RESUMEN
Infections of the lung cause observable sickness thought to be secondary to inflammation. Signs of sickness are crucial to alert others via behavioral-immune responses to limit contact with contagious individuals. Gram-negative bacteria produce exopolysaccharide (EPS) that provides microbial protection; however, the impact of EPS on sickness remains uncertain. Using genome-engineered Pseudomonas aeruginosa (P. aeruginosa) strains, we compared EPS-producers versus non-producers and a virulent Escherichia coli (E. coli) lung infection model in male and female mice. EPS-negative P. aeruginosa and virulent E. coli infection caused severe sickness, behavioral alterations, inflammation, and hypothermia mediated by TLR4 detection of the exposed lipopolysaccharide (LPS) in lung TRPV1+ sensory neurons. However, inflammation did not account for sickness. Stimulation of lung nociceptors induced acute stress responses in the paraventricular hypothalamic nuclei by activating corticotropin-releasing hormone neurons responsible for sickness behavior and hypothermia. Thus, EPS-producing biofilm pathogens evade initiating a lung-brain sensory neuronal response that results in sickness.
Asunto(s)
Infecciones por Escherichia coli , Escherichia coli , Pulmón , Polisacáridos Bacterianos , Infecciones por Pseudomonas , Pseudomonas aeruginosa , Animales , Femenino , Masculino , Ratones , Biopelículas , Escherichia coli/fisiología , Hipotermia/metabolismo , Hipotermia/patología , Inflamación/metabolismo , Inflamación/patología , Pulmón/microbiología , Pulmón/patología , Neumonía/microbiología , Neumonía/patología , Pseudomonas aeruginosa/fisiología , Células Receptoras Sensoriales , Polisacáridos Bacterianos/metabolismo , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Infecciones por Pseudomonas/metabolismo , Infecciones por Pseudomonas/microbiología , Infecciones por Pseudomonas/patología , Nociceptores/metabolismoRESUMEN
Ribonucleotide reductases (RNRs) catalyze the de novo conversion of nucleotides to deoxynucleotides in all organisms, controlling their relative ratios and abundance. In doing so, they play an important role in fidelity of DNA replication and repair. RNRs' central role in nucleic acid metabolism has resulted in five therapeutics that inhibit human RNRs. In this review, we discuss the structural, dynamic, and mechanistic aspects of RNR activity and regulation, primarily for the human and Escherichia coli class Ia enzymes. The unusual radical-based organic chemistry of nucleotide reduction, the inorganic chemistry of the essential metallo-cofactor biosynthesis/maintenance, the transport of a radical over a long distance, and the dynamics of subunit interactions all present distinct entry points toward RNR inhibition that are relevant for drug discovery. We describe the current mechanistic understanding of small molecules that target different elements of RNR function, including downstream pathways that lead to cell cytotoxicity. We conclude by summarizing novel and emergent RNR targeting motifs for cancer and antibiotic therapeutics.
Asunto(s)
Antibacterianos/química , Antineoplásicos/química , Infecciones por Escherichia coli/tratamiento farmacológico , Neoplasias/tratamiento farmacológico , Nucleótidos/metabolismo , Ribonucleótido Reductasas/química , Antibacterianos/uso terapéutico , Antineoplásicos/uso terapéutico , Biocatálisis , Descubrimiento de Drogas/métodos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Escherichia coli/efectos de los fármacos , Escherichia coli/enzimología , Escherichia coli/genética , Infecciones por Escherichia coli/enzimología , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Humanos , Simulación del Acoplamiento Molecular , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/patología , Nucleótidos/química , Oxidación-Reducción , Estructura Secundaria de Proteína , Subunidades de Proteína/antagonistas & inhibidores , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Ribonucleótido Reductasas/antagonistas & inhibidores , Ribonucleótido Reductasas/genética , Ribonucleótido Reductasas/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-ActividadRESUMEN
The rapid emergence of antibiotic-resistant infections is prompting increased interest in phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the successful development of phage therapies. Through natural evolution and structural modeling, we identified host-range-determining regions (HRDRs) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional diversity while minimizing disruptions to the overall tail fiber structure, resulting in synthetic "phagebodies." We showed that mutating HRDRs yields phagebodies with altered host-ranges, and select phagebodies enable long-term suppression of bacterial growth in vitro, by preventing resistance appearance, and are functional in vivo using a murine model. We anticipate that this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications.
