RESUMO
Clostridioides difficile is one of the leading causes of antibiotic-associated diarrhea. Gut microbiota-derived secondary bile acids and commensal Clostridia that carry the bile acid-inducible (bai) operon are associated with protection from C. difficile infection (CDI), although the mechanism is not known. In this study, we hypothesized that commensal Clostridia are important for providing colonization resistance against C. difficile due to their ability to produce secondary bile acids, as well as potentially competing against C. difficile for similar nutrients. To test this hypothesis, we examined the abilities of four commensal Clostridia carrying the bai operon (Clostridium scindens VPI 12708, C. scindens ATCC 35704, Clostridium hiranonis, and Clostridium hylemonae) to convert cholate (CA) to deoxycholate (DCA) in vitro, and we determined whether the amount of DCA produced was sufficient to inhibit the growth of a clinically relevant C. difficile strain. We also investigated the competitive relationships between these commensals and C. difficile using an in vitro coculture system. We found that inhibition of C. difficile growth by commensal Clostridia supplemented with CA was strain dependent, correlated with the production of â¼2 mM DCA, and increased the expression of bai operon genes. We also found that C. difficile was able to outcompete all four commensal Clostridia in an in vitro coculture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics. Future studies dissecting the regulation of the bai operon in vitro and in vivo and how this affects CDI will be important.IMPORTANCE Commensal Clostridia carrying the bai operon, such as C. scindens, have been associated with protection against CDI; however, the mechanism for this protection is unknown. Herein, we show four commensal Clostridia that carry the bai operon and affect C. difficile growth in a strain-dependent manner, with and without the addition of cholate. Inhibition of C. difficile by commensals correlated with the efficient conversion of cholate to deoxycholate, a secondary bile acid that inhibits C. difficile germination, growth, and toxin production. Competition studies also revealed that C. difficile was able to outcompete the commensals in an in vitro coculture system. These studies are instrumental in understanding the relationship between commensal Clostridia and C. difficile in the gut, which is vital for designing targeted bacterial therapeutics.
Assuntos
Ácidos e Sais Biliares/metabolismo , Clostridiales/fisiologia , Clostridioides difficile/fisiologia , Infecções por Clostridium/microbiologia , Clostridium histolyticum/fisiologia , Antibiose , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Clostridiales/genética , Clostridioides difficile/crescimento & desenvolvimento , Infecções por Clostridium/metabolismo , Clostridium histolyticum/genética , Humanos , Óperon , Especificidade da Espécie , SimbioseRESUMO
Efficacy of glucokinase activation on glycemic control is limited to a short-term period. One reason might be related to excess glucose signaling by glucokinase activation toward ß-cells. In this study, we investigated the effect of glucokinase haploinsufficiency on glucose tolerance as well as ß-cell function and mass using a mouse model of type 2 diabetes. Our results showed that in db/db mice with glucokinase haploinsufficiency, glucose tolerance was ameliorated by augmented insulin secretion associated with the increase in ß-cell mass when compared with db/db mice. Gene expression profiling and immunohistochemical and metabolomic analyses revealed that glucokinase haploinsufficiency in the islets of db/db mice was associated with lower expression of stress-related genes, greater expression of transcription factors involved in the maintenance and maturation of ß-cell function, less mitochondrial damage, and a superior metabolic pattern. These effects of glucokinase haploinsufficiency could preserve ß-cell mass under diabetic conditions. These findings verified our hypothesis that optimizing excess glucose signaling in ß-cells by inhibiting glucokinase could prevent ß-cell insufficiency, leading to improving glucose tolerance in diabetes status by preserving ß-cell mass. Therefore, glucokinase inactivation in ß-cells, paradoxically, could be a potential strategy for the treatment of type 2 diabetes.
