RESUMO
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.
Assuntos
Toxinas Bacterianas , Clostridium perfringens , Gangrena Gasosa , Fosfolipases Tipo C , Clostridium perfringens/genética , Clostridium perfringens/crescimento & desenvolvimento , Clostridium perfringens/metabolismo , Clostridium perfringens/fisiologia , Camundongos , Animais , Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Linhagem Celular , Gangrena Gasosa/microbiologia , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Diferenciação Celular , Células Musculares/microbiologia , Células Musculares/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Percepção de QuorumRESUMO
A 42-year-old man, with up-to-date COVID-19 vaccination, experienced symptomatic SARS-CoV-2 infection in December 2021. Mutation tests suggested a non-Omicron variant. After his recovery, and 24 days after the first positive SARS-CoV-2 test, he had onset of symptomatic infection with the BA.1.1 (Omicron) variant, which was confirmed by whole-genome sequencing.
Assuntos
COVID-19 , SARS-CoV-2 , Adulto , COVID-19/diagnóstico , Vacinas contra COVID-19 , Genoma Viral , Humanos , Pennsylvania , SARS-CoV-2/genéticaRESUMO
UNLABELLED: Vibrio cholerae is an aquatic organism and facultative human pathogen that colonizes the small intestine. In the small intestine, V. cholerae is exposed to a variety of antimicrobial compounds, including bile. V. cholerae resistance to bile is multifactorial and includes alterations in the membrane permeability barrier that are mediated by ToxR, a membrane-associated transcription factor. ToxR has also been shown to be required for activation of the LysR family transcription factor leuO in response to cyclic dipeptides. LeuO has been implicated in the regulation of multiple V. cholerae phenotypes, including biofilm production and virulence. In this study, we investigated the effects of bile on leuO expression. We show that leuO transcription increased in response to bile and bile salts but not in response to other detergents. The bile-dependent increase in leuO expression was dependent on ToxR, which was found to bind directly to the leuO promoter. The periplasmic domain of ToxR was required for basal leuO expression and for the bile-dependent induction of both leuO and ompU transcription. V. cholerae mutants that did not express leuO exhibited increased bile susceptibility, suggesting that LeuO contributes to bile resistance. Our collective results demonstrate that ToxR activates leuO expression in response to bile and that LeuO is a component of the ToxR-dependent responses that contribute to bile resistance. IMPORTANCE: The success of Vibrio cholerae as a human pathogen is dependent upon its ability to rapidly adapt to changes in its growth environment. Growth in the human gastrointestinal tract requires the expression of genes that provide resistance to host antimicrobial compounds, including bile. In this work, we show for the first time that the LysR family regulator LeuO mediates responses in V. cholerae that contribute to bile resistance.
Assuntos
Proteínas de Bactérias/metabolismo , Ácidos e Sais Biliares/farmacologia , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica/fisiologia , Fatores de Transcrição/metabolismo , Vibrio cholerae/efeitos dos fármacos , Vibrio cholerae/metabolismo , Proteínas de Bactérias/genética , Proteínas de Ligação a DNA/genética , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Fatores de Transcrição/genética , Vibrio cholerae/genéticaRESUMO
The human short PLUNC1 (SPLUNC1) protein has been identified as a component of the pulmonary antimicrobial response based on its structural similarity to the bactericidal/permeability-increasing (BPI) protein. Using a genetically modified mouse model, we recently verified the antimicrobial activity of SPLUNC1 against Pseudomonas aeruginosa in vivo. To further define the mechanism of epithelial SPLUNC1-mediated antibacterial action, we carried out studies to determine how SPLUNC1 protects the host from acute respiratory infections. P. aeruginosa treated with recombinant human SPLUNC1 protein showed decreased growth in vitro. This antibacterial activity was due to growth inhibition, as a consequence of a SPLUNC1-induced increase in bacterial cell permeability. Removal of SPLUNC1 allowed the recovery of P. aeruginosa and suggested no permanent cell injury or direct killing of bacteria. Further investigation showed coating of bacterial cells by SPLUNC1. We suggest that this "bacterial cell coating" is necessary for the bacteriostatic function of SPLUNC1. Additionally, we demonstrated a novel role for SPLUNC1 as a chemoattractant that facilitated migration of macrophages and neutrophils. Taking the findings together, we propose synergistic roles for human SPLUNC1 as an antibacterial agent with bacteriostatic and chemotactic activities.
