scholarly journals Inorganic Phosphate and Sodium Ions Are Cogerminants for Spores of Clostridium perfringens Type A Food Poisoning-Related Isolates

2009 ◽  
Vol 75 (19) ◽  
pp. 6299-6305 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
Pathima Udompijitkul ◽  
Mahfuzur R. Sarker
Keyword(s):  
Clostridium Perfringens ◽  
Inorganic Phosphate ◽  
Dipicolinic Acid ◽  
Food Poisoning ◽  
Type A ◽  
Gastrointestinal Diseases ◽  
Wild Type ◽  
Similar Extent ◽  
Active Growth ◽  
Food Borne

ABSTRACT Clostridium perfringens type A isolates carrying a chromosomal copy of the enterotoxin (cpe) gene are involved in the majority of food poisoning (FP) outbreaks, while type A isolates carrying a plasmid-borne cpe gene are involved in C. perfringens-associated non-food-borne (NFB) gastrointestinal diseases. To cause diseases, C. perfringens spores must germinate and return to active growth. Previously, we showed that only spores of FP isolates were able to germinate with K+ ions. We now found that the spores of the majority of FP isolates, but none of the NFB isolates, germinated with the cogerminants Na+ and inorganic phosphate (NaPi) at a pH of ∼6.0. Spores of gerKA-KC and gerAA mutants germinated to a lesser extent and released less dipicolinic acid (DPA) than did wild-type spores with NaPi. Although gerKB spores germinated to a similar extent as wild-type spores with NaPi, their rate of germination was lower. Similarly, gerO and gerO gerQ mutant spores germinated slower and released less DPA than did wild-type spores with NaPi. In contrast, gerQ spores germinated to a slightly lesser extent than wild-type spores but released all of their DPA during NaPi germination. In sum, this study identified NaPi as a novel nutrient germinant for spores of most FP isolates and provided evidence that proteins encoded by the gerKA-KC operon, gerAA, and gerO are required for NaPi-induced spore germination.

scholarly journals Clostridium perfringens Spore Germination: Characterization of Germinants and Their Receptors

2007 ◽  
Vol 190 (4) ◽  
pp. 1190-1201 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
J. Antonio Torres ◽  
Peter Setlow ◽  
Mahfuzur R. Sarker
Keyword(s):  
Clostridium Perfringens ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Food Poisoning ◽  
Wild Type ◽  
Active Growth ◽  
Food Borne ◽  
High Concentrations ◽  
Auxiliary Role

ABSTRACT Clostridium perfringens food poisoning is caused by type A isolates carrying a chromosomal enterotoxin (cpe) gene (C-cpe), while C. perfringens-associated non-food-borne gastrointestinal (GI) diseases are caused by isolates carrying a plasmid-borne cpe gene (P-cpe). C. perfringens spores are thought to be the important infectious cell morphotype, and after inoculation into a suitable host, these spores must germinate and return to active growth to cause GI disease. We have found differences in the germination of spores of C-cpe and P-cpe isolates in that (i) while a mixture of l-asparagine and KCl was a good germinant for spores of C-cpe and P-cpe isolates, KCl and, to a lesser extent, l-asparagine triggered spore germination in C-cpe isolates only; and (ii) l-alanine or l-valine induced significant germination of spores of P-cpe but not C-cpe isolates. Spores of a gerK mutant of a C-cpe isolate in which two of the proteins of a spore nutrient germinant receptor were absent germinated slower than wild-type spores with KCl, did not germinate with l-asparagine, and germinated poorly compared to wild-type spores with the nonnutrient germinants dodecylamine and a 1:1 chelate of Ca2+ and dipicolinic acid. In contrast, spores of a gerAA mutant of a C-cpe isolate that lacked another component of a nutrient germinant receptor germinated at the same rate as that of wild-type spores with high concentrations of KCl, although they germinated slightly slower with a lower KCl concentration, suggesting an auxiliary role for GerAA in C. perfringens spore germination. In sum, this study identified nutrient germinants for spores of both C-cpe and P-cpe isolates of C. perfringens and provided evidence that proteins encoded by the gerK operon are required for both nutrient-induced and non-nutrient-induced spore germination.

scholarly journals Organization of the Plasmid cpe Locus in Clostridium perfringens Type A Isolates

