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INTRODUCTION: The application of large language models such as generative pre-trained transformers (GPTs) has been promising in medical education, and its performance has been tested for different medical exams. This study aims to assess the performance of GPTs in responding to a set of sample questions of short-answer management problems (SAMPs) from the certification exam of the College of Family Physicians of Canada (CFPC). METHOD: Between August 8th and 25th, 2023, we used GPT-3.5 and GPT-4 in five rounds to answer a sample of 77 SAMPs questions from the CFPC website. Two independent certified family physician reviewers scored AI-generated responses twice: first, according to the CFPC answer key (ie, CFPC score), and second, based on their knowledge and other references (ie, Reviews' score). An ordinal logistic generalised estimating equations (GEE) model was applied to analyse repeated measures across the five rounds. RESULT: According to the CFPC answer key, 607 (73.6%) lines of answers by GPT-3.5 and 691 (81%) by GPT-4 were deemed accurate. Reviewer's scoring suggested that about 84% of the lines of answers provided by GPT-3.5 and 93% of GPT-4 were correct. The GEE analysis confirmed that over five rounds, the likelihood of achieving a higher CFPC Score Percentage for GPT-4 was 2.31 times more than GPT-3.5 (OR: 2.31; 95% CI: 1.53 to 3.47; p<0.001). Similarly, the Reviewers' Score percentage for responses provided by GPT-4 over 5 rounds were 2.23 times more likely to exceed those of GPT-3.5 (OR: 2.23; 95% CI: 1.22 to 4.06; p=0.009). Running the GPTs after a one week interval, regeneration of the prompt or using or not using the prompt did not significantly change the CFPC score percentage. CONCLUSION: In our study, we used GPT-3.5 and GPT-4 to answer complex, open-ended sample questions of the CFPC exam and showed that more than 70% of the answers were accurate, and GPT-4 outperformed GPT-3.5 in responding to the questions. Large language models such as GPTs seem promising for assisting candidates of the CFPC exam by providing potential answers. However, their use for family medicine education and exam preparation needs further studies.
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Certificación , Canadá , Humanos , Evaluación Educacional/métodos , Médicos de Familia/educación , Competencia Clínica , Medicina Familiar y Comunitaria/educaciónRESUMEN
Clostridium perfringens is the causative agent of human clostridial myonecrosis; the major toxins involved in this disease are α-toxin and perfringolysin O. The RevSR two-component regulatory system has been shown to be involved in regulating virulence in a mouse myonecrosis model. Previous microarray and RNAseq analysis of a revR mutant implied that factors other than the major toxins may play a role in virulence. The RNAseq data showed that the expression of the gene encoding the EngCP endo α-N-acetylgalactosaminidase (CPE0693) was significantly down-regulated in a revR mutant. Enzymes from this family have been identified in several Gram-positive pathogens and have been postulated to contribute to their virulence. In this study, we constructed an engCP mutant of C. perfringens and showed that it was significantly less virulent than its wild-type parent strain. Virulence was restored by complementation in trans with the wild-type engCP gene. We also demonstrated that purified EngCP was able to hydrolyse α-dystroglycan derived from C2C12 mouse myotubes. However, EngCP had little effect on membrane permeability in mice, suggesting that EngCP may play a role other than the disruption of the structural integrity of myofibres. Glycan array analysis indicated that EngCP could recognise structures containing the monosaccharide N-acetlygalactosamine at 4C, but could recognise structures terminating in galactose, glucose and N-acetylglucosamine under conditions where EngCP was enzymatically active. In conclusion, we have obtained evidence that EngCP is required for virulence in C. perfringens and, although classical exotoxins are important for disease, we have now shown that an O-glycosidase also plays an important role in the disease process.