Assuntos
Glicoproteínas/imunologia , Glicoproteínas/metabolismo , Fosfoproteínas/imunologia , Fosfoproteínas/metabolismo , Infecções por Pseudomonas/imunologia , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/imunologia , Pseudomonas aeruginosa/metabolismo , Animais , Antibacterianos/imunologia , Antibacterianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Proteínas Sanguíneas/imunologia , Proteínas Sanguíneas/metabolismo , Linhagem Celular Tumoral , Células HL-60 , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Permeabilidade , Ligação Proteica/imunologia , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Infecções Respiratórias/imunologia , Infecções Respiratórias/metabolismoRESUMO
Clostridium perfringens type B or D isolates, which cause enterotoxemias or enteritis in livestock, produce epsilon toxin (ETX). ETX is exceptionally potent, earning it a listing as a CDC class B select toxin. Most C. perfringens strains also express up to three different sialidases, although the possible contributions of those enzymes to type B or D pathogenesis remain unclear. Type D isolate CN3718 was found to carry two genes (nanI and nanJ) encoding secreted sialidases and one gene (nanH) encoding a cytoplasmic sialidase. Construction in CN3718 of single nanI, nanJ and nanH null mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells with NanI sialidase, or with culture supernatants of BMC206 (an isogenic CN3718 etx null mutant that still produces sialidases) enhanced the subsequent binding and cytotoxic effects of purified ETX. Complementation of BMC207 (an etx/nanH/nanI/nanJ null mutant) showed this effect is mainly attributable to NanI production. Contact between BMC206 and certain mammalian cells (e.g., enterocyte-like Caco-2 cells) resulted in more rapid sialidase production and this effect involved increased transcription of BMC206 nanI gene. BMC206 was shown to adhere to some (e.g. Caco-2 cells), but not all mammalian cells, and this effect was dependent upon sialidase, particularly NanI, expression. Finally, the sialidase activity of NanI (but not NanJ or NanH) could be enhanced by trypsin. Collectively these in vitro findings suggest that, during type D disease originating in the intestines, trypsin may activate NanI, which (in turn) could contribute to intestinal colonization by C. perfringens type D isolates and also increase ETX action.
Assuntos
Toxinas Bacterianas/metabolismo , Infecções por Clostridium/metabolismo , Clostridium perfringens/metabolismo , Interações Hospedeiro-Parasita/fisiologia , Neuraminidase/metabolismo , Animais , Toxinas Bacterianas/genética , Southern Blotting , Western Blotting , Células CACO-2 , Adesão Celular , Infecções por Clostridium/genética , Clostridium perfringens/genética , Ativação Enzimática/genética , Técnicas de Inativação de Genes , Humanos , Microscopia de Fluorescência , Neuraminidase/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tripsina/metabolismoRESUMO
Clostridium perfringens enterotoxin (encoded by the cpe gene) contributes to several important human, and possibly veterinary, enteric diseases. The current study investigated whether cpe locus organization in type C or D isolates resembles one of the three (one chromosomal and two plasmid-borne) cpe loci commonly found amongst type A isolates. Multiplex PCR assays capable of detecting sequences in those type A cpe loci failed to amplify products from cpe-positive type C and D isolates, indicating these isolates possess different cpe locus arrangements. Therefore, restriction fragments containing the cpe gene were cloned and sequenced from two type C isolates and one type D isolate. The obtained cpe locus sequences were then used to construct an overlapping PCR assay to assess cpe locus diversity amongst other cpe-positive type C and D isolates. All seven surveyed cpe-positive type C isolates had a plasmid-borne cpe locus partially resembling the cpe locus of type A isolates carrying a chromosomal cpe gene. In contrast, all eight type D isolates shared the same plasmid-borne cpe locus, which differed substantially from the cpe locus present in other C. perfringens by containing two copies of an ORF with 67% identity to a transposase gene (COG4644) found in Tn1546, but not previously associated with the cpe gene. These results identify greater diversity amongst cpe locus organization than previously appreciated, providing new insights into cpe locus evolution. Finally, evidence for cpe gene mobilization was found for both type C and D isolates, which could explain their cpe plasmid diversity.