Assuntos
Glucoquinase/metabolismo , Glucose/metabolismo , Metaboloma/fisiologia , Animais , Clostridium histolyticum/fisiologia , Glucoquinase/genética , Glucose/genética , Teste de Tolerância a Glucose , Imuno-Histoquímica , Células Secretoras de Insulina/metabolismo , Metaboloma/genética , Camundongos , Reação em Cadeia da Polimerase em Tempo RealRESUMO
In this study, for the first time, Clostridium histolyticum lethal factor was purified by ammonium sulfate precipitation of culture broth, centrifugation through an Amicon filter device and hydrophobic interaction chromatography. The purified lethal factor was devoid of proteolytic activity. At a concentration of 150 ng mL(-1) the lethal factor killed 50% of HeLa cells within 24 h of exposure. Abrogation of actin filaments, activation of caspases, fragmentation of nuclear DNA as well as ultrastructural changes indicated that the cell death occurred by apoptosis. The apoptotic action of the lethal factor is in agreement with changes induced in animal tissues by administration of C. histolyticum culture medium.
Assuntos
Apoptose , Toxinas Bacterianas/toxicidade , Clostridium histolyticum/fisiologia , Citoesqueleto de Actina/metabolismo , Toxinas Bacterianas/isolamento & purificação , Caspases/metabolismo , Sobrevivência Celular , Fragmentação do DNA , Células HeLa , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de FluorescênciaRESUMO
Clostridium histolyticum vacuolating cytotoxin was partially purified from culture broth using ammonium sulfate precipitation, gel filtration and hydrophobic interaction chromatography. The toxin caused vacuolization of HeLa cells visible under a light microscope after 2 h and distinct after 8 h. Transmission electron microscopy revealed the presence of numerous vacuoles, condensation of the mitochondrial matrix, increased cytoplasm density and increased amounts of heterochromatin. Apoptosis was not detected either by electron microscopy or by an apoptosis/necrosis discrimination assay with fluorescein-labeled annexin V and propidium iodide, or DNA fragmentation assay. Calcium ion influx was detected by flow cytometry after labeling cells with Fluo-4 AM. Vacuolation of HeLa cells by C. histolyticum cytotoxin was inhibited by bafilomycin A1, suggesting involvement of H+ -ATPase in the formation of vacuoles.
Assuntos
Clostridium histolyticum/química , Citotoxinas/isolamento & purificação , Citotoxinas/farmacologia , Vacúolos/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Cálcio/metabolismo , Infecções por Clostridium/microbiologia , Clostridium histolyticum/fisiologia , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Células HeLa , Humanos , Marcação In Situ das Extremidades Cortadas , Macrolídeos/farmacologia , Microscopia Eletrônica de Transmissão , Vermelho Neutro/farmacocinética , Sódio/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/microbiologia , Vacúolos/fisiologiaRESUMO
Evidence shows that nutritional and environmental stress stimuli during postnatal period influence brain development and interactions between gut and brain. In this study we show that in rats, prevention of weaning from maternal milk results in depressive-like behavior, which is accompanied by changes in the gut bacteria and host metabolism. Depressive-like behavior was studied using the forced-swim test on postnatal day (PND) 25 in rats either weaned on PND 21, or left with their mother until PND 25 (non-weaned). Non-weaned rats showed an increased immobility time consistent with a depressive phenotype. Fluorescence in situ hybridization showed non-weaned rats to harbor significantly lowered Clostridium histolyticum bacterial groups but exhibit marked stress-induced increases. Metabonomic analysis of urine from these animals revealed significant differences in the metabolic profiles, with biochemical phenotypes indicative of depression in the non-weaned animals. In addition, non-weaned rats showed resistance to stress-induced modulation of oxytocin receptors in amygdala nuclei, which is indicative of passive stress-coping mechanism. We conclude that delaying weaning results in alterations to the gut microbiota and global metabolic profiles which may contribute to a depressive phenotype and raise the issue that mood disorders at early developmental ages may reflect interplay between mammalian host and resident bacteria.