Asunto(s)
Bacteriófago T3/genética , Infecciones por Escherichia coli/terapia , Escherichia coli/virología , Terapia de Fagos/métodos , Enfermedades Cutáneas Bacterianas/terapia , Proteínas de la Cola de los Virus/genética , Animales , Farmacorresistencia Bacteriana , Especificidad del Huésped , Ratones , Mutagénesis Sitio-DirigidaRESUMEN
Intergenerational inheritance of immune traits linked to epigenetic modifications has been demonstrated in plants and invertebrates. Here we provide evidence for transmission of trained immunity across generations to murine progeny that survived a sublethal systemic infection with Candida albicans or a zymosan challenge. The progeny of trained mice exhibited cellular, developmental, transcriptional and epigenetic changes associated with the bone marrow-resident myeloid effector and progenitor cell compartment. Moreover, the progeny of trained mice showed enhanced responsiveness to endotoxin challenge, alongside improved protection against systemic heterologous Escherichia coli and Listeria monocytogenes infections. Sperm DNA of parental male mice intravenously infected with the fungus C. albicans showed DNA methylation differences linked to immune gene loci. These results provide evidence for inheritance of trained immunity in mammals, enhancing protection against infections.
Asunto(s)
Candida albicans/inmunología , Candidiasis/inmunología , Infecciones por Escherichia coli/inmunología , Escherichia coli/inmunología , Herencia , Inmunidad Innata/genética , Listeria monocytogenes/inmunología , Listeriosis/inmunología , Células Mieloides/inmunología , Animales , Candida albicans/patogenicidad , Candidiasis/genética , Candidiasis/metabolismo , Candidiasis/microbiología , Células Cultivadas , Metilación de ADN , Modelos Animales de Enfermedad , Epigénesis Genética , Escherichia coli/patogenicidad , Infecciones por Escherichia coli/genética , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Interacciones Huésped-Patógeno , Listeria monocytogenes/patogenicidad , Listeriosis/genética , Listeriosis/metabolismo , Listeriosis/microbiología , Masculino , Ratones Transgénicos , Células Mieloides/metabolismo , Células Mieloides/microbiología , Espermatozoides/inmunología , Espermatozoides/metabolismo , Transcripción GenéticaRESUMEN
The full neutrophil heterogeneity and differentiation landscape remains incompletely characterized. Here, we profiled >25,000 differentiating and mature mouse neutrophils using single-cell RNA sequencing to provide a comprehensive transcriptional landscape of neutrophil maturation, function and fate decision in their steady state and during bacterial infection. Eight neutrophil populations were defined by distinct molecular signatures. The three mature peripheral blood neutrophil subsets arise from distinct maturing bone marrow neutrophil subsets. Driven by both known and uncharacterized transcription factors, neutrophils gradually acquire microbicidal capability as they traverse the transcriptional landscape, representing an evolved mechanism for fine-tuned regulation of an effective but balanced neutrophil response. Bacterial infection reprograms the genetic architecture of neutrophil populations, alters dynamic transitions between subpopulations and primes neutrophils for augmented functionality without affecting overall heterogeneity. In summary, these data establish a reference model and general framework for studying neutrophil-related disease mechanisms, biomarkers and therapeutic targets at single-cell resolution.
