RESUMO
Rebyota is a rectally administered fecal microbiota suspension for prevention of recurrence of Clostridioides difficile infection. The mechanism of action of Rebyota probably involves competitive exclusion of C. difficile by donor microbes with reduced toxin production; other factors may include restoration of protective taxa and modulation of the recipient's microbiome by phage, donor microbes, or metabolites.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Microbiota , Humanos , Transplante de Microbiota Fecal , Fezes , Infecções por Clostridium/terapia , RecidivaRESUMO
The emergence of hypervirulent clade 2 Clostridioides difficile is associated with severe symptoms and accounts for >20% of global infections. TcdB is a dominant virulence factor of C. difficile, and clade 2 strains exclusively express two TcdB variants (TcdB2 and TcdB4) that use unknown receptors distinct from the classic TcdB. Here, we performed CRISPR/Cas9 screens for TcdB4 and identified tissue factor pathway inhibitor (TFPI) as its receptor. Using cryo-EM, we determined a complex structure of the full-length TcdB4 with TFPI, defining a common receptor-binding region for TcdB. Residue variations within this region divide major TcdB variants into 2 classes: one recognizes Frizzled (FZD), and the other recognizes TFPI. TFPI is highly expressed in the intestinal glands, and recombinant TFPI protects the colonic epithelium from TcdB2/4. These findings establish TFPI as a colonic crypt receptor for TcdB from clade 2 C. difficile and reveal new mechanisms for CDI pathogenesis.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Proteínas de Bactérias/química , Toxinas Bacterianas/química , Clostridioides difficile/genética , Lipoproteínas/genéticaRESUMO
Due to the rapid emergence of antibiotic-resistant bacteria, there is a growing need to discover new antibiotics. To address this challenge, we trained a deep neural network capable of predicting molecules with antibacterial activity. We performed predictions on multiple chemical libraries and discovered a molecule from the Drug Repurposing Hub-halicin-that is structurally divergent from conventional antibiotics and displays bactericidal activity against a wide phylogenetic spectrum of pathogens including Mycobacterium tuberculosis and carbapenem-resistant Enterobacteriaceae. Halicin also effectively treated Clostridioides difficile and pan-resistant Acinetobacter baumannii infections in murine models. Additionally, from a discrete set of 23 empirically tested predictions from >107 million molecules curated from the ZINC15 database, our model identified eight antibacterial compounds that are structurally distant from known antibiotics. This work highlights the utility of deep learning approaches to expand our antibiotic arsenal through the discovery of structurally distinct antibacterial molecules.
Assuntos
Antibacterianos/farmacologia , Descoberta de Drogas/métodos , Aprendizado de Máquina , Tiadiazóis/farmacologia , Acinetobacter baumannii/efeitos dos fármacos , Animais , Antibacterianos/química , Quimioinformática/métodos , Clostridioides difficile/efeitos dos fármacos , Bases de Dados de Compostos Químicos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Mycobacterium tuberculosis/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Tiadiazóis/químicaRESUMO
Clostridium difficile infection (CDI) is facilitated by alteration of the microbiome following antibiotic administration. Antimicrobial therapy directed against the pathogen can treat CDI. Unfortunately, â¼20% of successfully treated patients will suffer recurrence. Bezlotoxumab, a human monoclonal antibody, binds to C. difficile toxin B (TcdB), reducing recurrence presumably by limiting epithelial damage and facilitating microbiome recovery.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Anticorpos Neutralizantes/uso terapêutico , Clostridioides difficile/fisiologia , Enterocolite Pseudomembranosa/tratamento farmacológico , Anticorpos Amplamente Neutralizantes , Microbioma Gastrointestinal , Humanos , Intestinos/efeitos dos fármacos , Prevenção SecundáriaRESUMO
Infections caused by Gram-negative pathogens are increasingly prevalent and are typically treated with broad-spectrum antibiotics, resulting in disruption of the gut microbiome and susceptibility to secondary infections1-3. There is a critical need for antibiotics that are selective both for Gram-negative bacteria over Gram-positive bacteria, as well as for pathogenic bacteria over commensal bacteria. Here we report the design and discovery of lolamicin, a Gram-negative-specific antibiotic targeting the lipoprotein transport system. Lolamicin has activity against a panel of more than 130 multidrug-resistant clinical isolates, shows efficacy in multiple mouse models of acute pneumonia and septicaemia infection, and spares the gut microbiome in mice, preventing secondary infection with Clostridioides difficile. The selective killing of pathogenic Gram-negative bacteria by lolamicin is a consequence of low sequence homology for the target in pathogenic bacteria versus commensals; this doubly selective strategy can be a blueprint for the development of other microbiome-sparing antibiotics.