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Clostridium perfringens/enzimología , Clostridium perfringens/patogenicidad , Gangrena Gaseosa/microbiología , Factores de Virulencia/genética , alfa-N-Acetilgalactosaminidasa/genética , Animales , Permeabilidad de la Membrana Celular , Clostridium perfringens/genética , Femenino , Regulación Bacteriana de la Expresión Génica , Ratones , Ratones Endogámicos BALB C , Análisis de Secuencia de ARN , alfa-N-Acetilgalactosaminidasa/metabolismoRESUMEN
To obtain an insight into host-pathogen interactions in clostridial myonecrosis, we carried out comparative transcriptome analysis of both the bacterium and the host in a murine Clostridium perfringens infection model, which is the first time that such an investigation has been conducted. Analysis of the host transcriptome from infected muscle tissues indicated that many genes were upregulated compared to the results seen with mock-infected mice. These genes were enriched for host defense pathways, including Toll-like receptor (TLR) and Nod-like receptor (NLR) signaling components. Real-time PCR confirmed that host TLR2 and NLRP3 inflammasome genes were induced in response to C. perfringens infection. Comparison of the transcriptome of C. perfringens cells from the infected tissues with that from broth cultures showed that host selective pressure induced a global change in C. perfringens gene expression. A total of 33% (923) of C. perfringens genes were differentially regulated, including 10 potential virulence genes that were upregulated relative to their expression in vitro These genes encoded putative proteins that may be involved in the synthesis of cell wall-associated macromolecules, in adhesion to host cells, or in protection from host cationic antimicrobial peptides. This report presents the first successful expression profiling of coregulated transcriptomes of bacterial and host genes during a clostridial myonecrosis infection and provides new insights into disease pathogenesis and host-pathogen interactions.IMPORTANCEClostridium perfringens is the causative agent of traumatic clostridial myonecrosis, or gas gangrene. In this study, we carried out transcriptional analysis of both the host and the bacterial pathogen in a mouse myonecrosis infection. The results showed that in comparison to mock-infected control tissues, muscle tissues from C. perfringens-infected mice had a significantly altered gene expression profile. In particular, the expression of many genes involved in the innate immune system was upregulated. Comparison of the expression profiles of C. perfringens cells isolated from the infected tissues with those from equivalent broth cultures identified many potential virulence genes that were significantly upregulated in vivo These studies have provided a new understanding of the range of factors involved in host-pathogen interactions in a myonecrosis infection.
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Clostridium perfringens/patogenicidad , Gangrena Gaseosa/genética , Gangrena Gaseosa/inmunología , Animales , Femenino , Gangrena Gaseosa/microbiología , Interacciones Huésped-Patógeno , Inmunidad Innata/genética , Inmunidad Innata/fisiología , Inflamasomas/metabolismo , Ratones , Ratones Endogámicos BALB C , Transcriptoma/genética , Virulencia/genéticaRESUMEN
The extracellular bloodstream form parasite Trypanosoma brucei is supremely adapted to escape the host innate and adaptive immune system. Evasion is mediated through an antigenically variable Variant Surface Glycoprotein (VSG) coat, which is recycled at extraordinarily high rates. Blocking VSG synthesis triggers a precytokinesis arrest where stalled cells persist for days in vitro with superficially intact VSG coats, but are rapidly cleared within hours in mice. We therefore investigated the role of VSG synthesis in trypanosome phagocytosis by activated mouse macrophages. T. brucei normally effectively evades macrophages, and induction of VSG RNAi resulted in little change in phagocytosis of the arrested cells. Halting VSG synthesis resulted in stalled cells which swam directionally rather than tumbling, with a significant increase in swim velocity. This is possibly a consequence of increased rigidity of the cells due to a restricted surface coat in the absence of VSG synthesis. However if VSG RNAi was induced in the presence of anti-VSG221 antibodies, phagocytosis increased significantly. Blocking VSG synthesis resulted in reduced clearance of anti-VSG antibodies from the trypanosome surface, possibly as a consequence of the changed motility. This was particularly marked in cells in the G2/ M cell cycle stage, where the half-life of anti-VSG antibody increased from 39.3 ± 4.2 seconds to 99.2 ± 15.9 seconds after induction of VSG RNAi. The rates of internalisation of bulk surface VSG, or endocytic markers like transferrin, tomato lectin or dextran were not significantly affected by the VSG synthesis block. Efficient elimination of anti-VSG-antibody complexes from the trypanosome cell surface is therefore essential for trypanosome evasion of macrophages. These experiments highlight the essentiality of high rates of VSG recycling for the rapid removal of host opsonins from the parasite surface, and identify this process as a key parasite virulence factor during a chronic infection.