2002 ◽  
Vol 70 (8) ◽  
pp. 4261-4272 ◽  
Author(s):  
Kazuaki Miyamoto ◽  
Ganes Chakrabarti ◽  
Yosiharu Morino ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Horizontal Transfer ◽  
Food Poisoning ◽  
Type A ◽  
Gastrointestinal Diseases ◽  
Open Reading Frame ◽  
Enterotoxin Gene ◽  
Genetic Element ◽  
Reading Frame ◽  
Food Borne

ABSTRACT Clostridium perfringens type A isolates causing food poisoning have a chromosomal enterotoxin gene (cpe), while C. perfringens type A isolates responsible for non-food-borne human gastrointestinal diseases carry a plasmid cpe gene. In the present study, the plasmid cpe locus of the type A non-food-borne-disease isolate F4969 was sequenced to design primers and probes for comparative PCR and Southern blot studies of the cpe locus in other type A isolates. Those analyses determined that the region upstream of the plasmid cpe gene is highly conserved among type A isolates carrying a cpe plasmid. The organization of the type A plasmid cpe locus was also found to be unique, as it contains IS1469 sequences located similarly to those in the chromosomal cpe locus but lacks the IS1470 sequences found upstream of IS1469 in the chromosomal cpe locus. Instead of those upstream IS1470 sequences, a partial open reading frame potentially encoding cytosine methylase (dcm) was identified upstream of IS1469 in the plasmid cpe locus of all type A isolates tested. Similar dcm sequences were also detected in several cpe-negative C. perfringens isolates carrying plasmids but not in type A isolates carrying a chromosomal cpe gene. Contrary to previous reports, sequences homologous to IS1470, rather than IS1151, were found downstream of the plasmid cpe gene in most type A isolates tested. Those IS1470-like sequences reside in about the same position but are oppositely oriented and defective relative to the IS1470 sequences found downstream of the chromosomal cpe gene. Collectively, these and previous results suggest that the cpe plasmid of many type A isolates originated from integration of a cpe-containing genetic element near the dcm sequences of a C. perfringens plasmid. The similarity of the plasmid cpe locus in many type A isolates is consistent with horizontal transfer of a common cpe plasmid among C. perfringens type A strains.

scholarly journals Complete Sequencing and Diversity Analysis of the Enterotoxin-Encoding Plasmids in Clostridium perfringens Type A Non-Food-Borne Human Gastrointestinal Disease Isolates

2006 ◽  
Vol 188 (4) ◽  
pp. 1585-1598 ◽  
Author(s):  
Kazuaki Miyamoto ◽  
Derek J. Fisher ◽  
Jihong Li ◽  
Sameera Sayeed ◽  
Shigeru Akimoto ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Gastrointestinal Disease ◽  
Food Poisoning ◽  
Variable Region ◽  
Type A ◽  
Gastrointestinal Diseases ◽  
Open Reading Frames ◽  
Toxin Gene ◽  
Virulence Plasmids ◽  
Food Borne

ABSTRACT Enterotoxin-producing Clostridium perfringens type A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carrying cpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lacking cpb2 and IS1151 but carrying cpe and IS1470-like sequences. By completely sequencing these two cpe plasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an ∼35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon Tn916. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the ∼43.6-kb pCPF5603 variable region contains a functional cpb2 gene and several metabolic genes. Diversity studies indicated that other type A plasmid cpe +/IS1151 SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmid cpe +/IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in the cpe plasmids of other type A SPOR/AAD isolates, as well as in representative C. perfringens type B to D isolates carrying other virulence plasmids, possibly suggesting that most or all C. perfringens virulence plasmids transfer conjugatively.

scholarly journals Evidence That the Enterotoxin Gene Can Be Episomal in Clostridium perfringens Isolates Associated with Non-Food-Borne Human Gastrointestinal Diseases

1998 ◽  
Vol 36 (1) ◽  
pp. 30-36 ◽  
Author(s):  
Renee E. Collie ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Gel Electrophoresis ◽  
Fragment Length Polymorphism ◽  
Food Poisoning ◽  
Type A ◽  
Gastrointestinal Diseases ◽  
Pulsed Field Gel Electrophoresis ◽  
Transmission Mechanisms ◽  
Food Borne ◽  
Antibiotic Associated Diarrhea