Assuntos
Clostridium perfringens/isolamento & purificação , Genes Bacterianos , Sequência de Bases , Southern Blotting , Clostridium perfringens/classificação , Clostridium perfringens/genética , Primers do DNA , Eletroforese em Gel de Campo Pulsado , Enterotoxinas , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
The important veterinary pathogen Clostridium perfringens type B is unique for producing the two most lethal C. perfringens toxins, i.e., epsilon-toxin and beta-toxin. Our recent study (K. Miyamoto, J. Li, S. Sayeed, S. Akimoto, and B. A. McClane, J. Bacteriol. 190:7178-7188, 2008) showed that most, if not all, type B isolates carry a 65-kb epsilon-toxin-encoding plasmid. However, this epsilon-toxin plasmid did not possess the cpb gene encoding beta-toxin, suggesting that type B isolates carry at least one additional virulence plasmid. Therefore, the current study used Southern blotting of pulsed-field gels to localize the cpb gene to approximately 90-kb plasmids in most type B isolates, although a few isolates carried a approximately 65-kb cpb plasmid distinct from their etx plasmid. Overlapping PCR analysis then showed that the gene encoding the recently discovered TpeL toxin is located approximately 3 kb downstream of the plasmid-borne cpb gene. As shown earlier for their epsilon-toxin-encoding plasmids, the beta-toxin-encoding plasmids of type B isolates were found to carry a tcp locus, suggesting that they are conjugative. Additionally, IS1151-like sequences were identified upstream of the cpb gene in type B isolates. These IS1151-like sequences may mobilize the cpb gene based upon detection of possible cpb-containing circular transposition intermediates. Most type B isolates also possessed a third virulence plasmid that carries genes encoding urease and lambda-toxin. Collectively, these findings suggest that type B isolates are among the most plasmid dependent of all C. perfringens isolates for virulence, as they usually carry three potential virulence plasmids.
Assuntos
Toxinas Bacterianas/genética , Toxinas Bacterianas/metabolismo , Clostridium perfringens/classificação , Clostridium perfringens/patogenicidade , Variação Genética , Plasmídeos/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/fisiologia , VirulênciaRESUMO
Clostridium perfringens type C isolates cause enterotoxemias and enteritis in humans and livestock. While the major disease signs and lesions of type C disease are usually attributed to beta toxin (CPB), these bacteria typically produce several different lethal toxins. Since understanding of disease pathogenesis and development of improved vaccines is hindered by the lack of small animal models mimicking the lethality caused by type C isolates, in this study we developed two mouse models of C. perfringens type C-induced lethality. When inoculated into BALB/c mice by intragastric gavage, 7 of 14 type C isolates were lethal, whereas when inoculated intraduodenally, these strains were all lethal in these mice. Clinical signs in intragastrically and intraduodenally challenged mice were similar and included respiratory distress, abdominal distension, and neurological alterations. At necropsy, the small, and occasionally the large, intestine was dilated and gas filled in most mice developing a clinical response. Histological changes in the gut were relatively mild, consisting of attenuation of the mucosa with villus blunting. Inactivation of the CPB-encoding gene rendered the highly virulent type C strain CN3685 avirulent in the intragastric model and nearly nonlethal in the intraduodenal model. In contrast, inactivation of the genes encoding alpha toxin and perfringolysin O only slightly decreased the lethality of CN3685. Mice could be protected against lethality by intravenous passive immunization with a CPB antibody prior to intragastric challenge. This study proves that CPB is a major contributor to the systemic effects of type C infections and provides new mouse models for investigating the pathogenesis of type C-induced lethality.