Asunto(s)
Infecciones por Escherichia coli/inmunología , Escherichia coli/fisiología , Neutrófilos/fisiología , Peritonitis/inmunología , Análisis de la Célula Individual/métodos , Animales , Diferenciación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Femenino , Perfilación de la Expresión Génica , Homeostasis , Humanos , Ratones , Análisis de Secuencia de ARNRESUMEN
Females have an overall advantage over males in resisting Gram-negative bacteremias, thus hinting at sexual dimorphism of immunity during infections. Here, through intravital microscopy, we observed a sex-biased difference in the capture of blood-borne bacteria by liver macrophages, a process that is critical for the clearance of systemic infections. Complement opsonization was indispensable for the capture of enteropathogenic Escherichia coli (EPEC) in male mice; however, a faster complement component 3-independent process involving abundant preexisting antibodies to EPEC was detected in female mice. These antibodies were elicited predominantly in female mice at puberty in response to estrogen regardless of microbiota-colonization conditions. Estrogen-driven antibodies were maternally transferrable to offspring and conferred protection during infancy. These antibodies were conserved in humans and recognized specialized oligosaccharides integrated into the bacterial lipopolysaccharide and capsule. Thus, an estrogen-driven, innate antibody-mediated immunological strategy conferred protection to females and their offspring.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Infecciones por Escherichia coli/inmunología , Inmunidad Innata/inmunología , Caracteres Sexuales , Animales , Escherichia coli Enteropatógena , Estrógenos/inmunología , Femenino , Humanos , Lactante , Macrófagos del Hígado/inmunología , Masculino , Intercambio Materno-Fetal/inmunología , Ratones , EmbarazoRESUMEN
Host-pathogen interactions involve a series of attacks and counter-attacks. Miao et al. show that, although some invading bacteria can take shelter in lysosomes by neutralizing their pH, this respite is temporary, as host cells can expel them in exosomes.
Asunto(s)
Infecciones por Escherichia coli/inmunología , Lisosomas/microbiología , Canales Catiónicos TRPC/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Infecciones Urinarias/inmunología , Escherichia coli Uropatógena/fisiología , AnimalesRESUMEN
Vertebrate cells have evolved elaborate cell-autonomous defense programs to monitor subcellular compartments for infection and to evoke counter-responses. These programs are activated by pathogen-associated pattern molecules and by various strategies intracellular pathogens employ to alter cellular microenvironments. Here, we show that, when uropathogenic E. coli (UPEC) infect bladder epithelial cells (BECs), they are targeted by autophagy but avoid degradation because of their capacity to neutralize lysosomal pH. This change is detected by mucolipin TRP channel 3 (TRPML3), a transient receptor potential cation channel localized to lysosomes. TRPML3 activation then spontaneously initiates lysosome exocytosis, resulting in expulsion of exosome-encased bacteria. These studies reveal a cellular default system for lysosome homeostasis that has been co-opted by the autonomous defense program to clear recalcitrant pathogens.
Asunto(s)
Infecciones por Escherichia coli/inmunología , Lisosomas/microbiología , Canales Catiónicos TRPC/metabolismo , Canales de Potencial de Receptor Transitorio/metabolismo , Infecciones Urinarias/inmunología , Escherichia coli Uropatógena/fisiología , Animales , Autofagia , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Exocitosis , Lisosomas/enzimología , Lisosomas/metabolismo , Ratones , Vejiga Urinaria/inmunología , Vejiga Urinaria/microbiología , Vejiga Urinaria/patología , Infecciones Urinarias/microbiología , Infecciones Urinarias/patologíaRESUMEN
Gut bacteria are known to affect immune cell development, but most intestinal lymphocytes have no direct contact with luminal bacteria. Two studies by Atarashi et al. and Sano et al. shed light on how bacterial adhesion can cue intestinal epithelial cells to direct differentiation of gut T cells.