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Evasión Inmune/inmunología , Macrófagos/inmunología , Fagocitosis/inmunología , Tripanosomiasis Africana/inmunología , Glicoproteínas Variantes de Superficie de Trypanosoma/biosíntesis , Animales , Anticuerpos Antiprotozoarios/inmunología , Modelos Animales de Enfermedad , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Ratones , Imagen de Lapso de Tiempo , Trypanosoma brucei brucei/inmunología , Glicoproteínas Variantes de Superficie de Trypanosoma/inmunologíaRESUMEN
The ability of a pathogenic bacterium to scavenge iron from its host is important for its growth and survival during an infection. Our studies on C. perfringens gas gangrene strain JIR325, a derivative of strain 13, showed that it is capable of utilizing both human hemoglobin and ferric chloride, but not human holo-transferrin, as an iron source for in vitro growth. Analysis of the C. perfringens strain 13 genome sequence identified a putative heme acquisition system encoded by an iron-regulated surface gene region that we have named the Cht (Clostridium perfringens heme transport) locus. This locus comprises eight genes that are co-transcribed and includes genes that encode NEAT domain-containing proteins (ChtD and ChtE) and a putative sortase (Srt). The ChtD, ChtE and Srt proteins were shown to be expressed in JIR325 cells grown under iron-limited conditions and were localized to the cell envelope. Moreover, the NEAT proteins, ChtD and ChtE, were found to bind heme. Both chtDE and srt mutants were constructed, but these mutants were not defective in hemoglobin or ferric chloride utilization. They were, however, attenuated for virulence when tested in a mouse myonecrosis model, although the virulence phenotype could not be restored via complementation and, as is common with such systems, secondary mutations were identified in these strains. In summary, this study provides evidence for the functional redundancies that occur in the heme transport pathways of this life threatening pathogen.
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Clostridium perfringens/metabolismo , Hemo/metabolismo , Western Blotting , Electroforesis en Gel de Poliacrilamida , Unión Proteica , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transcripción GenéticaRESUMEN
Clostridium difficile is a global health burden and the leading cause of antibiotic-associated diarrhoea worldwide, causing severe gastrointestinal disease and death. Three well characterised toxins are encoded by this bacterium in two genetic loci, specifically, TcdB (toxin B) and TcdA (toxin A) in the Pathogenicity Locus (PaLoc) and binary toxin (CDT) in the genomically distinct CDT locus (CdtLoc). Toxin production is controlled by regulators specific to each locus. The orphan response regulator, CdtR, encoded within the CdtLoc, up-regulates CDT production. Until now there has been no suggestion that CdtR influences TcdA and TcdB production since it is not carried by all PaLoc-containing strains and CdtLoc is not linked genetically to PaLoc. Here we show that, in addition to CDT, CdtR regulates TcdA and TcdB production but that this effect is strain dependent. Of clinical relevance, CdtR increased the production of TcdA, TcdB and CDT in two epidemic ribotype 027 human strains, modulating their virulence in a mouse infection model. Strains traditionally from animal lineages, notably ribotype 078 strains, are increasingly being isolated from humans and their genetic and phenotypic analysis is critical for future studies on this important pathogen. Here we show that CdtR-mediated toxin regulation did not occur in other strain backgrounds, including a ribotype 078 animal strain. The finding that toxin gene regulation is strain dependent highlights the regulatory diversity between C. difficile isolates and the importance of studying virulence regulation in diverse lineages and clinically relevant strains. Our work provides the first evidence that TcdA, TcdB and CDT production is linked by a common regulatory mechanism and that CdtR may act as a global regulator of virulence in epidemic 027 strains.
