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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.

ABSTRACTClostridium perfringensenterotoxin (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. perfringensdisplayed a spo0Aphenotype, 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 Piwas able to counteract the inhibitory effect of glucose at the onset of sporulation and induced spo0Aexpression, indicating that Piacts 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.

ABSTRACTRecent epidemiological studies suggested that cpb2-positive Clostridium perfringensisolates are associated with gastrointestinal (GI) diseases in horses. These putative relationships, indicated by PCR genotyping, were tested in the present study by further genotyping and phenotyping of 23 cpb2-positive C. perfringensisolates from horses with GI disease (referred to hereafter as horse GI disease isolates). Our beta2-toxin (CPB2) Western blot analyses demonstrated that all of the tested isolates were unable to produce detectable levels of CPB2. However, Southern blot and nucleotide sequencing analyses identified intact cpb2open reading frames in all of our surveyed horse GI disease isolates. Furthermore, reverse transcriptase PCR and Northern blot analyses showed that cpb2genes in all of our surveyed horse GI disease isolates were transcriptionally active, i.e., an ~1.2-kb cpb2-specific mRNA was identified in total RNA from our surveyed isolates. The levels of cpb2mRNA in CWC245 (a high-CPB2-producing pig strain) and our surveyed horse GI disease isolates differed to such an extent (35-fold) that this difference could be considered as a major cause of the difference in levels of CPB2 production by CWC245 and horse GI disease isolates. This finding received further support from our observation that the complementing strain 106902(pMRS140), which produced significantly higher levels of mRNA than strain 106902, produced high levels of CPB2. Collectively, our results indicated that there is a positive correlation between cpb2transcription levels and the amount of CPB2 produced by a C. perfringenscell and that decreased transcription and/or message instability may be involved, at least in part, in the low CPB2 production noted for horse GI disease isolates in comparison to that noted for pig GI disease isolate CWC245.

ABSTRACTAlthough Clostridium perfringensis recognized as an important cause of clostridial enteric diseases, only limited knowledge exists concerning the association of particular C. perfringenstoxinotypes (type A to E) with gastrointestinal (GI) diseases in domestic animals. Some C. perfringensisolates also produce the newly discovered beta2-toxin (CPB2). Recent epidemiological studies suggested that C. perfringensisolates carrying the gene encoding CPB2 (cpb2) are strongly associated with clostridial GI diseases in domestic animals, including necrotic enteritis in piglets and typhlocolitis in horses. These putative relationships, obtained by PCR genotyping, were tested in the present study by further genotyping and phenotyping of 29 cpb2-positive C. perfringensisolates from pigs with GI disease (pig GI disease isolates). PCR and restriction fragment length polymorphism analysis reconfirmed the presence of cpb2gene sequences in all the disease isolates included in the study. Furthermore, genotyping by pulsed-field gel electrophoresis analyses showed that the pig GI disease isolates included in this study all carry a plasmid cpb2gene, yet no clonal relationships were detected between the cpb2-positive pig GI disease isolates surveyed. Finally, CPB2-specific Western blotting demonstrated CPB2 expression by all of the cpb2-positive isolates surveyed. The CPB2 proteins made by five of these pig GI disease isolates were shown to have the same deduced amino acid sequences as the biologically active CPB2 protein made by the original type C isolate, CWC245. Collectively, our present results support a significant association between CPB2-positive C. perfringensisolates and diarrhea in piglets.