Assuntos
Antibacterianos , Descoberta de Drogas , Microbioma Gastrointestinal , Bactérias Gram-Negativas , Infecções por Bactérias Gram-Negativas , Simbiose , Animais , Feminino , Humanos , Masculino , Camundongos , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Linhagem Celular , Clostridioides difficile/efeitos dos fármacos , Infecções por Clostridium/microbiologia , Infecções por Clostridium/tratamento farmacológico , Modelos Animais de Doenças , Desenho de Fármacos , Farmacorresistência Bacteriana Múltipla , Microbioma Gastrointestinal/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Infecções por Bactérias Gram-Negativas/microbiologia , Lipoproteínas/metabolismo , Camundongos Endogâmicos C57BL , Transporte Proteico/efeitos dos fármacos , Sepse/microbiologia , Sepse/tratamento farmacológico , Especificidade por Substrato , Simbiose/efeitos dos fármacosRESUMO
Iron is indispensable for almost all forms of life but toxic at elevated levels1-4. To survive within their hosts, bacterial pathogens have evolved iron uptake, storage and detoxification strategies to maintain iron homeostasis1,5,6. Recent studies showed that three Gram-negative environmental anaerobes produce iron-containing ferrosome granules7,8. However, it remains unclear whether ferrosomes are generated exclusively by Gram-negative bacteria. The Gram-positive bacterium Clostridioides difficile is the leading cause of nosocomial and antibiotic-associated infections in the USA9. Here we report that C. difficile undergoes an intracellular iron biomineralization process and stores iron in membrane-bound ferrosome organelles containing non-crystalline iron phosphate biominerals. We found that a membrane protein (FezA) and a P1B6-ATPase transporter (FezB), repressed by both iron and the ferric uptake regulator Fur, are required for ferrosome formation and play an important role in iron homeostasis during transition from iron deficiency to excess. Additionally, ferrosomes are often localized adjacent to cellular membranes as shown by cryo-electron tomography. Furthermore, using two mouse models of C. difficile infection, we demonstrated that the ferrosome system is activated in the inflamed gut to combat calprotectin-mediated iron sequestration and is important for bacterial colonization and survival during C. difficile infection.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Compostos Férricos , Interações entre Hospedeiro e Microrganismos , Ferro , Organelas , Animais , Camundongos , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/imunologia , Clostridioides difficile/metabolismo , Infecções por Clostridium/imunologia , Infecções por Clostridium/metabolismo , Infecções por Clostridium/microbiologia , Ferro/metabolismo , Organelas/metabolismo , Homeostase , Compostos Férricos/metabolismo , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Microscopia Crioeletrônica , Tomografia com Microscopia Eletrônica , Modelos Animais de Doenças , Complexo Antígeno L1 Leucocitário/metabolismo , Viabilidade Microbiana , Inflamação/metabolismo , Inflamação/microbiologia , Intestinos/metabolismo , Intestinos/microbiologiaRESUMO
Clostridioides difficile infection (CDI) is a major cause of healthcare-associated gastrointestinal infections1,2. The exaggerated colonic inflammation caused by C. difficile toxins such as toxin B (TcdB) damages tissues and promotes C. difficile colonization3-6, but how TcdB causes inflammation is unclear. Here we report that TcdB induces neurogenic inflammation by targeting gut-innervating afferent neurons and pericytes through receptors, including the Frizzled receptors (FZD1, FZD2 and FZD7) in neurons and chondroitin sulfate proteoglycan 4 (CSPG4) in pericytes. TcdB stimulates the secretion of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) from neurons and pro-inflammatory cytokines from pericytes. Targeted delivery of the TcdB enzymatic domain, through fusion with a detoxified diphtheria toxin, into peptidergic sensory neurons that express exogeneous diphtheria toxin receptor (an approach we term toxogenetics) is sufficient to induce neurogenic inflammation and recapitulates major colonic histopathology associated with CDI. Conversely, mice lacking SP, CGRP or the SP receptor (neurokinin 1 receptor) show reduced pathology in both models of caecal TcdB injection and CDI. Blocking SP or CGRP signalling reduces tissue damage and C. difficile burden in mice infected with a standard C. difficile strain or with hypervirulent strains expressing the TcdB2 variant. Thus, targeting neurogenic inflammation provides a host-oriented therapeutic approach for treating CDI.