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Clostridioides difficile/metabolismo , Enterocolitis Seudomembranosa/metabolismo , Regulación Bacteriana de la Expresión Génica/fisiología , Factores de Virulencia/biosíntesis , Virulencia/fisiología , ADP Ribosa Transferasas/biosíntesis , Animales , Proteínas Bacterianas/biosíntesis , Toxinas Bacterianas/biosíntesis , Western Blotting , Modelos Animales de Enfermedad , Enterotoxinas/biosíntesis , Masculino , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena de la PolimerasaRESUMEN
Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium that causes human gas gangrene (clostridial myonecrosis) and food poisoning. Early studies showed that virulence was regulated by the VirSR two-component signal transduction system. However, our identification of the RevR orphan response regulator indicated that more than one system was involved in controlling virulence. To further characterize this virulence-associated regulator, gel mobility shift experiments, coupled with DNase I footprinting, were used to identify the RevR DNA binding sequence. Bioinformatics analysis suggested that an orphan sensor histidine kinase, CPE1757 (renamed RevS), was the cognate sensor of RevR. Interaction between RevS and RevR was demonstrated by use of a bacterial two-hybrid system and validated by protein-protein interaction studies using biolayer interferometry. To assess the involvement of RevS in virulence regulation, the revS gene was inactivated by Targetron insertion. When isogenic wild-type, revS and complemented revS strains were tested in a mouse myonecrosis model, the revS mutant was found to be attenuated in virulence, which was similar to the attenuation observed previously with the revR mutant. However, transcriptional analysis of selected RevR-regulated genes in the revS mutant revealed a different pattern of expression to a revR mutant, suggesting that the RevSR system is more complex than originally thought. Taken together, the results have led to the identification and characterization of the two essential parts of a new regulatory network that is involved in the regulation of virulence in C. perfringens.
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Clostridium perfringens/fisiología , Regulación Bacteriana de la Expresión Génica , Histidina Quinasa/metabolismo , Transducción de Señal , Factores de Transcripción/metabolismo , Animales , Sitios de Unión , Clostridium perfringens/genética , Huella de ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Femenino , Técnicas de Inactivación de Genes , Prueba de Complementación Genética , Histidina Quinasa/genética , Ratones Endogámicos BALB C , Mutagénesis Insercional , Unión Proteica , Mapeo de Interacción de Proteínas , Factores de Transcripción/genética , Técnicas del Sistema de Dos Híbridos , VirulenciaRESUMEN
We conducted a retrospective study to assess the accuracy of fine-needle aspiration cytology (FNAC) in the diagnosis of Warthin tumor and to evaluate the subsequent risk of conservative nonsurgical management. We reviewed the records of 75 patients (76 tumors) with a parotid mass that had been diagnosed as a Warthin tumor by FNAC. This patient population was made up of 64 men and 11 women, aged 46 to 93 years (mean: 67). Of the 76 tumors, 40 were treated with surgical excision and 36 with conservative measures. Histology of the 40 excised parotid masses revealed that 38 (95%) were indeed Warthin tumors, 1 (2.5%) was a low-grade adenocarcinoma, and 1 was benign-not otherwise specified. None of the 36 tumors underwent malignant transformation either clinically or on repeat FNAC (if performed) during a follow-up of 4 to 120 months (mean: 55.5 ± 32.2). We conclude that conservative management of Warthin tumors confidently diagnosed on FNAC may be an option for patients who are unwilling or unable to undergo surgical excision.
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Adenocarcinoma/patología , Adenolinfoma/patología , Tratamiento Conservador , Glándula Parótida/cirugía , Neoplasias de la Parótida/patología , Adenocarcinoma/diagnóstico , Adenolinfoma/diagnóstico , Adenolinfoma/terapia , Anciano , Anciano de 80 o más Años , Biopsia con Aguja Fina , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias de la Parótida/diagnóstico , Neoplasias de la Parótida/terapia , Estudios Retrospectivos , Sensibilidad y EspecificidadRESUMEN
Bacterial pathogens have adopted numerous mechanisms for acquiring iron from host proteins during an infection, including the direct acquisition of ferric iron from heme-associated proteins or from iron-scavenging siderophores. Ferric iron then is transported into the cytosol, where it can be utilized by the bacterial pathogen. Under anaerobic conditions bacteria can also transport ferrous iron using the transmembrane complex FeoAB, but little is known about iron transport systems in anaerobic bacteria such as the pathogenic clostridia. In this study we sought to characterize the iron acquisition process in Clostridium perfringens. Bioinformatic analysis of the Clostridium perfringens strain 13 genome sequence revealed that it has seven potential iron acquisition systems: three siderophore-mediated systems, one ferric citrate uptake system, two heme-associated acquisition systems and one ferrous iron uptake system (FeoAB). The relative level of expression of these systems was determined using quantitative real-time RT-PCR assays that were specific for one gene from each system. Each of these genes was expressed, with the feoAB genes generating the most abundant iron-uptake related transcripts. To further examine the role of this system in the growth of C. perfringens, insertional inactivation was used to isolate a chromosomal feoB mutant. Growth of this mutant in the presence and absence of iron revealed that it had altered growth properties and a markedly reduced total iron and manganese content compared to the wild type; effects that were reversed upon complementation with the wild-type feoB gene. These studies suggest that under anaerobic conditions FeoB is the major protein required for the uptake of iron into the cell and that it may play an important role in the pathogenesis of C. perfringens infections.