Assuntos
Clostridium perfringens/patogenicidade , Modelos Animais de Doenças , Enterotoxemia/patologia , Enterotoxemia/fisiopatologia , Animais , Antitoxinas/uso terapêutico , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidade , Proteínas de Ligação ao Cálcio/genética , Duodeno/microbiologia , Deleção de Genes , Proteínas Hemolisinas/genética , Imunização Passiva/métodos , Mucosa Intestinal/patologia , Intestino Grosso/patologia , Intestino Delgado/patologia , Camundongos , Camundongos Endogâmicos BALB C , Estômago/microbiologia , Análise de Sobrevida , Fosfolipases Tipo C/genéticaRESUMO
Clostridium perfringens type B and D isolates produce epsilon-toxin, the third most potent clostridial toxin. The epsilon-toxin gene (etx) is plasmid borne in type D isolates, but etx genetics have been poorly studied in type B isolates. This study reports the first sequencing of any etx plasmid, i.e., pCP8533etx, from type B strain NCTC8533. This etx plasmid is 64.7 kb, carries tcp conjugative transfer genes, and encodes additional potential virulence factors including beta2-toxin, sortase, and collagen adhesin but not beta-toxin. Interestingly, nearly 80% of pCP8533etx open reading frames (ORFs) are also present on pCPF5603, an enterotoxin-encoding plasmid from type A isolate F5603. Pulsed-field gel electrophoresis and overlapping PCR indicated that a pCP8533etx-like etx plasmid is also present in most, if not all, other type B isolates and some beta2-toxin-positive, cpe-negative type D isolates, while other type D isolates carry different etx plasmids. Sequences upstream of the etx gene vary between type B isolates and some type D isolates that do not carry a pCP8533etx-like etx plasmid. However, nearly all type B and D isolates have an etx locus with an upstream IS1151, and those etx loci typically reside near a dcm ORF. These results suggest that pCPF5603 and pCP8533etx evolved from insertion of mobile genetic elements carrying enterotoxin or etx genes, respectively, onto a common progenitor plasmid.
Assuntos
Clostridium perfringens/genética , Enterotoxinas/metabolismo , Variação Genética , Plasmídeos/genética , Southern Blotting , Clostridium perfringens/metabolismo , Eletroforese em Gel de Campo Pulsado , Modelos Genéticos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Análise de Sequência de DNARESUMO
Enterotoxemia caused by Clostridium perfringens type D in sheep is believed to result from the action of epsilon toxin (ETX). However, the sole role of ETX in the intestinal changes of the acute and chronic forms of enterotoxemia in goats remains controversial, and the synergistic action of other C. perfringens toxins has been suggested previously. The current study examined 2 goats that were found dead without premonitory clinical signs. Gross lesions at necropsy consisted of multifocal fibrinonecrotic enterocolitis, edematous lungs, and excess pleural fluid. Histologically, there were multifocal fibrinonecrotic and ulcerative ileitis and colitis, edema of the colonic serosa, and proteinaceous interstitial edema of the lungs. Clostridium perfringens type D carrying the genes for enterotoxin (CPE) and beta2 toxin (CPB2) was cultured from intestinal content and feces of 1 of 2 goats, while C. perfringens type D CPB2-positive was isolated from the other animal. When multiple colonies of the primary isolations from both animals were tested by Western blot, most of the isolates expressed CPB2, and only a few isolates from the first case expressed CPE. Alpha toxin and ETX were detected in ileal and colonic contents and feces of both animals by antigen capture enzyme-linked immunosorbent assay. CPB2, but not CPE, was identified in the small and large intestines of both goats by immunohistochemistry. These findings indicate that CPB2 may have contributed to the necrotic changes observed in the intestine, possibly assisting ETX transit across the intestinal mucosa.