Assuntos
Toxinas Bacterianas , Clostridioides difficile , Inflamação Neurogênica , Neurônios Aferentes , Pericitos , Animais , Camundongos , Toxinas Bacterianas/administração & dosagem , Toxinas Bacterianas/farmacologia , Peptídeo Relacionado com Gene de Calcitonina/antagonistas & inibidores , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Clostridioides difficile/patogenicidade , Infecções por Clostridium/microbiologia , Inflamação Neurogênica/induzido quimicamente , Inflamação Neurogênica/microbiologia , Inflamação Neurogênica/patologia , Pericitos/efeitos dos fármacos , Pericitos/microbiologia , Pericitos/patologia , Receptores da Neurocinina-1/metabolismo , Substância P/antagonistas & inibidores , Substância P/metabolismo , Neurônios Aferentes/efeitos dos fármacos , Neurônios Aferentes/microbiologia , Neurônios Aferentes/patologia , Mediadores da Inflamação/metabolismo , Ceco/efeitos dos fármacos , Ceco/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
Enteric pathogens are exposed to a dynamic polymicrobial environment in the gastrointestinal tract1. This microbial community has been shown to be important during infection, but there are few examples illustrating how microbial interactions can influence the virulence of invading pathogens2. Here we show that expansion of a group of antibiotic-resistant, opportunistic pathogens in the gut-the enterococci-enhances the fitness and pathogenesis of Clostridioides difficile. Through a parallel process of nutrient restriction and cross-feeding, enterococci shape the metabolic environment in the gut and reprogramme C. difficile metabolism. Enterococci provide fermentable amino acids, including leucine and ornithine, which increase C. difficile fitness in the antibiotic-perturbed gut. Parallel depletion of arginine by enterococci through arginine catabolism provides a metabolic cue for C. difficile that facilitates increased virulence. We find evidence of microbial interaction between these two pathogenic organisms in multiple mouse models of infection and patients infected with C. difficile. These findings provide mechanistic insights into the role of pathogenic microbiota in the susceptibility to and the severity of C. difficile infection.
Assuntos
Clostridioides difficile , Enterococcus , Interações Microbianas , Animais , Humanos , Camundongos , Antibacterianos/farmacologia , Arginina/deficiência , Arginina/metabolismo , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidade , Clostridioides difficile/fisiologia , Modelos Animais de Doenças , Farmacorresistência Bacteriana , Enterococcus/efeitos dos fármacos , Enterococcus/metabolismo , Enterococcus/patogenicidade , Enterococcus/fisiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Intestinos/efeitos dos fármacos , Intestinos/metabolismo , Intestinos/microbiologia , Leucina/metabolismo , Ornitina/metabolismo , Virulência , Suscetibilidade a DoençasRESUMO
Fidaxomicin (Fdx) is widely used to treat Clostridioides difficile (Cdiff) infections, but the molecular basis of its narrow-spectrum activity in the human gut microbiome remains unknown. Cdiff infections are a leading cause of nosocomial deaths1. Fidaxomicin, which inhibits RNA polymerase, targets Cdiff with minimal effects on gut commensals, reducing recurrence of Cdiff infection2,3. Here we present the cryo-electron microscopy structure of Cdiff RNA polymerase in complex with fidaxomicin and identify a crucial fidaxomicin-binding determinant of Cdiff RNA polymerase that is absent in most gut microbiota such as Proteobacteria and Bacteroidetes. By combining structural, biochemical, genetic and bioinformatic analyses, we establish that a single residue in Cdiff RNA polymerase is a sensitizing element for fidaxomicin narrow-spectrum activity. Our results provide a blueprint for targeted drug design against an important human pathogen.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Clostridioides , Infecções por Clostridium/tratamento farmacológico , Infecções por Clostridium/microbiologia , Microscopia Crioeletrônica , RNA Polimerases Dirigidas por DNA , Fidaxomicina/química , Fidaxomicina/farmacologia , Fidaxomicina/uso terapêutico , HumanosRESUMO
The obligate anaerobic, enteric pathogen Clostridioides difficile persists in the intestinal tract by forming antibiotic-resistant endospores that contribute to relapsing and recurrent infections. Despite the importance of sporulation for C. difficile pathogenesis, environmental cues and molecular mechanisms that regulate sporulation initiation remain ill-defined. Here, by using RIL-seq to globally capture the Hfq-dependent RNA-RNA interactome, we discovered a network of small RNAs that bind to mRNAs encoding sporulation-related genes. We show that two of these small RNAs, SpoX and SpoY, regulate translation of the master regulator of sporulation, Spo0A, in an opposing manner, which ultimately leads to altered sporulation rates. Infection of antibiotic-treated mice with SpoX and SpoY deletion mutants revealed a global effect on gut colonization and intestinal sporulation. Our work uncovers an elaborate RNA-RNA interactome controlling the physiology and virulence of C. difficile and identifies a complex post-transcriptional layer in the regulation of spore formation in this important human pathogen.