Asunto(s)
Adhesión Bacteriana , Citrobacter rodentium/fisiología , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Escherichia coli/inmunología , Escherichia coli O157/fisiología , Microbioma Gastrointestinal , Interleucinas/metabolismo , Mucosa Intestinal/inmunología , Intestinos/inmunología , Receptores de Interleucina/metabolismo , Proteína Amiloide A Sérica/metabolismo , Células Th17/inmunología , Animales , HumanosRESUMEN
Intestinal Th17 cells are induced and accumulate in response to colonization with a subgroup of intestinal microbes such as segmented filamentous bacteria (SFB) and certain extracellular pathogens. Here, we show that adhesion of microbes to intestinal epithelial cells (ECs) is a critical cue for Th17 induction. Upon monocolonization of germ-free mice or rats with SFB indigenous to mice (M-SFB) or rats (R-SFB), M-SFB and R-SFB showed host-specific adhesion to small intestinal ECs, accompanied by host-specific induction of Th17 cells. Citrobacter rodentium and Escherichia coli O157 triggered similar Th17 responses, whereas adhesion-defective mutants of these microbes failed to do so. Moreover, a mixture of 20 bacterial strains, which were selected and isolated from fecal samples of a patient with ulcerative colitis on the basis of their ability to cause a robust induction of Th17 cells in the mouse colon, also exhibited EC-adhesive characteristics.
Asunto(s)
Adhesión Bacteriana , Citrobacter rodentium/fisiología , Infecciones por Enterobacteriaceae/inmunología , Infecciones por Escherichia coli/inmunología , Escherichia coli O157/fisiología , Mucosa Intestinal/inmunología , Células Th17/inmunología , Animales , Infecciones Bacterianas/inmunología , Células Epiteliales/inmunología , Células Epiteliales/microbiología , Células Epiteliales/ultraestructura , Heces/microbiología , Humanos , Inmunoglobulina A/inmunología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Microscopía Electrónica de Rastreo , Ratas , Ratas Endogámicas F344 , Especificidad de la EspecieRESUMEN
The gut microbiome has major roles in modulating host physiology. One such function is colonization resistance, or the ability of the microbial collective to protect the host against enteric pathogens1-3, including enterohaemorrhagic Escherichia coli (EHEC) serotype O157:H7, an attaching and effacing (AE) food-borne pathogen that causes severe gastroenteritis, enterocolitis, bloody diarrhea and acute renal failure4,5 (haemolytic uremic syndrome). Although gut microorganisms can provide colonization resistance by outcompeting some pathogens or modulating host defence provided by the gut barrier and intestinal immune cells6,7, this phenomenon remains poorly understood. Here, we show that activation of the neurotransmitter receptor dopamine receptor D2 (DRD2) in the intestinal epithelium by gut microbial metabolites produced upon dietary supplementation with the essential amino acid L-tryptophan protects the host against Citrobacter rodentium, a mouse AE pathogen that is widely used as a model for EHEC infection8,9. We further find that DRD2 activation by these tryptophan-derived metabolites decreases expression of a host actin regulatory protein involved in C. rodentium and EHEC attachment to the gut epithelium via formation of actin pedestals. Our results reveal a noncanonical colonization resistance pathway against AE pathogens that features an unconventional role for DRD2 outside the nervous system in controlling actin cytoskeletal organization in the gut epithelium. Our findings may inspire prophylactic and therapeutic approaches targeting DRD2 with dietary or pharmacological interventions to improve gut health and treat gastrointestinal infections, which afflict millions globally.