Assuntos
Toxinas Bacterianas/isolamento & purificação , Infecções por Clostridium/veterinária , Clostridium perfringens/isolamento & purificação , Colite Ulcerativa/veterinária , Enterocolite/veterinária , Doenças das Cabras/microbiologia , Animais , Infecções por Clostridium/diagnóstico , Colite Ulcerativa/microbiologia , Enterocolite/microbiologia , Feminino , CabrasRESUMO
We investigated the frequency of Clostridium perfringens in the normal fecal flora of healthy North Americans. About half of 43 subjects were colonized with C. perfringens at levels of approximately 10(6)cfu/g feces. Only type A strains were recovered. Spores sometimes outnumbered vegetative cells. Several genotypes were found. Some donors carried two genotypes, some only one. We found no alpha, beta2 or enterotoxin in the stools of any donors. Though some isolates carried toxin genes (e.g. cpe and cpb2) on plasmids, we saw no indication that healthy humans are the reservoir for the chromosomally-borne cpe recovered from cases of C. perfringens food poisoning.
Assuntos
Toxinas Bacterianas/genética , Clostridium perfringens/genética , Proteínas de Ligação ao Cálcio/genética , Portador Sadio/microbiologia , Clostridium perfringens/isolamento & purificação , Contagem de Colônia Microbiana , Enterotoxinas/genética , Fezes/microbiologia , Feminino , Genótipo , Humanos , Masculino , América do Norte , Plasmídeos , Esporos Bacterianos/isolamento & purificação , Fosfolipases Tipo C/genéticaRESUMO
Clostridium perfringens type C isolates, which cause enteritis necroticans in humans and enteritis and enterotoxaemias of domestic animals, typically produce (at minimum) beta toxin (CPB), alpha toxin (CPA) and perfringolysin O (PFO) during log-phase growth. To assist development of improved vaccines and therapeutics, we evaluated the contribution of these three toxins to the intestinal virulence of type C disease isolate CN3685. Similar to natural type C infection, log-phase vegetative cultures of wild-type CN3685 caused haemorrhagic necrotizing enteritis in rabbit ileal loops. When isogenic toxin null mutants were prepared using TargeTron technology, even a double cpa/pfoA null mutant of CN3685 remained virulent in ileal loops. However, two independent cpb null mutants were completely attenuated for virulence in this animal model. Complementation of a cpb mutant restored its CPB production and intestinal virulence. Additionally, pre-incubation of wild-type CN3685 with a CPB-neutralizing monoclonal antibody rendered the strain avirulent for causing intestinal pathology. Finally, highly purified CPB reproduced the intestinal damage of wild-type CN3685 and that damage was prevented by pre-incubating purified CPB with a CPB monoclonal antibody. These results indicate that CPB is both required and sufficient for CN3685-induced enteric pathology, supporting a key role for this toxin in type C intestinal pathogenesis.
Assuntos
Toxinas Bacterianas/metabolismo , Infecções por Clostridium/veterinária , Clostridium perfringens/patogenicidade , Doenças do Íleo/veterinária , Animais , Anticorpos Antibacterianos/imunologia , Anticorpos Monoclonais/imunologia , Antitoxinas/imunologia , Toxinas Bacterianas/genética , Toxinas Bacterianas/imunologia , Toxinas Bacterianas/toxicidade , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Infecções por Clostridium/microbiologia , Clostridium perfringens/classificação , Clostridium perfringens/imunologia , Modelos Animais de Doenças , Feminino , Genótipo , Proteínas Hemolisinas/genética , Proteínas Hemolisinas/metabolismo , Humanos , Doenças do Íleo/microbiologia , Doenças do Íleo/patologia , Íleo/microbiologia , Íleo/patologia , Masculino , Mutagênese Insercional , Fenótipo , Coelhos , Doenças dos Ovinos/microbiologia , Fosfolipases Tipo C/genética , Fosfolipases Tipo C/metabolismo , Fatores de Virulência/metabolismoRESUMO
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.
Assuntos
Clostridium perfringens/genética , Enterotoxinas/genética , Microbiologia do Solo , Southern Blotting , Western Blotting , Clostridium perfringens/isolamento & purificação , Clostridium perfringens/metabolismo , Utensílios de Alimentação e Culinária , Eletroforese em Gel de Campo Pulsado , Enterotoxinas/biossíntese , Microbiologia de Alimentos , Genótipo , Humanos , Pennsylvania , Reação em Cadeia da PolimeraseRESUMO
Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native epsilon-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional epsilon-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.