Assuntos
Clostridioides difficile , Clostridioides , Animais , Humanos , Camundongos , Clostridioides/genética , Clostridioides/metabolismo , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Antibacterianos , RNA/metabolismo , Esporos Bacterianos/genética , Esporos Bacterianos/metabolismo , Proteínas de Bactérias/metabolismo , Regulação Bacteriana da Expressão GênicaRESUMO
Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years, vancomycin resistance has emerged as a serious problem in several gram-positive pathogens, but high-level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here, we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified 2 distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity traits. Moreover, our data identify potential mutational routes to resistance that should be considered in genomic surveillance.
Assuntos
Antibacterianos , Clostridioides difficile , Resistência a Vancomicina , Vancomicina , Clostridioides difficile/efeitos dos fármacos , Clostridioides difficile/genética , Clostridioides difficile/patogenicidade , Resistência a Vancomicina/genética , Vancomicina/farmacologia , Antibacterianos/farmacologia , Aptidão Genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Humanos , Transdução de Sinais , Mutação , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Esporos Bacterianos/efeitos dos fármacos , Esporos Bacterianos/genéticaRESUMO
Several enteric pathogens can gain specific metabolic advantages over other members of the microbiota by inducing host pathology and inflammation. The pathogen Clostridium difficile is responsible for a toxin-mediated colitis that causes 450,000 infections and 15,000 deaths in the United States each year1; however, the molecular mechanisms by which C. difficile benefits from this pathology remain unclear. To understand how the metabolism of C. difficile adapts to the inflammatory conditions that its toxins induce, here we use RNA sequencing to define, in a mouse model, the metabolic states of wild-type C. difficile and of an isogenic mutant that lacks toxins. By combining bacterial and mouse genetics, we demonstrate that C. difficile uses sorbitol derived from both diet and host. Host-derived sorbitol is produced by the enzyme aldose reductase, which is expressed by diverse immune cells and is upregulated during inflammation-including during toxin-mediated disease induced by C. difficile. This work highlights a mechanism by which C. difficile can use a host-derived nutrient that is generated during toxin-induced disease by an enzyme that has not previously been associated with infection.
Assuntos
Toxinas Bacterianas/metabolismo , Clostridioides difficile/metabolismo , Clostridioides difficile/patogenicidade , Infecções por Clostridium/metabolismo , Infecções por Clostridium/microbiologia , Interações Hospedeiro-Patógeno , Sorbitol/metabolismo , Aldeído Redutase/metabolismo , Animais , Toxinas Bacterianas/biossíntese , Toxinas Bacterianas/genética , Clostridioides difficile/genética , Infecções por Clostridium/enzimologia , Colite/enzimologia , Colite/metabolismo , Colite/microbiologia , Feminino , Regulação Bacteriana da Expressão Gênica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MutaçãoRESUMO
Diet is a major factor that shapes the gut microbiome1, but the consequences of diet-induced changes in the microbiome for host pathophysiology remain poorly understood. We conducted a randomized human intervention study using a very-low-calorie diet (NCT01105143). Although metabolic health was improved, severe calorie restriction led to a decrease in bacterial abundance and restructuring of the gut microbiome. Transplantation of post-diet microbiota to mice decreased their body weight and adiposity relative to mice that received pre-diet microbiota. Weight loss was associated with impaired nutrient absorption and enrichment in Clostridioides difficile, which was consistent with a decrease in bile acids and was sufficient to replicate metabolic phenotypes in mice in a toxin-dependent manner. These results emphasize the importance of diet-microbiome interactions in modulating host energy balance and the need to understand the role of diet in the interplay between pathogenic and beneficial symbionts.