Asunto(s)
Citrobacter rodentium , Mucosa Intestinal , Receptores de Dopamina D2 , Triptófano , Animales , Femenino , Humanos , Masculino , Ratones , Citoesqueleto de Actina/efectos de los fármacos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Carga Bacteriana/efectos de los fármacos , Citrobacter rodentium/crecimiento & desarrollo , Citrobacter rodentium/metabolismo , Citrobacter rodentium/patogenicidad , Suplementos Dietéticos , Modelos Animales de Enfermedad , Infecciones por Enterobacteriaceae/microbiología , Infecciones por Enterobacteriaceae/prevención & control , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/prevención & control , Escherichia coli O157/patogenicidad , Escherichia coli O157/fisiología , Mucosa Intestinal/citología , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Receptores de Dopamina D2/metabolismo , Triptófano/administración & dosificación , Triptófano/metabolismo , Triptófano/farmacologíaRESUMEN
Macrophages tightly scale their core metabolism after being activated, but the precise regulation of the mitochondrial electron-transport chain (ETC) and its functional implications are currently unknown. Here we found that recognition of live bacteria by macrophages transiently decreased assembly of the ETC complex I (CI) and CI-containing super-complexes and switched the relative contributions of CI and CII to mitochondrial respiration. This was mediated by phagosomal NADPH oxidase and the reactive oxygen species (ROS)-dependent tyrosine kinase Fgr. It required Toll-like receptor signaling and the NLRP3 inflammasome, which were both connected to bacterial viability-specific immune responses. Inhibition of CII during infection with Escherichia coli normalized serum concentrations of interleukin 1ß (IL-1ß) and IL-10 to those in mice treated with dead bacteria and impaired control of bacteria. We have thus identified ETC adaptations as an early immunological-metabolic checkpoint that adjusts innate immune responses to bacterial infection.
Asunto(s)
Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Infecciones por Escherichia coli/inmunología , Escherichia coli K12/inmunología , Macrófagos/inmunología , Mitocondrias/metabolismo , Animales , Células Cultivadas , Metabolismo Energético/genética , Interacciones Huésped-Parásitos , Inmunidad Innata/genética , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Fagocitosis , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The acute phase of sepsis is characterized by a strong inflammatory reaction. At later stages in some patients, immunoparalysis may be encountered, which is associated with a poor outcome. By transcriptional and metabolic profiling of human patients with sepsis, we found that a shift from oxidative phosphorylation to aerobic glycolysis was an important component of initial activation of host defense. Blocking metabolic pathways with metformin diminished cytokine production and increased mortality in systemic fungal infection in mice. In contrast, in leukocytes rendered tolerant by exposure to lipopolysaccharide or after isolation from patients with sepsis and immunoparalysis, a generalized metabolic defect at the level of both glycolysis and oxidative metabolism was apparent, which was restored after recovery of the patients. Finally, the immunometabolic defects in humans were partially restored by therapy with recombinant interferon-γ, which suggested that metabolic processes might represent a therapeutic target in sepsis.
Asunto(s)
Citocinas/inmunología , Endotoxemia/inmunología , Metabolismo Energético/inmunología , Tolerancia Inmunológica/inmunología , Inmunidad Innata/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Sepsis/inmunología , Adenosina Trifosfato/metabolismo , Adulto , Animales , Antifúngicos/uso terapéutico , Aspergilosis/tratamiento farmacológico , Aspergilosis/inmunología , Aspergilosis/metabolismo , Candidiasis Invasiva/tratamiento farmacológico , Candidiasis Invasiva/inmunología , Candidiasis Invasiva/metabolismo , Endotoxemia/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Femenino , Glucólisis , Humanos , Immunoblotting , Interferón gamma/uso terapéutico , Ácido Láctico/metabolismo , Leucocitos/inmunología , Leucocitos/metabolismo , Lipopolisacáridos/inmunología , Macrófagos/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Monocitos/metabolismo , NAD/metabolismo , Fosforilación Oxidativa , Consumo de Oxígeno , Estudios Prospectivos , Sepsis/tratamiento farmacológico , Sepsis/metabolismo , Transcriptoma , Adulto JovenRESUMEN
Differentiating neutrophils undergo large-scale changes in nuclear morphology. How such alterations in structure are established and modulated upon exposure to microbial agents is largely unknown. Here, we found that prior to encounter with bacteria, an armamentarium of inflammatory genes was positioned in a transcriptionally passive environment suppressing premature transcriptional activation. Upon microbial exposure, however, human neutrophils rapidly (<3 h) repositioned the ensemble of proinflammatory genes toward the transcriptionally permissive compartment. We show that the repositioning of genes was closely associated with the swift recruitment of cohesin across the inflammatory enhancer landscape, permitting an immediate transcriptional response upon bacterial exposure. We found that activated enhancers, marked by increased deposition of H3K27Ac, were highly enriched for cistromic elements associated with PU.1, CEBPB, TFE3, JUN, and FOSL2 occupancy. These data reveal how upon microbial challenge the cohesin machinery is recruited to an activated enhancer repertoire to instruct changes in chromatin folding, nuclear architecture, and to activate an inflammatory gene program.