Assuntos
Toxinas Bacterianas/genética , Clostridium perfringens/genética , Conjugação Genética , Plasmídeos , Animais , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/toxicidade , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Cães , Eletroforese em Gel de Campo Pulsado , Injeções Intravenosas , Camundongos , MutagêneseRESUMO
Clostridium perfringens type D isolates cause enterotoxemia in sheep, goats, and probably cattle. While the major disease signs and lesions of type D animal disease are usually attributed to epsilon toxin, a class B select agent, these bacteria typically produce several lethal toxins. Understanding of disease pathogenesis and development of improved vaccines are hindered by the lack of a small-animal model mimicking natural disease caused by type D isolates. Addressing this need, we developed an oral challenge mouse model of C. perfringens type D enterotoxemia. When BALB/c mice with a sealed anus were inoculated by intragastric gavage with type D isolates, 7 of 10 type D isolates were lethal, as defined by spontaneous death or severe clinical signs necessitating euthanasia. The lethalities of the seven type D isolates varied between 14 and 100%. Clinical signs in the lethally challenged mice included seizures, convulsions, hyperexcitability, and/or depression. Mild intestinal gas distention and brain edema were observed at necropsy in a few mice, while histology showed multifocal acute tubular necrosis of the kidney and edema in the lungs of most challenged mice that developed a clinical response. When the lethality of type D isolates in this model was compared with in vitro toxin production, only a limited correlation was observed. However, mice could be protected against lethality by intravenous passive immunization with an epsilon toxin antibody prior to oral challenge. This study provides an economical new model for studying the pathogenesis of C. perfringens type D infections.
Assuntos
Infecções por Clostridium/microbiologia , Clostridium perfringens/patogenicidade , Modelos Animais de Doenças , Enterotoxemia/microbiologia , Administração Oral , Animais , Anticorpos Antibacterianos/administração & dosagem , Anticorpos Antibacterianos/uso terapêutico , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais/uso terapêutico , Toxinas Bacterianas/biossíntese , Infecções por Clostridium/imunologia , Infecções por Clostridium/mortalidade , Clostridium perfringens/imunologia , Clostridium perfringens/isolamento & purificação , Duodeno/metabolismo , Duodeno/microbiologia , Enterotoxemia/metabolismo , Enterotoxemia/mortalidade , Imunização Passiva , Intubação Gastrointestinal , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Clostridium perfringens type D isolates are important in biodefense and also cause natural enterotoxemias in sheep, goats, and occasionally cattle. In these isolates, the gene (etx) encoding epsilon-toxin is thought to reside on poorly characterized large plasmids. Type D isolates sometimes also produce other potentially plasmid-encoded toxins, including C. perfringens enterotoxin and beta2 toxin, encoded by the cpe and cbp2 genes, respectively. In the current study we demonstrated that the etx, cpe, and cpb2 genes are carried on plasmids in type D isolates and characterized the toxin-encoding plasmids to obtain insight into their genetic organization, potential transferability, and diversity. Southern blotting of pulsed-field gels showed that the etx gene of type D isolates can be present on at least five different plasmids, whose sizes range from 48 to 110 kb. The etx plasmids also typically carried IS1151 and tcp open reading frames (ORFs) known to mediate conjugative transfer of C. perfringens plasmid pCW3. PCR studies revealed that other than their tcp ORFs, etx plasmids of type D isolates do not carry substantial portions of the conserved or variable regions in the cpe plasmids of type A isolates. Southern blotting also demonstrated that in type D isolates the cpe and cpb2 genes are sometimes present on the etx plasmid. Collectively, these findings confirmed that the virulence of type D isolates is heavily plasmid dependent and indicated that (i) a single type D isolate can carry multiple virulence plasmids, (ii) a single type D virulence plasmid can carry up to three different toxin genes, and (iii) many etx plasmids should be capable of conjugative transfer.