ABSTRACTClostridium perfringensenterotoxin is the major virulence factor involved in the pathogenesis of C. perfringenstype A food poisoning and several non-food-borne human gastrointestinal illnesses. The enterotoxin gene,cpe, is located on the chromosome of food-poisoning isolates but is found on a large plasmid in non-food-borne gastrointestinal disease isolates and in veterinary isolates. To evaluate whether the cpeplasmid encodes its own conjugative transfer, a C. perfringensstrain carrying pMRS4969, a plasmid in which a 0.4-kb segment internal to thecpegene had been replaced by the chloramphenicol resistance gene catP, was used as a donor in matings with several cpe-negative C. perfringensisolates. Chloramphenicol resistance was transferred at frequencies ranging from 2.0 × 10−2to 4.6 × 10−4transconjugants per donor cell. The transconjugants were characterized by PCR, pulsed-field gel electrophoresis, and Southern hybridization analyses. The results demonstrated that the entire pMRS4969 plasmid had been transferred to the recipient strain. Plasmid transfer required cell-to-cell contact and was DNase resistant, indicating that transfer occurred by a conjugation mechanism. In addition, several fragments of the prototype C. perfringenstetracycline resistance plasmid, pCW3, hybridized with pMRS4969, suggesting that pCW3 shares some similarity to pMRS4969. The clinical significance of these findings is that if conjugative transfer of the cpeplasmid occurred in vivo, it would have the potential to convertcpe-negative C. perfringensstrains in normal intestinal flora into strains capable of causing gastrointestinal disease.

ABSTRACTClostridium perfringenstype A isolates producing enterotoxin (CPE) are an important cause of food poisoning and non-food-borne human gastrointestinal (GI) diseases, including antibiotic-associated diarrhea (AAD). Recent studies suggest thatC. perfringenstype A food poisoning is caused by C. perfringensisolates carrying a chromosomal cpegene, while CPE-associated non-food-borne GI diseases, such as AAD, are caused by plasmid cpeisolates. Those putative relationships, obtained predominantly with European isolates, were tested in the current study by examining 34 cpe-positive,C. perfringensfecal isolates from North American cases of food poisoning or AAD. These North American disease isolates were all classified as type A using a multiplex PCR assay. Furthermore, restriction fragment length polymorphism and pulsed-field gel electrophoresis genotyping analyses showed the North American AAD isolates included in this collection all have a plasmid cpegene, but the North American food poisoning isolates all carry a chromosomal cpegene. Western blotting demonstrated CPE expression by nearly all of these disease isolates, confirming their virulence potential. These findings with North American isolates provide important new evidence that, regardless of geographic origin or date of isolation, plasmid cpeisolates cause most CPE-associated AAD cases and chromosomal cpeisolates cause most C. perfringenstype A food poisoning cases. These findings hold importance for the development of assays for distinguishing cases of CPE-associated food-borne and non-food-borne human GI illnesses and also identify potential epidemiologic tools for determining the reservoirs for these illnesses.

Gene transfer in seven pathogenic enteric bacteria was studied using an RP4::mini‐Mu element, the plasmid pULB113. From the E. coliK‐12 host strain the plasmid could be efficiently transferred to these enteric bacteria, but its transfer back to E. coliK‐12 was not as efficient, being detected only in Shigella dysenteriae1, S. flexneriand the ‘smooth’ variant of S. sonnei.In these three species, transposition of chromosomal fragments into the plasmid to produce R‐prime plasmid was also detected at a frequency of approximately 10−5. Transposition was random as suggested by the recovery at approximately the same frequency (10−5to 10−6) of R‐primes involving 20 different auxotrophic markers from widely separated chromosomal locations. Formation of R‐prime plasmids expressing toxicity in the E. coliK‐12 recipient strain was also efficient in S. dysenteriae1 but the toxin‐activity was rapidly lost from these R‐primes. In our experiments, the plasmid pULB113 incorporated relatively small amounts of chromosomal DNA as determined by restriction endonuclease digestion. For a Thy+ R‐prime that we analyzed, the amount of cloned DNA was approximately 15 kb.