Asunto(s)
Núcleo Celular/inmunología , Cromatina/inmunología , Infecciones por Escherichia coli/inmunología , Neutrófilos/inmunología , Activación Transcripcional/genética , Activación Transcripcional/inmunología , Células Cultivadas , Escherichia coli , Histonas/metabolismo , HumanosRESUMEN
The execution of shock following high dose E. coli lipopolysaccharide (LPS) or bacterial sepsis in mice required pro-apoptotic caspase-8 in addition to pro-pyroptotic caspase-11 and gasdermin D. Hematopoietic cells produced MyD88- and TRIF-dependent inflammatory cytokines sufficient to initiate shock without any contribution from caspase-8 or caspase-11. Both proteases had to be present to support tumor necrosis factor- and interferon-ß-dependent tissue injury first observed in the small intestine and later in spleen and thymus. Caspase-11 enhanced the activation of caspase-8 and extrinsic cell death machinery within the lower small intestine. Neither caspase-8 nor caspase-11 was individually sufficient for shock. Both caspases collaborated to amplify inflammatory signals associated with tissue damage. Therefore, combined pyroptotic and apoptotic signaling mediated endotoxemia independently of RIPK1 kinase activity and RIPK3 function. These observations bring to light the relevance of tissue compartmentalization to disease processes in vivo where cytokines act in parallel to execute diverse cell death pathways.
Asunto(s)
Caspasa 8/metabolismo , Caspasas/metabolismo , Infecciones por Escherichia coli/enzimología , Infecciones por Escherichia coli/fisiopatología , Choque Séptico/enzimología , Choque Séptico/fisiopatología , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Caspasa 8/genética , Caspasas/genética , Caspasas Iniciadoras , Células Cultivadas , Femenino , Inflamación/metabolismo , Inflamación/patología , Factor 3 Regulador del Interferón/genética , Interferón beta/sangre , Interferón beta/metabolismo , Intestino Delgado/patología , Péptidos y Proteínas de Señalización Intracelular , Lipopolisacáridos/toxicidad , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Unión a Fosfato , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Transducción de Señal , Bazo/patología , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/sangre , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Rab GTPases are frequent targets of vacuole-living bacterial pathogens for appropriate trafficking of the vacuole. Here we discover that bacterial effectors including VirA from nonvacuole Shigella flexneri and EspG from extracellular Enteropathogenic Escherichia coli (EPEC) harbor TBC-like dual-finger motifs and exhibits potent RabGAP activities. Specific inactivation of Rab1 by VirA/EspG disrupts ER-to-Golgi trafficking. S. flexneri intracellular persistence requires VirA TBC-like GAP activity that mediates bacterial escape from autophagy-mediated host defense. Rab1 inactivation by EspG severely blocks host secretory pathway, resulting in inhibited interleukin-8 secretion from infected cells. Crystal structures of VirA/EspG-Rab1-GDP-aluminum fluoride complexes highlight TBC-like catalytic role for the arginine and glutamine finger residues and reveal a 3D architecture distinct from that of the TBC domain. Structure of Arf6-EspG-Rab1 ternary complex illustrates a pathogenic signaling complex that rewires host Arf signaling to Rab1 inactivation. Structural distinctions of VirA/EspG further predict a possible extensive presence of TBC-like RabGAP effectors in counteracting various host defenses.