Assuntos
Bactérias/isolamento & purificação , Bactérias/metabolismo , Restrição Calórica , Dieta Redutora , Microbioma Gastrointestinal/fisiologia , Adiposidade , Animais , Bactérias/crescimento & desenvolvimento , Bactérias/patogenicidade , Toxinas Bacterianas/metabolismo , Ácidos e Sais Biliares/metabolismo , Peso Corporal , Clostridioides difficile/crescimento & desenvolvimento , Clostridioides difficile/isolamento & purificação , Clostridioides difficile/metabolismo , Metabolismo Energético , Humanos , Absorção Intestinal , Masculino , Camundongos , Nutrientes/metabolismo , Simbiose , Redução de PesoRESUMO
Antibiotics are used to fight pathogens but also target commensal bacteria, disturbing the composition of gut microbiota and causing dysbiosis and disease1. Despite this well-known collateral damage, the activity spectrum of different antibiotic classes on gut bacteria remains poorly characterized. Here we characterize further 144 antibiotics from a previous screen of more than 1,000 drugs on 38 representative human gut microbiome species2. Antibiotic classes exhibited distinct inhibition spectra, including generation dependence for quinolones and phylogeny independence for ß-lactams. Macrolides and tetracyclines, both prototypic bacteriostatic protein synthesis inhibitors, inhibited nearly all commensals tested but also killed several species. Killed bacteria were more readily eliminated from in vitro communities than those inhibited. This species-specific killing activity challenges the long-standing distinction between bactericidal and bacteriostatic antibiotic classes and provides a possible explanation for the strong effect of macrolides on animal3-5 and human6,7 gut microbiomes. To mitigate this collateral damage of macrolides and tetracyclines, we screened for drugs that specifically antagonized the antibiotic activity against abundant Bacteroides species but not against relevant pathogens. Such antidotes selectively protected Bacteroides species from erythromycin treatment in human-stool-derived communities and gnotobiotic mice. These findings illluminate the activity spectra of antibiotics in commensal bacteria and suggest strategies to circumvent their adverse effects on the gut microbiota.
Assuntos
Antibacterianos/efeitos adversos , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Animais , Antibacterianos/classificação , Bactérias/classificação , Bactérias Anaeróbias/efeitos dos fármacos , Bacteroides/efeitos dos fármacos , Clostridioides difficile/efeitos dos fármacos , Dicumarol/farmacologia , Eritromicina/farmacologia , Fezes/microbiologia , Feminino , Vida Livre de Germes , Humanos , Macrolídeos/farmacologia , Masculino , Camundongos , Microbiota/efeitos dos fármacos , Simbiose/efeitos dos fármacos , Tetraciclinas/farmacologiaRESUMO
Most bacteria are surrounded by a cell wall that contains peptidoglycan (PG), a large polymer composed of glycan strands held together by short peptide cross-links. There are two major types of cross-links, termed 4-3 and 3-3 based on the amino acids involved. 4-3 cross-links are created by penicillin-binding proteins, while 3-3 cross-links are created by L,D-transpeptidases (LDTs). In most bacteria, the predominant mode of cross-linking is 4-3, and these cross-links are essential for viability, while 3-3 cross-links comprise only a minor fraction and are not essential. However, in the opportunistic intestinal pathogen Clostridioides difficile, about 70% of the cross-links are 3-3. We show here that 3-3 cross-links and LDTs are essential for viability in C. difficile. We also show that C. difficile has five LDTs, three with a YkuD catalytic domain as in all previously known LDTs and two with a VanW catalytic domain, whose function was until now unknown. The five LDTs exhibit extensive functional redundancy. VanW domain proteins are found in many gram-positive bacteria but scarce in other lineages. We tested seven non-C. difficile VanW domain proteins and confirmed LDT activity in three cases. In summary, our findings uncover a previously unrecognized family of PG cross-linking enzymes, assign a catalytic function to VanW domains, and demonstrate that 3-3 cross-linking is essential for viability in C. difficile, the first time this has been shown in any bacterial species. The essentiality of LDTs in C. difficile makes them potential targets for antibiotics that kill C. difficile selectively.