Clones containing the Bacteroides fragilis leuB‐complementing gene were isolated by screening of a B. fragilisgenomic library constructed in Escherichia coli. One recombinant clone, designated pOT865, with the smallest DNA insert (4.5 kb) could complement three independent leuBmutations in E. coliand the leuB‐complementing determinant in pOT865 was localized to a region of 1.5‐kb DNA. The results of Southern blot analysis suggested that a single copy of the cloned gene was present in the B. fragilisgenome. The cloned fragment appeared to contain a sequence that could function as a promoter in E. coliand direct the synthesis of a 42‐kDa protein. These results suggest that the cloned segment contains the structural gene for β‐isopropylmalate dehydrogenase (leuB).

As an approach to the development of attenuated strains, thymine-requiring (Thy−) and temperature-sensitive (Ts−) single and double mutants of Shigella flexneriY have been isolated by u.v. mutagenesis and selection. The mutants retained the 140 MDa plasmid, were Sereny-negative and avirulent in ‘conditioned’ rabbits and in monkeys. Rabbits and monkeys immunized orally with two doses of the mutants were solidly protected against disease and lethality when challenged with the homologous virulent strain. The immunized animals had higher levels of antibodies reactive to the lipopolysaccharide of the wild type strain and also a shorter duration of shedding of the challenge bacteria. It was concluded that Thy−and Ts−mutants of Shigellaspp. are highly attenuated and immunogenic to be considered for further studies towards the development of vaccine candidates.

ABSTRACTSeveral Clostridium perfringensgenotype E isolates, all associated with hemorrhagic enteritis of neonatal calves, were identified by multiplex PCR. These genotype E isolates were demonstrated to express α and ι toxins, but, despite carrying sequences for the gene (cpe) encoding C. perfringensenterotoxin (CPE), were unable to express CPE. These silent cpesequences were shown to be highly conserved among type E isolates. However, relative to the functionalcpegene of type A isolates, these silent type Ecpesequences were found to contain nine nonsense and two frameshift mutations and to lack the initiation codon, promoters, and ribosome binding site. The type E animal enteritis isolates carrying these silent cpesequences do not appear to be clonally related, and their silent type E cpesequences are always located, near the ι toxin genes, on episomal DNA. These findings suggest that the highly conserved, silent cpesequences present in most or all type E isolates may have resulted from the recent horizontal transfer of an episome, which also carries ι toxin genes, to several different type A C. perfringensisolates.

ABSTRACTExtracellular Yersiniadisables the immune system of its host by injecting effector Yop proteins into host cells. We show that a Yersinia enterocoliticanonpolar lcrGmutant is severely impaired in the translocation of YopE, YopH, YopM, YpkA/YopO, and YopP into eukaryotic cells. LcrG is thus required for efficient internalization of all the known Yop effectors.

The complete nucleotide sequence of the gene (leuB) coding for β‐isopropylmaiate dehydrogenase of Bacteroides fragiliswas determined. An open reading frame of 1,061 nucleotides was detected that could encode a polypeptide of 353 amino acid residues with a calculated molecular mass of 39,179 Da. The deduced amino acid sequence of the β‐isopropylmalate dehydrogenase from B. fragilisshowed substantial sequence similarity with the β‐isopropylmalate dehydrogenases from other bacteria.

ABSTRACTCodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringenstype D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codYmutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codYgene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codYmutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringensvirulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection.IMPORTANCEClostridium perfringensis a major human and livestock pathogen because it produces many potent toxins. C. perfringenstype D strains cause intestinal infections by producing toxins, especially epsilon toxin (ETX). Previous studies identified CodY as a regulator of certain virulence properties in other Gram-positive bacteria. Our study now demonstrates that CodY is a global regulator of virulence-associated properties for type D strain CN3718. It promotes production of ETX, attachment of CN3718 vegetative cells or spores to host enterocyte-like Caco-2 cells, and spore germination; the last two effects may assist intestinal colonization. In contrast, CodY represses sporulation. These results provide the first evidence that CodY can function as a global regulator of C. perfringensvirulence-associated properties and that this strain coordinately regulates its virulence-associated properties using CodY to increase ETX production, host cell attachment, and spore germination but to repress sporulation, as would be optimal during type D intestinal infection.