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Proteínas Bacterianas/genética , Clostridium perfringens/genética , Proteínas de Transporte de Membrana/genética , Proteínas Bacterianas/metabolismo , Toxinas Bacterianas/biosíntesis , Clostridium perfringens/metabolismo , Hierro/metabolismo , Manganeso/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Mutación , Transcripción GenéticaRESUMEN
BACKGROUND: Clostridium perfringens causes toxin-mediated diseases, including gas gangrene (clostridial myonecrosis) and food poisoning in humans. The production of the toxins implicated in gas gangrene, α-toxin and perfringolysin O, is regulated by the VirSR two-component regulatory system. In addition, RevR, an orphan response regulator, has been shown to affect virulence in the mouse myonecrosis model. RevR positively regulates the expression of genes that encode hydrolytic enzymes, including hyaluronidases and sialidases. RESULTS: To further characterize the VirSR and RevR regulatory networks, comparative transcriptomic analysis was carried out with strand-specific RNA-seq on C. perfringens strain JIR325 and its isogenic virR and revR regulatory mutants. Using the edgeR analysis package, 206 genes in the virR mutant and 67 genes in the revR mutant were found to be differentially expressed. Comparative analysis revealed that VirR acts as a global negative regulator, whilst RevR acts as a global positive regulator. Therefore, about 95 % of the differentially expressed genes were up-regulated in the virR mutant, whereas 81 % of the differentially expressed genes were down-regulated in the revR mutant. Importantly, we identified 23 genes that were regulated by both VirR and RevR, 18 of these genes, which included the sporulation-specific spoIVA, sigG and sigF genes, were regulated positively and negatively by RevR and VirR, respectively. Furthermore, analysis of the mapped RNA-seq reads visualized as depth of coverage plots showed that there were 93 previously unannotated transcripts in intergenic regions. These transcripts potentially encode small RNA molecules. CONCLUSION: In conclusion, using strand-specific RNA-seq analysis, this study has identified differentially expressed chromosomal and pCP13 native plasmid-encoded genes, antisense transcripts, and transcripts within intergenic regions that are controlled by the VirSR or RevR regulatory systems.
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Proteínas Bacterianas/genética , Clostridium perfringens/genética , Mutación , Análisis de Secuencia de ARN/métodos , Perfilación de la Expresión Génica/métodos , Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Anotación de Secuencia MolecularRESUMEN
Among many other virulence factors, Clostridium perfringens produces three sialidases NanH, NanI and NanJ. NanH lacks a secretion signal peptide and is predicted to be an intracellular enzyme, while NanI and NanJ are secreted. Previously, we had identified part of an operon encoding NanE (epimerase) and NanA (sialic acid lyase) enzymes. Further analysis of the entire operon suggests that it encodes a complete pathway for the transport and metabolism of sialic acid along with a putative transcriptional regulator, NanR. The addition of 30 mM N-acetyl neuraminic acid (Neu5Ac) to a semi-defined medium significantly enhanced the growth yield of strain 13, suggesting that Neu5Ac can be used as a nutrient. C. perfringens strain 13 lacks a nanH gene, but has NanI- and NanJ-encoding genes. Analysis of nanI, nanJ, and nanInanJ mutants constructed by homologous recombination revealed that the expression of the major sialidase, NanI, was induced by the addition of Neu5Ac to the medium, and that in separate experiments, the same was true of a nanI-gusA transcriptional fusion. For the nanI and nanJ genes, primer extension identified three and two putative transcription start sites, respectively. Gel mobility shift assays using purified NanR and DNA from the promoter regions of the nanI and nanE genes showed high affinity, specific binding by NanR. We propose that NanR is a global regulator of sialic acid-associated genes and that it responds, in a positive feedback loop, to the concentration of sialic acid in the cell.