Assuntos
Toxinas Bacterianas/genética , Clostridium perfringens/classificação , Clostridium perfringens/patogenicidade , Enterotoxinas/genética , Variação Genética , Plasmídeos/genética , Animais , Southern Blotting , Clostridium perfringens/genética , Clostridium perfringens/isolamento & purificação , Conjugação Genética , Elementos de DNA Transponíveis , Eletroforese em Gel de Campo Pulsado , Transferência Genética Horizontal , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , VirulênciaRESUMO
Clostridium perfringens is capable of producing up to 15 toxins, including alpha-toxin (CPA), beta-toxin (CPB), epsilon-toxin (ETX), enterotoxin, beta2-toxin (CPB2), and perfringolysin O. Type B isolates, which must produce CPA, CPB, and ETX, are associated with animal illnesses characterized by sudden death or acute neurological signs, with or without intestinal damage. Type B pathogenesis in ruminants is poorly understood, with some animals showing lesions and clinical signs similar to those caused by either type C or type D infections. It is unknown whether host or environmental conditions are dominant for determining the outcome of type B disease or if disease outcomes are determined by variable characteristics of type B isolates. To help clarify this issue, 19 type B isolates were evaluated for toxin production during late-log-phase growth via quantitative Western blotting and by biological activity assays. Most type B isolates produced CPB levels similar to those produced by type C isolates in vitro and have the potential to produce genotype C-like disease. The lethality of type B isolate supernatants administered intravenously to mice was evaluated with or without prior trypsin treatment, and monoclonal antibody neutralization studies also were performed. Correlation analyses comparing toxin levels in type B supernatants versus lethality and neutralization studies both found that the main contributor to lethality without pretreatment with trypsin was CPB, whereas neutralization studies indicated that CPB and ETX were both important after trypsin pretreatment. At least part of the CPB produced by type B isolates remained active after trypsin treatment. However, the overall lethalities of most supernatants were lower after trypsin pretreatment. Also, there was a significant association between ETX, CPB2, and CPA production in vitro among type B isolates. However, our results suggest that both CPB and ETX are likely the most important contributors to the pathogenesis of C. perfringens type B infections in domestic animals.
Assuntos
Toxinas Bacterianas/toxicidade , Clostridium perfringens/patogenicidade , Animais , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/biossíntese , Clostridium perfringens/isolamento & purificação , Feminino , Injeções Intravenosas , Dose Letal Mediana , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência MolecularRESUMO
OBJECTIVE: To create, array, and characterize a pooled, high-coverage, genomic library composed of multiple biofilm-forming clinical strains of the opportunistic pathogen, Pseudomonas aeruginosa (PA). Twelve strains were obtained from patients with otorrhea, otitis media, and cystic fibrosis as a resource for investigating: difference in the transcriptomes of planktonic and biofilm envirovars; the size of the PA supragenome and determining the number of virulence genes available at the population level; and the distributed genome hypothesis. METHODS: High molecular weight genomic DNAs from 12 clinical PA strains were individually hydrodynamically sheared to produce mean fragment sizes of approximately 1.5 kb. Equimolar amounts of the 12 sheared genomic DNAs were then pooled and used in the construction of a genomic library with approximately 250,000 clones that was arrayed and subjected to quality control analyses. RESULTS: Restriction endonuclease and sequence analyses of 686 clones picked at random from the library demonstrated that >75% of the clones contained inserts larger than 0.5 kb with the desired mean insert size of 1.4 kb. Thus, this library provides better than 4.5x coverage for each of the genomes from the 12 components clinical PA isolates. Our sequencing effort ( approximately 1 million nucleotides to date) reveals that 13% of the clones present in this library are not represented in the genome of the reference P. aeruginosa strain PA01. CONCLUSIONS: Our data suggests that reliance on a single laboratory strain, such as PA01, as being representative of a pathogenic bacterial species will fail to identify many important genes, and that to obtain a complete picture of complex phenomena, including bacterial pathogenesis and the genetics of biofilm development will require characterization of the P. aeruginosa population-based supra-genome.