Asunto(s)
Factores de Ribosilacion-ADP/metabolismo , Escherichia coli Enteropatógena/patogenicidad , Proteínas de Escherichia coli/metabolismo , Proteínas Activadoras de GTPasa/metabolismo , Shigella flexneri/patogenicidad , Factores de Virulencia/metabolismo , Secuencia de Aminoácidos , Animales , Autofagia , Disentería Bacilar/inmunología , Disentería Bacilar/microbiología , Escherichia coli Enteropatógena/metabolismo , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/química , Fibroblastos/metabolismo , Interleucina-8/inmunología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Alineación de Secuencia , Shigella flexneri/metabolismo , Virulencia , Factores de Virulencia/químicaRESUMEN
Glucuronidation is a detoxification process to eliminate endo- and xeno-biotics and neurotransmitters from the host circulation. Glucuronosyltransferase binds these compounds to glucuronic acid (GlcA), deactivating them and allowing their elimination through the gastrointestinal (GI) tract. However, the microbiota produces ß-glucuronidases that release GlcA and reactivate these compounds. Enteric pathogens such as enterohemorrhagic Escherichia coli (EHEC) and Citrobacter rodentium sense and utilize galacturonic acid (GalA), an isomer of GlcA, to outcompete the microbiota promoting gut colonization. However, the role of GlcA in pathogen colonization has not been explored. Here, we show that treatment of mice with a microbial ß-glucuronidase inhibitor (GUSi) decreased C. rodentium's colonization of the GI tract, without modulating bacterial virulence or host inflammation. Metagenomic studies indicated that GUSi did not change the composition of the intestinal microbiota in these animals. GlcA confers an advantage for pathogen expansion through its utilization as a carbon source. Congruently mutants unable to catabolize GlcA depict lower GI colonization compared to wild type and are not sensitive to GUSi. Germfree mice colonized with a commensal E. coli deficient for ß-glucuronidase production led to a decrease of C. rodentium tissue colonization, compared to animals monocolonized with an E. coli proficient for production of this enzyme. GlcA is not sensed as a signal and doesn't activate virulence expression but is used as a metabolite. Because pathogens can use GlcA to promote their colonization, inhibitors of microbial ß-glucuronidases could be a unique therapeutic against enteric infections without disturbing the host or microbiota physiology.
Asunto(s)
Infecciones por Escherichia coli , Microbiota , Animales , Ratones , Escherichia coli/genética , Ácido Glucurónico , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Virulencia/fisiologíaRESUMEN
This study supports the development of predictive bacteriophage (phage) therapy: the concept of phage cocktail selection to treat a bacterial infection based on machine learning (ML) models. For this purpose, ML models were trained on thousands of measured interactions between a panel of phage and sequenced bacterial isolates. The concept was applied to Escherichia coli associated with urinary tract infections. This is an important common infection in humans and companion animals from which multidrug-resistant (MDR) bloodstream infections can originate. The global threat of MDR infection has reinvigorated international efforts into alternatives to antibiotics including phage therapy. E. coli exhibit extensive genome-level variation due to horizontal gene transfer via phage and plasmids. Associated with this, phage selection for E. coli is difficult as individual isolates can exhibit considerable variation in phage susceptibility due to differences in factors important to phage infection including phage receptor profiles and resistance mechanisms. The activity of 31 phage was measured on 314 isolates with growth curves in artificial urine. Random Forest models were built for each phage from bacterial genome features, and the more generalist phage, acting on over 20% of the bacterial population, exhibited F1 scores of >0.6 and could be used to predict phage cocktails effective against previously untested strains. The study demonstrates the potential of predictive ML models which integrate bacterial genomics with phage activity datasets allowing their use on data derived from direct sequencing of clinical samples to inform rapid and effective phage therapy.