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Proteínas Bacterianas/genética , Infecciones por Clostridium/microbiología , Clostridium perfringens/enzimología , Clostridium perfringens/genética , Regulación Bacteriana de la Expresión Génica , Ácido N-Acetilneuramínico/metabolismo , Neuraminidasa/genética , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Clostridium perfringens/crecimiento & desarrollo , Clostridium perfringens/metabolismo , Genes Bacterianos , Humanos , Datos de Secuencia Molecular , Neuraminidasa/metabolismo , Operón , Regiones Promotoras Genéticas , Sitio de Iniciación de la Transcripción , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
The most common animal models used to study Clostridium perfringens infections in humans and animals are reviewed here. The classical C. perfringens-mediated histotoxic disease of humans is clostridial myonecrosis or gas gangrene and the use of a mouse myonecrosis model coupled with genetic studies has contributed greatly to our understanding of disease pathogenesis. Similarly, the use of a chicken model has enhanced our understanding of type A-mediated necrotic enteritis in poultry and has led to the identification of NetB as the primary toxin involved in disease. C. perfringens type A food poisoning is a highly prevalent bacterial illness in the USA and elsewhere. Rabbits and mice are the species most commonly used to study the action of enterotoxin, the causative toxin. Other animal models used to study the effect of this toxin are rats, non-human primates, sheep and cattle. In rabbits and mice, CPE produces severe necrosis of the small intestinal epithelium along with fluid accumulation. C. perfringens type D infection has been studied by inoculating epsilon toxin (ETX) intravenously into mice, rats, sheep, goats and cattle, and by intraduodenal inoculation of whole cultures of this microorganism in mice, sheep, goats and cattle. Molecular Koch's postulates have been fulfilled for enterotoxigenic C. perfringens type A in rabbits and mice, for C. perfringens type A necrotic enteritis and gas gangrene in chickens and mice, respectively, for C. perfringens type C in mice, rabbits and goats, and for C. perfringens type D in mice, sheep and goats.
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Infecciones por Clostridium/microbiología , Clostridium perfringens/patogenicidad , Gangrena Gaseosa/microbiología , Animales , Bovinos , Pollos , Modelos Animales de Enfermedad , Enterotoxinas/metabolismo , Cabras , Humanos , Ratones , Primates , Conejos , Ratas , OvinosRESUMEN
Clostridium septicum is the causative agent of atraumatic gas gangrene, with α-toxin, an extracellular pore-forming toxin, essential for disease. How C. septicum modulates the host's innate immune response is poorly defined, although α-toxin-intoxicated muscle cells undergo cellular oncosis, characterised by mitochondrial dysfunction and release of reactive oxygen species. Nonetheless, the signalling events that occur prior to the initiation of oncosis are poorly characterised. Our aims were to characterise the ability of α-toxin to activate the host mitogen activated protein kinase (MAPK) signalling pathway both in vitro and in vivo. Treatment of Vero cells with purified α-toxin activated the extracellular-signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 arms of the MAPK pathway and stimulated the release of TNF-α in a dose-dependent manner. Studies using inhibitors of all three MAPK components suggested that activation of ERK occurred in a Ras-c-Raf dependent manner, whereas activation of JNK and p38 occurred by a Ras-independent mechanism. Toxin-mediated activation was dependent on efficient receptor binding and pore formation and on an influx of extracellular calcium ions. In the mouse myonecrosis model we showed that the MAPK pathway was activated in tissues of infected mice, implying that it has an important role in the disease process.
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Toxinas Bacterianas/toxicidad , Clostridium septicum/química , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Quinasas Quinasa Quinasa PAM/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Proteínas Citotóxicas Formadoras de Poros/toxicidad , Proteínas Proto-Oncogénicas c-raf/metabolismo , Animales , Toxinas Bacterianas/genética , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Relación Dosis-Respuesta a Droga , Ratones , Músculo Esquelético/enzimología , Proteínas Citotóxicas Formadoras de Poros/genética , Sepsis/enzimología , Sepsis/microbiología , Bazo/enzimología , Células VeroRESUMEN
Prolonged ingestion of a cholesterol- or saturated fatty acid-enriched diet induces chronic, often systemic, auto-inflammatory responses resulting in significant health problems worldwide. In vivo information regarding the local and direct inflammatory effect of these dietary components in the intestine and, in particular, on the intestinal epithelium is lacking. Here we report that both mice and zebrafish exposed to high-fat (HFDs) or high-cholesterol (HCDs) diets develop acute innate inflammatory responses within hours, reflected in the localized interleukin-1ß-dependent accumulation of myeloid cells in the intestine. Acute HCD-induced intestinal inflammation is dependent on cholesterol uptake via Niemann-Pick C1-like 1 and inflammasome activation involving apoptosis-associated Speck-like protein containing a caspase recruitment domain, which leads to Caspase-1 activity in intestinal epithelial cells. Extended exposure to HCD results in localized, inflammation-dependent, functional dysregulation as well as systemic pathologies. Our model suggests that dietary cholesterol initiates intestinal inflammation in epithelial cells.