Clostridium perfringens enterotoxin (CPE) is responsible for the diarrheal and cramping symptoms of human C. perfringens type A food poisoning. CPE-producing C. perfringens isolates have also recently been associated with several non-food-borne human gastrointestinal (GI) illnesses, including antibiotic-associated diarrhea and sporadic diarrhea. The current study has used restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) analyses to compare the genotypes of 43 cpe-positive C. perfringensisolates obtained from diverse sources. All North American and European food-poisoning isolates examined in this study were found to carry a chromosomal cpe, while all non-food-borne human GI disease isolates characterized in this study were determined to carry their cpe on an episome. Collectively, these results provide the first evidence that distinct subpopulations ofcpe-positive C. perfringens isolates may be responsible for C. perfringens type A food poisoning versus CPE-associated non-food-borne human GI diseases. If these putative associations are confirmed in additional surveys, cpe RFLP and PFGE genotyping assays may facilitate the differential diagnosis of food-borne versus non-food-borne CPE-associated human GI illnesses and may also be useful epidemiologic tools for identifying reservoirs or transmission mechanisms for the subpopulations ofcpe-positive isolates specifically responsible for CPE-associated food-borne versus non-food-borne human GI diseases.

Effect of the cortex-lytic enzyme SleC from non-food-borne Clostridium perfringens on the germination properties of SleC-lacking spores of a food poisoning isolate

2010 ◽  
Vol 56 (11) ◽  
pp. 952-958 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
Mahfuzur R. Sarker
Keyword(s):  
Clostridium Perfringens ◽  
Spore Germination ◽  
Type Strain ◽  
Wild Type Strain ◽  
Dipicolinic Acid ◽  
Food Poisoning ◽  
Lytic Enzyme ◽  
Wild Type ◽  
Lytic Enzymes ◽  
Food Borne

The hallmark of bacterial spore germination is peptidoglycan cortex hydrolysis by cortex-lytic enzymes. In spores of Clostridium perfringens wild-type strain SM101, which causes food poisoning, the sole essential cortex-lytic enzyme SleC is activated by a unique serine protease CspB. Interestingly, the non-food-borne wild-type strain F4969 encodes a significantly divergent SleC variant (SleCF4969) and 3 serine proteases (CspA, CspB, and CspC). Consequently, in this study we evaluated the functional compatibility of SleCF4969and SleCSM101by complementing the germination phenotypes of SM101ΔsleC spores with sleCF4969. Our results show that although pro-SleCF4969was processed into mature SleCF4969in the SM101ΔsleC spores, it partially restored spore germination with nutrient medium, with a mixture of l-asparagine and KCl, or with a 1:1 chelate of Ca2+and dipicolinic acid. While the amount of dipicolinic acid released was lower, the amount of hexosamine-containing material released during germination of SM101ΔsleC(sleCF4969) spores was similar to the amount released during germination of SM101 wild-type spores. The viability of SM101ΔsleC(sleCF4969) spores was 8- and 3-fold lower than that of SM101 and F4969 spores, respectively. Together, these data indicate that the peptidoglycan cortex hydrolysis machinery in the food poisoning isolate SM101 is functionally divergent than that in the non-food-borne isolate F4969.

scholarly journals Prevalence of Enterotoxigenic Clostridium perfringens Isolates in Pittsburgh (Pennsylvania) Area Soils and Home Kitchens

2007 ◽  
Vol 73 (22) ◽  
pp. 7218-7224 ◽  
Author(s):  
Jihong Li ◽  
Sameera Sayeed ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Type A ◽  
Gastrointestinal Diseases ◽  
The United States ◽  
Soil Samples ◽  
Promoter Regions ◽  
Food Borne ◽  
Upstream Promoter ◽  
Type C

ABSTRACT In the United States and Europe, food poisoning due to Clostridium perfringens type A is predominantly caused by C. perfringens isolates carrying a chromosomal enterotoxin gene (cpe). Neither the reservoir for these isolates nor the point in the food chain where these bacteria contaminate foods is currently understood. Therefore, the current study investigated whether type A isolates carrying a chromosomal cpe gene are present in two potential reservoirs, i.e., soil and home kitchen surfaces. No C. perfringens isolates were recovered from home kitchen surfaces, but most surveyed soil samples contained C. perfringens. The recovered soil isolates were predominantly type A, but some type C, D, and E soil isolates were also identified. All cpe-positive isolates recovered from soil were genotyped as type A, with their cpe genes on cpe plasmids rather than the chromosome. However, two cpe-positive soil isolates did not carry a classical cpe plasmid. Both of those atypical cpe-positive soil isolates were sporulation capable yet failed to produce C. perfringens enterotoxin, possibly because of differences in their upstream promoter regions. Collectively these results suggest that neither soil nor home kitchen surfaces represent major reservoirs for type A isolates with chromosomal cpe that cause food poisoning, although soil does appear to be a reservoir for cpe-positive isolates causing non-food-borne gastrointestinal diseases.