Asunto(s)
Bacteriófagos , Infecciones por Escherichia coli , Terapia de Fagos , Infecciones Urinarias , Humanos , Animales , Escherichia coli/genética , Infecciones por Escherichia coli/microbiología , Bacteriófagos/genética , Antibacterianos/farmacología , Infecciones Urinarias/tratamiento farmacológicoRESUMEN
Urinary tract infection (UTI) commonly afflicts people with diabetes. This augmented infection risk is partly due to deregulated insulin receptor (IR) signaling in the kidney collecting duct. The collecting duct is composed of intercalated cells (ICs) and principal cells (PCs). Evidence suggests that ICs contribute to UTI defenses. Here, we interrogate how IR deletion in ICs impacts antibacterial defenses against uropathogenic Escherichia coli. We also explore how IR deletion affects immune responses in neighboring PCs with intact IR expression. To accomplish this objective, we profile the transcriptomes of IC and PC populations enriched from kidneys of wild-type and IC-specific IR knock-out mice that have increased UTI susceptibility. Transcriptomic analysis demonstrates that IR deletion suppresses IC-integrated stress responses and innate immune defenses. To define how IR shapes these immune defenses, we employ murine and human kidney cultures. When challenged with bacteria, murine ICs and human kidney cells with deregulated IR signaling cannot engage central components of the integrated stress response-including activating transcriptional factor 4 (ATF4). Silencing ATF4 impairs NFkB activation and promotes infection. In turn, NFkB silencing augments infection and suppresses antimicrobial peptide expression. In diabetic mice and people with diabetes, collecting duct cells show reduced IR expression, impaired integrated stress response engagement, and compromised immunity. Collectively, these translational data illustrate how IR orchestrates collecting duct antibacterial responses and the communication between ICs and PCs.
Asunto(s)
Ratones Noqueados , Receptor de Insulina , Infecciones Urinarias , Escherichia coli Uropatógena , Animales , Humanos , Ratones , Infecciones por Escherichia coli/inmunología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Inmunidad Innata , Riñón/metabolismo , Túbulos Renales Colectores/metabolismo , Ratones Endogámicos C57BL , Receptor de Insulina/metabolismo , Transducción de Señal , Infecciones Urinarias/microbiología , Infecciones Urinarias/metabolismo , Infecciones Urinarias/inmunología , Escherichia coli Uropatógena/inmunologíaRESUMEN
The presence of bacteria in the bloodstream is associated with severe clinical outcomes. In mice, intravenous inoculation of Escherichia coli can lead to the formation of macroscopic abscesses in the liver. Abscesses are regions of severe necrosis and consist of millions of bacteria surrounded by inflammatory immune cells. Liver abscess susceptibility varies widely across strains of mice, but the host factors governing this variation are unknown. Here, we profiled hepatic transcriptomes in mice with varying susceptibility to liver abscess formation. We found that transcripts from endogenous retroviruses (ERVs) are robustly induced in the liver by E. coli infection and ERV expression positively correlates with the frequency of abscess formation. Hypothesizing that ERV-encoded reverse transcriptase may generate cytoplasmic DNA and heighten inflammatory responses, we tested whether nucleoside/nucleotide reverse transcriptase inhibitors (NRTIs) influence abscess formation. Strikingly, a single NRTI dose administered immediately following E. coli inoculation prevented abscess formation, leading to a concomitant 100,000-fold reduction in bacterial burden. We provide evidence that NRTIs inhibit abscess formation by preventing the tissue necrosis that facilitates bacterial replication. Together, our findings suggest that endogenous reverse transcriptases drive inflammatory responses during bacterial bloodstream infection to drive abscess formation. The high efficacy of NRTIs in preventing abscess formation suggests that the consequences of reverse transcription on inflammation should be further examined, particularly in infectious diseases where inflammation drives negative clinical outcomes, such as sepsis.