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Colesterol en la Dieta/efectos adversos , Dieta Alta en Grasa , Hígado Graso/etiología , Inflamación/inmunología , Mucosa Intestinal/inmunología , Animales , Benzoxazoles/farmacología , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Caspasa 1/genética , Caspasa 1/inmunología , Ácidos Grasos/metabolismo , Hígado Graso/genética , Hígado Graso/inmunología , Hígado Graso/patología , Femenino , Inmunidad Innata , Inflamasomas/efectos de los fármacos , Inflamación/etiología , Inflamación/genética , Inflamación/patología , Interleucina-1beta/biosíntesis , Interleucina-1beta/metabolismo , Mucosa Intestinal/patología , Intestinos/inmunología , Intestinos/patología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/inmunología , Ratones , Ratones Endogámicos BALB C , Células Mieloides/inmunología , Células Mieloides/patología , FN-kappa B/genética , FN-kappa B/inmunología , Proteína con Dominio Pirina 3 de la Familia NLR , Triazoles/farmacología , Pez CebraRESUMEN
Clostridium perfringens is a Gram-positive rod that is widely distributed in nature and is the etiological agent of several human and animal diseases. The complete genome sequence of C. perfringens strain 13 has been determined and multiple two-component signal transduction systems identified. One of these systems, designated here as the MalNO system, was analyzed in this study. Microarray analysis was used to carry out functional analysis of a malO mutant. The results, which were confirmed by quantitative reverse-transcriptase PCR, indicated that genes putatively involved in the uptake and metabolism of maltose were up-regulated in the malO mutant. These effects were reversed by complementation with the wild-type malO gene. Growth of these isogenic strains in medium with and without maltose showed that the malO mutant recovered more quickly from maltose deprivation when compared to the wild-type and complemented strains, leading to the conclusion that the MalNO system regulates maltose utilization in C. perfringens. It is postulated that this regulatory network may allow this soil bacterium and opportunistic pathogen to respond to environmental conditions where there are higher concentrations of maltose or maltodextrins, such as in the presence of decaying plant material in rich soil.
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Clostridium perfringens/genética , Clostridium perfringens/metabolismo , Regulación Bacteriana de la Expresión Génica , Maltosa/metabolismo , Transducción de Señal , Eliminación de Gen , Perfilación de la Expresión Génica , Prueba de Complementación Genética , Análisis por Micromatrices , Reacción en Cadena en Tiempo Real de la Polimerasa , Microbiología del SueloRESUMEN
TnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451 and Tn4453 in Clostridium perfringens and Clostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a C. perfringens plc virR double mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host C. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clonación Molecular/métodos , Clostridium perfringens/genética , ADN Bacteriano/genética , Escherichia coli/genética , Genoma Bacteriano , Recombinasas/metabolismo , Proteínas Bacterianas/genética , Clostridium perfringens/enzimología , ADN Nucleotidiltransferasas , Escherichia coli/metabolismo , Prueba de Complementación Genética , Recombinasas/genética , Recombinación GenéticaRESUMEN
Gas gangrene is a potentially fatal disease that is primarily caused by the ubiquitous, anaerobic bacteria Clostridium perfringens and Clostridium septicum. Treatment is limited to antibiotic therapy, debridement of the infected tissue, and, in severe cases, amputation. The need for new treatment approaches is compelling. Opioid-based analgesics such as buprenorphine and morphine also have immunomodulatory properties, usually leading to faster disease progression. However, here we show that mice pretreated with buprenorphine and morphine do not die from clostridial myonecrosis. Treatment with buprenorphine after the onset of infection also arrested disease development. Protection against myonecrotic disease was specific to C. perfringens-mediated myonecrosis; buprenorphine did not protect against disease caused by C. septicum infection even though infections due to both species are very similar. These data provide the first evidence of a protective role for opioids during infection and suggest that new therapeutic strategies may be possible for the treatment of C. perfringens-mediated myonecrosis.