scholarly journals Characterization of Clostridium perfringens Spores That Lack SpoVA Proteins and Dipicolinic Acid

2008 ◽  
Vol 190 (13) ◽  
pp. 4648-4659 ◽  
Author(s):  
Daniel Paredes-Sabja ◽  
Barbara Setlow ◽  
Peter Setlow ◽  
Mahfuzur R. Sarker
Keyword(s):  
Water Content ◽  
Uv Radiation ◽  
Clostridium Perfringens ◽  
Spore Germination ◽  
Gastrointestinal Disease ◽  
Dipicolinic Acid ◽  
High Heat ◽  
Wild Type ◽  
Active Growth ◽  
Moist Heat

ABSTRACT Spores of Clostridium perfringens possess high heat resistance, and when these spores germinate and return to active growth, they can cause gastrointestinal disease. Work with Bacillus subtilis has shown that the spore's dipicolinic acid (DPA) level can markedly influence both spore germination and resistance and that the proteins encoded by the spoVA operon are essential for DPA uptake by the developing spore during sporulation. We now find that proteins encoded by the spoVA operon are also essential for the uptake of Ca2+ and DPA into the developing spore during C. perfringens sporulation. Spores of a spoVA mutant had little, if any, Ca2+ and DPA, and their core water content was approximately twofold higher than that of wild-type spores. These DPA-less spores did not germinate spontaneously, as DPA-less B. subtilis spores do. Indeed, wild-type and spoVA C. perfringens spores germinated similarly with a mixture of l-asparagine and KCl (AK), KCl alone, or a 1:1 chelate of Ca2+ and DPA (Ca-DPA). However, the viability of C. perfringens spoVA spores was 20-fold lower than the viability of wild-type spores. Decoated wild-type and spoVA spores exhibited little, if any, germination with AK, KCl, or exogenous Ca-DPA, and their colony-forming efficiency was 103- to 104-fold lower than that of intact spores. However, lysozyme treatment rescued these decoated spores. Although the levels of DNA-protective α/β-type, small, acid-soluble spore proteins in spoVA spores were similar to those in wild-type spores, spoVA spores exhibited markedly lower resistance to moist heat, formaldehyde, HCl, hydrogen peroxide, nitrous acid, and UV radiation than wild-type spores did. In sum, these results suggest the following. (i) SpoVA proteins are essential for Ca-DPA uptake by developing spores during C. perfringens sporulation. (ii) SpoVA proteins and Ca-DPA release are not required for C. perfringens spore germination. (iii) A low spore core water content is essential for full resistance of C. perfringens spores to moist heat, UV radiation, and chemicals.

scholarly journals Evaluating the Involvement of Alternative Sigma Factors SigF and SigG in Clostridium perfringens Sporulation and Enterotoxin Synthesis

2010 ◽  
Vol 78 (10) ◽  
pp. 4286-4293 ◽  
Author(s):  
Jihong Li ◽  
Bruce A. McClane
Keyword(s):  
Bacillus Subtilis ◽  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Sigma Factors ◽  
Wild Type ◽  
Alternative Sigma Factors ◽  
Food Borne ◽  
Food Borne Illness ◽  
Bacterial Food ◽  
Null Mutants

ABSTRACT Clostridium perfringens type A food poisoning is the second most commonly identified bacterial food-borne illness. Sporulation contributes to this disease in two ways: (i) most food-poisoning strains form exceptionally resistant spores to facilitate their survival of food-associated stresses, and (ii) the enterotoxin (CPE) responsible for the symptoms of this food poisoning is synthesized only during sporulation. In Bacillus subtilis, four alternative sigma factors mediate sporulation. The same four sigma factors are encoded by C. perfringens genomes, and two (SigE and SigK) have previously been shown to be necessary for sporulation and CPE production by SM101, a transformable derivative of a C. perfringens food-poisoning strain (K. H. Harry, R. Zhou, L. Kroos, and S. B. Melville, J. Bacteriol. 2009, 191:2728-2742). However, the importance of SigF and SigG for C. perfringens sporulation or CPE production had not yet been assessed. In the current study, after confirming that sporulating wild-type SM101 cultures produce SigF (from a tricistronic operon) and SigG, we prepared isogenic sigF- or sigG-null mutants. Whereas SM101 formed heat-resistant, phase-refractile spores, spore formation was blocked in the sigF- and sigG-null mutants. Complementation fully restored sporulation by both mutants. By use of these mutants and complementing strains, CPE production was shown to be SigF dependent but SigG independent. This finding apparently involved regulation of the production of SigE and SigK, which Harry et al. showed to be necessary for CPE synthesis, by SigF. By combining these findings with those previous results, it is now apparent that all four alternative sigma factors are necessary for C. perfringens sporulation, but only SigE, SigF, and SigK are needed for CPE synthesis.