Assuntos
Proteínas de Bactérias , Parede Celular , Clostridioides difficile , Peptidoglicano , Clostridioides difficile/enzimologia , Clostridioides difficile/metabolismo , Peptidoglicano/metabolismo , Parede Celular/metabolismo , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/química , Peptidoglicano Glicosiltransferase/metabolismo , Peptidoglicano Glicosiltransferase/química , Peptidoglicano Glicosiltransferase/genéticaRESUMO
Interleukin 22 (IL-22) promotes intestinal barrier integrity, stimulating epithelial cells to enact defense mechanisms against enteric infections, including the production of antimicrobial peptides. IL-22 binding protein (IL-22BP) is a soluble decoy encoded by the Il22ra2 gene that decreases IL-22 bioavailability, attenuating IL-22 signaling. The impact of IL-22BP on gut microbiota composition and functioning is poorly understood. We found that Il22ra2-/- mice are better protected against Clostridioides difficile and Citrobacter rodentium infections. This protection relied on IL-22-induced antimicrobial mechanisms before the infection occurred, rather than during the infection itself. Indeed, the gut microbiota of Il22ra2-/- mice mitigated infection of wild-type (WT) mice when transferred via cohousing or by cecal microbiota transplantation. Indicator species analysis of WT and Il22ra2-/- mice with and without cohousing disclosed that IL22BP deficiency yields a gut bacterial composition distinct from that of WT mice. Manipulation of dietary fiber content, measurements of intestinal short-chain fatty acids and oral treatment with acetate disclosed that resistance to C. difficile infection is related to increased production of acetate by Il22ra2-/--associated microbiota. Together, these findings suggest that IL-22BP represents a potential therapeutic target for those at risk for or with already manifest infection with this and perhaps other enteropathogens.
Assuntos
Citrobacter rodentium , Clostridioides difficile , Infecções por Enterobacteriaceae , Microbioma Gastrointestinal , Interleucina 22 , Camundongos Knockout , Animais , Camundongos , Infecções por Enterobacteriaceae/imunologia , Infecções por Enterobacteriaceae/microbiologia , Infecções por Enterobacteriaceae/prevenção & controle , Receptores de Interleucina/metabolismo , Receptores de Interleucina/genética , Interleucinas/metabolismo , Camundongos Endogâmicos C57BL , Infecções por Clostridium/imunologia , Infecções por Clostridium/microbiologia , Infecções por Clostridium/prevenção & controleRESUMO
Spore formation is required for environmental survival and transmission of the human enteropathogenic Clostridioides difficile. In all bacterial spore formers, sporulation is regulated through activation of the master response regulator, Spo0A. However, the factors and mechanisms that directly regulate C. difficile Spo0A activity are not defined. In the well-studied Bacillus species, Spo0A is directly inactivated by Spo0E, a small phosphatase. To understand Spo0E function in C. difficile, we created a null mutation of the spo0E ortholog and assessed sporulation and physiology. The spo0E mutant produced significantly more spores, demonstrating Spo0E represses C. difficile sporulation. Unexpectedly, the spo0E mutant also exhibited increased motility and toxin production, and enhanced virulence in animal infections. We uncovered that Spo0E interacts with both Spo0A and the toxin and motility regulator, RstA. Direct interactions between Spo0A, Spo0E, and RstA constitute a previously unknown molecular switch that coordinates sporulation with motility and toxin production. Reinvestigation of Spo0E function in B. subtilis revealed that Spo0E induced motility, demonstrating Spo0E regulation of motility and sporulation among divergent species. Further, 3D structural analyses of Spo0E revealed specific and exclusive interactions between Spo0E and binding partners in C. difficile and B. subtilis that provide insight into the conservation of this regulatory mechanism among different species.
