTANCEClostridium perfringens type F strains cause the second most common bacterial foodborne illness in the United States. C. perfringens enterotoxin (CPE) is responsible for the diarrhea and cramping symptoms of this food poisoning (FP). Previous studies showed that NanI sialidase can enhance CPE activity in vitro While many type F FP strains do not produce NanI, they do consistently make NanH sialidase. This study shows that, like CPE, NanH is produced by sporulating type F FP strains and then released extracellularly when their sporulating cells lyse to release their mature spore. NanH was shown to enhance CPE cytotoxicity in vitro by increasing CPE binding to cultured Caco-2 cells. This enhancement could be important because many type F FP strains produce less CPE than necessary (in a purified form) to cause intestinal pathology in animal models. Therefore, NanH represents a potential accessory virulence factor for type F FP.

Evidence That VirS Is a Receptor for the Signaling Peptide of the Clostridium perfringens Agr-like Quorum Sensing System.

Since both the Agr (accessory gene regulator)-like quorum sensing (QS) system and VirS/VirR (VirS/R) two-component regulatory system of Clostridium perfringens positively regulate production of several toxins, including C. perfringens beta toxin (CPB), it has been hypothesized the VirS membrane sensor protein is an Agr-like QS signaling peptide (SP) receptor. To begin evaluating whether VirS is an SP receptor, this study sequenced the virS gene in C. perfringens strains CN3685 and CN1795 because it was reported that agrB mutants of both strains increase CPB production in response to the pentapeptide 5R, likely the natural SP, but only the CN3685 agrB mutant responds to 8R, which is 5R plus a 3-amino-acid tail. This sequencing identified differences between the predicted VirS extracellular loop 2 (ECL2) of CN3685 versus that of CN1795. To explore if those ECL2 differences explain strain-related variations in SP sensitivity and support VirS as an SP receptor, virS agrB double-null mutants of each strain were complemented to swap which VirS protein they produce. CPB Western blotting showed that this complementation changed the natural responsiveness of each strain to 8R. A pulldown experiment using biotin-5R demonstrated that VirS can bind SP. To further support VirS:SP binding and to identify a VirS binding site for SP, a 14-mer peptide corresponding to VirS ECL2 was synthesized. This ECL2 peptide inhibited 5R signaling to agrB mutant and wild-type strains. This inhibition was specific, since a single N to D substitution in the ECL2 peptide abrogated these effects. Collectively, these results support VirS as an important SP receptor and may assist development of therapeutics.IMPORTANCEC. perfringens beta toxin (CPB) is essential for the virulence of type C strains, a common cause of fatal necrotizing enteritis and enterotoxemia in humans and domestic animals. Production of CPB, as well as several other C. perfringens toxins, is positively regulated by both the Agr-like QS system and the VirS/R two-component regulatory system. This study presents evidence that the VirS membrane sensor protein is a receptor for the AgrD-derived SP and that the second extracellular loop of VirS is important for SP binding. Understanding interactions between SP and VirS improves knowledge of C. perfringens pathogenicity and may provide insights for designing novel strategies to reduce C. perfringens toxin production during infections.