scholarly journals Inorganic Phosphate Induces Spore Morphogenesis and Enterotoxin Production in the Intestinal Pathogen Clostridium perfringens

2006 ◽  
Vol 74 (6) ◽  
pp. 3651-3656 ◽  
Author(s):  
Valeria A. Philippe ◽  
Marcelo B. Méndez ◽  
I-Hsiu Huang ◽  
Lelia M. Orsaria ◽  
Mahfuzur R. Sarker ◽  
...  
Keyword(s):  
Clostridium Perfringens ◽  
Inorganic Phosphate ◽  
Food Poisoning ◽  
Inhibitory Effect ◽  
Physiological Signal ◽  
Food Borne ◽  
Intestinal Pathogen ◽  
Important Virulence Factor ◽  
Dna Partitioning ◽  
Effect Of Glucose

ABSTRACT Clostridium perfringens enterotoxin (CPE) is an important virulence factor for food poisoning and non-food borne gastrointestinal (GI) diseases. Although CPE production is strongly regulated by sporulation, the nature of the signal(s) triggering sporulation remains unknown. Here, we demonstrated that inorganic phosphate (Pi), and not pH, constitutes an environmental signal inducing sporulation and CPE synthesis. In the absence of Pi-supplementation, C. perfringens displayed a spo0A phenotype, i.e., absence of polar septation and DNA partitioning in cells that reached the stationary phase of growth. These results received support from our Northern blot analyses which demonstrated that Pi was able to counteract the inhibitory effect of glucose at the onset of sporulation and induced spo0A expression, indicating that Pi acts as a key signal triggering spore morphogenesis. In addition to being the first study reporting the nature of a physiological signal triggering sporulation in clostridia, these findings have relevance for the development of antisporulation drugs to prevent or treat CPE-mediated GI diseases in humans.

scholarly journals CodY Promotes Sporulation and Enterotoxin Production by Clostridium perfringens Type A Strain SM101

2017 ◽  
Vol 85 (3) ◽  
Author(s):  
Jihong Li ◽  
John C. Freedman ◽  
Daniel R. Evans ◽  
Bruce A. McClane
Keyword(s):  
Gene Expression ◽  
Clostridium Perfringens ◽  
Food Poisoning ◽  
Type A ◽  
Toxin Production ◽  
Null Mutant ◽  
Wild Type ◽  
Content Type ◽  
Type D ◽  
Epsilon Toxin

ABSTRACT Clostridium perfringens type D strains cause enterotoxemia and enteritis in livestock via epsilon toxin production. In type D strain CN3718, CodY was previously shown to increase the level of epsilon toxin production and repress sporulation. C. perfringens type A strains producing C. perfringens enterotoxin (CPE) cause human food poisoning and antibiotic-associated diarrhea. Sporulation is critical for C. perfringens type A food poisoning since spores contribute to transmission and resistance in the harsh food environment and sporulation is essential for CPE production. Therefore, the current study asked whether CodY also regulates sporulation and CPE production in SM101, a derivative of C. perfringens type A food-poisoning strain NCTC8798. An isogenic codY-null mutant of SM101 showed decreased levels of spore formation, along with lower levels of CPE production. A complemented strain recovered wild-type levels of both sporulation and CPE production. When this result was coupled with the earlier results obtained with CN3718, it became apparent that CodY regulation of sporulation varies among different C. perfringens strains. Results from quantitative reverse transcriptase PCR analysis clearly demonstrated that, during sporulation, codY transcript levels remained high in SM101 but rapidly declined in CN3718. In addition, abrB gene expression patterns varied significantly between codY-null mutants of SM101 and CN3718. Compared to the levels in their wild-type parents, the level of abrB gene expression decreased in the CN3718 codY-null mutant strain but significantly increased in the SM101 codY-null mutant strain, demonstrating CodY-dependent regulation differences in abrB expression between these two strains. This difference appears to be important since overexpression of the abrB gene in SM101 reduced the levels of sporulation and enterotoxin production, supporting the involvement of AbrB repression in regulating C. perfringens sporulation.

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