Assuntos
Proteínas de Bactérias , Clostridioides difficile , Regulação Bacteriana da Expressão Gênica , Esporos Bacterianos , Clostridioides difficile/patogenicidade , Clostridioides difficile/genética , Clostridioides difficile/metabolismo , Esporos Bacterianos/genética , Virulência , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Animais , Camundongos , Infecções por Clostridium/microbiologiaRESUMO
Keratinicyclins and keratinimicins are recently discovered glycopeptide antibiotics. Keratinimicins show broad-spectrum activity against Gram-positive bacteria, while keratinicyclins form a new chemotype by virtue of an unusual oxazolidinone moiety and exhibit specific antibiosis against Clostridioides difficile. Here we report the mechanism of action of keratinicyclin B (KCB). We find that steric constraints preclude KCB from binding peptidoglycan termini. Instead, KCB inhibits C. difficile growth by binding wall teichoic acids (WTAs) and interfering with cell wall remodeling. A computational model, guided by biochemical studies, provides an image of the interaction of KCB with C. difficile WTAs and shows that the same H-bonding framework used by glycopeptide antibiotics to bind peptidoglycan termini is used by KCB for interacting with WTAs. Analysis of KCB in combination with vancomycin (VAN) shows highly synergistic and specific antimicrobial activity, and that nanomolar combinations of the two drugs are sufficient for complete growth inhibition of C. difficile, while leaving common commensal strains unaffected.
Assuntos
Antibacterianos , Clostridioides difficile , Testes de Sensibilidade Microbiana , Clostridioides difficile/efeitos dos fármacos , Antibacterianos/farmacologia , Antibacterianos/química , Vancomicina/farmacologia , Vancomicina/química , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Ácidos Teicoicos/metabolismo , Peptidoglicano/metabolismo , Peptidoglicano/química , Quimioterapia Combinada , Peptídeos Cíclicos , LipopeptídeosRESUMO
In the human gut, the growth of the pathogen Clostridioides difficile is impacted by a complex web of interspecies interactions with members of human gut microbiota. We investigate the contribution of interspecies interactions on the antibiotic response of C. difficile to clinically relevant antibiotics using bottom-up assembly of human gut communities. We identify 2 classes of microbial interactions that alter C. difficile's antibiotic susceptibility: interactions resulting in increased ability of C. difficile to grow at high antibiotic concentrations (rare) and interactions resulting in C. difficile growth enhancement at low antibiotic concentrations (common). Based on genome-wide transcriptional profiling data, we demonstrate that metal sequestration due to hydrogen sulfide production by the prevalent gut species Desulfovibrio piger increases the minimum inhibitory concentration (MIC) of metronidazole for C. difficile. Competition with species that display higher sensitivity to the antibiotic than C. difficile leads to enhanced growth of C. difficile at low antibiotic concentrations due to competitive release. A dynamic computational model identifies the ecological principles driving this effect. Our results provide a deeper understanding of ecological and molecular principles shaping C. difficile's response to antibiotics, which could inform therapeutic interventions.
Assuntos
Clostridioides difficile , Infecções por Clostridium , Microbioma Gastrointestinal , Humanos , Antibacterianos/farmacologia , ClostridioidesRESUMO
Wnt signaling pathways are transmitted via 10 homologous frizzled receptors (FZD1-10) in humans. Reagents broadly inhibiting Wnt signaling pathways reduce growth and metastasis of many tumors, but their therapeutic development has been hampered by the side effect. Inhibitors targeting specific Wnt-FZD pair(s) enriched in cancer cells may reduce side effect, but the therapeutic effect of narrow-spectrum Wnt-FZD inhibitors remains to be established in vivo. Here, we developed a fragment of C. difficile toxin B (TcdBFBD), which recognizes and inhibits a subclass of FZDs, FZD1/2/7, and examined whether targeting this FZD subgroup may offer therapeutic benefits for treating breast cancer models in mice. Utilizing 2 basal-like and 1 luminal-like breast cancer models, we found that TcdBFBD reduces tumor-initiating cells and attenuates growth of basal-like mammary tumor organoids and xenografted tumors, without damaging Wnt-sensitive tissues such as bones in vivo. Furthermore, FZD1/2/7-positive cells are enriched in chemotherapy-resistant cells in both basal-like and luminal mammary tumors treated with cisplatin, and TcdBFBD synergizes strongly with cisplatin in inhibiting both tumor types. These data demonstrate the therapeutic value of narrow-spectrum Wnt signaling inhibitor in treating breast cancers.