Asunto(s)
Analgésicos Opioides/uso terapéutico , Buprenorfina/uso terapéutico , Clostridium perfringens , Gangrena Gaseosa/tratamiento farmacológico , Morfina/uso terapéutico , Animales , Femenino , Ratones , Ratones Endogámicos BALB C , Naltrexona/uso terapéuticoRESUMEN
The genus Clostridium comprises a large, heterogeneous group of obligate anaerobic, Gram-positive spore forming bacilli. Members of this genus are ubiquitous in the environment and although most species are considered saprophytic, several are pathogenic to both humans and animals. These bacteria cause a variety of diseases including neuroparalysis, gas gangrene, necrotic enteritis, food poisoning, toxic shock syndrome and pseudomembraneous colitis, which in most cases arise as a consequence of the production of potent exotoxins. Treatment options are often limited, underscoring the need for new treatment strategies and novel therapeutics. Understanding the fundamental mechanisms and signals that control toxin production in the pathogenic clostridia may lead to the identification of novel therapeutic targets that can be exploited in the development of new antimicrobial agents.
Asunto(s)
Toxinas Bacterianas/biosíntesis , Clostridium/metabolismo , Genes Bacterianos , Secuencia de Aminoácidos , Regulación Bacteriana de la Expresión Génica , Filogenia , Alineación de Secuencia , Transducción de SeñalRESUMEN
Clostridium perfringens is ubiquitous in nature and is often found as a commensal of the human and animal gastrointestinal tract. It is the primary etiological agent of clostridial myonecrosis, or gas gangrene, a serious infection that results in extensive tissue necrosis due to the action of one or more potent extracellular toxins. α-toxin and perfringolysin O are the major extracellular toxins involved in the pathogenesis of gas gangrene, but histotoxic strains of C. perfringens, such as strain 13, also produce many degradative enzymes such as collagenases, hyaluronidases, sialidases and the cysteine protease, α-clostripain. The production of many of these toxins is regulated either directly or indirectly by the global VirSR two-component signal transduction system. By isolating a chromosomal mutant and carrying out microarray analysis we have identified an orphan sensor histidine kinase, which we have named ReeS (regulator of extracellular enzymes sensor). Expression of the sialidase genes nanI and nanJ was down-regulated in a reeS mutant. Since complementation with the wild-type reeS gene restored nanI and nanJ expression to wild-type levels, as shown by quantitative reverse transcription-PCR and sialidase assays we concluded that ReeS positively regulates the expression of these sialidase genes. However, mutation of the reeS gene had no significant effect on virulence in the mouse myonecrosis model. Sialidase production in C. perfringens has been previously shown to be regulated by both the VirSR system and RevR. In this report, we have analyzed a previously unknown sensor histidine kinase, ReeS, and have shown that it also is involved in controlling the expression of sialidase genes, adding further complexity to the regulatory network that controls sialidase production in C. perfringens.
Asunto(s)
Clostridium perfringens/metabolismo , Neuraminidasa/biosíntesis , Proteínas Quinasas/metabolismo , Animales , Clostridium perfringens/citología , Clostridium perfringens/enzimología , Clostridium perfringens/genética , Espacio Extracelular/metabolismo , Femenino , Regulación Bacteriana de la Expresión Génica , Histidina Quinasa , Ratones , Ratones Endogámicos BALB C , Mutación , Neuraminidasa/genética , Proteínas Quinasas/genéticaRESUMEN
Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a ß-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a ß-pore-forming toxin. We carried out structural and functional studies of NetB to provide a mechanistic insight into its mode of action and to assist in the development of a necrotic enteritis vaccine. We determined the structure of the monomeric form of NetB to 1.8 Å, used both site-directed and random mutagenesis to identify key residues that are required for its biological activity, and analyzed pore formation by NetB and its substitution-containing derivatives in planar lipid bilayers.