Assuntos
Testes Genéticos/métodos , Biblioteca Genômica , Infecções por Pseudomonas/microbiologia , Pseudomonas aeruginosa/genética , Pseudomonas aeruginosa/isolamento & purificação , Pré-Escolar , DNA Bacteriano/análise , DNA Bacteriano/genética , Expressão Gênica , Genoma Bacteriano , Humanos , Mapeamento por Restrição , Análise de Sequência de DNARESUMO
The gram-positive anaerobe Clostridium perfringens produces a large arsenal of toxins that are responsible for histotoxic and enteric infections, including enterotoxemias, in humans and domestic animals. C. perfringens type C isolates, which cause rapidly fatal diseases in domestic animals and enteritis necroticans in humans, contain the genes for alpha toxin (plc), perfringolysin O (pfoA), beta toxin (cpb), and sometimes beta2 toxin (cpb2) and/or enterotoxin (cpe). Due to the economic impact of type C-induced diseases, domestic animals are commonly vaccinated with crude type C toxoid (prepared from inactivated culture supernatants) or bacterin/toxoid vaccines, and it is not clear which toxin(s) present in these vaccines actually elicits the protective immune response. To improve type C vaccines, it would be helpful to assess the contribution of each toxin present in type C supernatants to lethality. To address this issue, we surveyed a large collection of type C isolates to determine their toxin-producing abilities. When late-log-phase vegetative culture supernatants were analyzed by quantitative Western blotting or activity assays, most type C isolates produced at least three lethal toxins, alpha toxin, beta toxin, and perfringolysin O, and several isolates also produced beta2 toxin. In the mouse intravenous injection model, beta toxin was identified as the main lethal factor present in type C late-log-phase culture supernatants. This conclusion was based on monoclonal antibody neutralization studies and regression analyses in which the levels of alpha toxin, beta toxin, perfringolysin O, and beta2 toxin production were compared with lethality. Collectively, our results highlight the importance of beta toxin for type C-induced toxemia.
Assuntos
Toxinas Bacterianas/análise , Toxinas Bacterianas/toxicidade , Infecções por Clostridium/microbiologia , Clostridium perfringens/química , Clostridium perfringens/patogenicidade , Animais , Anticorpos Monoclonais/farmacologia , Toxinas Bacterianas/genética , Sequência de Bases , Western Blotting , Infecções por Clostridium/imunologia , Clostridium perfringens/genética , Morte , Modelos Animais de Doenças , Genes Bacterianos , Genótipo , Injeções Intravenosas , Camundongos , Dados de Sequência MolecularRESUMO
The distributed genome hypothesis (DGH) states that each strain within a bacterial species receives a unique distribution of genes from a population-based supragenome that is many times larger than the genome of any given strain. The observations that natural infecting populations are often polyclonal and that most chronic bacterial pathogens have highly developed mechanisms for horizontal gene transfer suggested the DGH and provided the means and the mechanisms to explain how chronic infections persist in the face of a mammalian host's adaptive defense mechanisms. Having previously established the validity of the DGH for obligate pathogens, we wished to evaluate its applicability to an opportunistic bacterial pathogen. This was accomplished by construction and analysis of a highly redundant pooled genomic library containing approximately 216,000 functional clones that was constructed from 12 low-passage clinical isolates of Pseudomonas aeruginosa, 6 otorrheic isolates and 6 from other body sites. Sequence analysis of 3,214 randomly picked clones (mean insert size, approximately 1.4 kb) from this library demonstrated that 348 (10.8%) of the clones were unique with respect to all genomic sequences of the P. aeruginosa prototype strain, PAO1. Hypothetical translations of the open reading frames within these unique sequences demonstrated protein homologies to a number of bacterial virulence factors and other proteins not previously identified in P. aeruginosa. PCR and reverse transcription-PCR-based assays were performed to analyze the distribution and expression patterns of a 70-open reading frame subset of these sequences among 11 of the clinical strains. These sequences were unevenly distributed among the clinical isolates, with nearly half (34/70) of the novel sequences being present in only one or two of the individual strains. Expression profiling revealed that a vast majority of these sequences are expressed, strongly suggesting they encode functional proteins.