RESUMO
Gasdermins (GSDMs) share a common functional domain structure and are best known for their capacity to form membrane pores. These pores are hallmarks of a specific form of cell death called pyroptosis and mediate the secretion of pro-inflammatory cytokines such as interleukin 1ß (IL1ß) and interleukin 18 (IL18). Thereby, Gasdermins have been implicated in various immune responses against cancer and infectious diseases such as acute Salmonella Typhimurium (S.Tm) gut infection. However, to date, we lack a comprehensive functional assessment of the different Gasdermins (GSDMA-E) during S.Tm infection in vivo. Here, we used epithelium-specific ablation, bone marrow chimeras, and mouse lines lacking individual Gasdermins, combinations of Gasdermins or even all Gasdermins (GSDMA1-3C1-4DE) at once and performed littermate-controlled oral S.Tm infections in streptomycin-pretreated mice to investigate the impact of all murine Gasdermins. While GSDMA, C, and E appear dispensable, we show that GSDMD i) restricts S.Tm loads in the gut tissue and systemic organs, ii) controls gut inflammation kinetics, and iii) prevents epithelium disruption by 72 h of the infection. Full protection requires GSDMD expression by both bone-marrow-derived lamina propria cells and intestinal epithelial cells (IECs). In vivo experiments as well as 3D-, 2D-, and chimeric enteroid infections further show that infected IEC extrusion proceeds also without GSDMD, but that GSDMD controls the permeabilization and morphology of the extruding IECs, affects extrusion kinetics, and promotes overall mucosal barrier capacity. As such, this work identifies a unique multipronged role of GSDMD among the Gasdermins for mucosal tissue defense against a common enteric pathogen.
Assuntos
Gasderminas , Infecções por Salmonella , Animais , Camundongos , Infecções por Salmonella/prevenção & controle , Salmonella typhimurium , Inflamação , Células Epiteliais , InflamassomosRESUMO
Recruitment of neutrophils into and across the gut mucosa is a cardinal feature of intestinal inflammation in response to enteric infections. Previous work using the model pathogen Salmonella enterica serovar Typhimurium (S.Tm) established that invasion of intestinal epithelial cells by S.Tm leads to recruitment of neutrophils into the gut lumen, where they can reduce pathogen loads transiently. Notably, a fraction of the pathogen population can survive this defense, re-grow to high density, and continue triggering enteropathy. However, the functions of intraluminal neutrophils in the defense against enteric pathogens and their effects on preventing or aggravating epithelial damage are still not fully understood. Here, we address this question via neutrophil depletion in different mouse models of Salmonella colitis, which differ in their degree of enteropathy. In an antibiotic pretreated mouse model, neutrophil depletion by an anti-Ly6G antibody exacerbated epithelial damage. This could be linked to compromised neutrophil-mediated elimination and reduced physical blocking of the gut-luminal S.Tm population, such that the pathogen density remained high near the epithelial surface throughout the infection. Control infections with a ssaV mutant and gentamicin-mediated elimination of gut-luminal pathogens further supported that neutrophils are protecting the luminal surface of the gut epithelium. Neutrophil depletion in germ-free and gnotobiotic mice hinted that the microbiota can modulate the infection kinetics and ameliorate epithelium-disruptive enteropathy even in the absence of neutrophil-protection. Together, our data indicate that the well-known protective effect of the microbiota is augmented by intraluminal neutrophils. After antibiotic-mediated microbiota disruption, neutrophils are central for maintaining epithelial barrier integrity during acute Salmonella-induced gut inflammation, by limiting the sustained pathogen assault on the epithelium in a critical window of the infection.
Assuntos
Neutrófilos , Infecções por Salmonella , Animais , Camundongos , Salmonella typhimurium , Células Epiteliais , Antibacterianos , Inflamação , Epitélio , Mucosa IntestinalRESUMO
Intestinal epithelial cell (IEC) NF-κB signaling regulates the balance between mucosal homeostasis and inflammation. It is not fully understood which signals tune this balance and how bacterial exposure elicits the process. Pure LPS induces epithelial NF-κB activation in vivo. However, we found that in mice, IECs do not respond directly to LPS. Instead, tissue-resident lamina propria intercrypt macrophages sense LPS via TLR4 and rapidly secrete TNF to elicit epithelial NF-κB signaling in their immediate neighborhood. This response pattern is relevant also during oral enteropathogen infection. The macrophage-TNF-IEC axis avoids responses to luminal microbiota LPS but enables crypt- or tissue-scale epithelial NF-κB responses in proportion to the microbial threat. Thereby, intercrypt macrophages fulfill important sentinel functions as first responders to Gram-negative microbes breaching the epithelial barrier. The tunability of this crypt response allows the induction of defense mechanisms at an appropriate scale according to the localization and intensity of microbial triggers.
Assuntos
Antibacterianos/metabolismo , Células Epiteliais/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/metabolismo , NF-kappa B/metabolismo , Fatores de Necrose Tumoral/metabolismo , Animais , Regulação da Expressão Gênica/fisiologia , Inflamação/metabolismo , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais/fisiologiaRESUMO
The intestinal epithelium is a complex structure that integrates digestive, immunological, neuroendocrine, and regenerative functions. Epithelial homeostasis is maintained by a coordinated cross-talk of different epithelial cell types. Loss of integrity of the intestinal epithelium plays a key role in inflammatory diseases and gastrointestinal infection. Here we show that the intestine-enriched miR-802 is a central regulator of intestinal epithelial cell proliferation, Paneth cell function, and enterocyte differentiation. Genetic ablation of mir-802 in the small intestine of mice leads to decreased glucose uptake, impaired enterocyte differentiation, increased Paneth cell function and intestinal epithelial proliferation. These effects are mediated in part through derepression of the miR-802 target Tmed9, a modulator of Wnt and lysozyme/defensin secretion in Paneth cells, and the downstream Wnt signaling components Fzd5 and Tcf4. Mutant Tmed9 mice harboring mutations in miR-802 binding sites partially recapitulate the augmented Paneth cell function of mice lacking miR-802. Our study demonstrates a broad miR-802 network that is important for the integration of signaling pathways of different cell types controlling epithelial homeostasis in the small intestine.
Assuntos
Diferenciação Celular/fisiologia , Enterócitos/metabolismo , Intestino Delgado/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Celulas de Paneth/metabolismo , Animais , Proliferação de Células , Feminino , Receptores Frizzled/metabolismo , Expressão Gênica , Células HEK293 , Homeostase/fisiologia , Humanos , Mucosa Intestinal/metabolismo , Intestinos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Salmonella typhimurium , Fator de Transcrição 4/metabolismo , Transcriptoma , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo , Via de Sinalização WntRESUMO
The gut epithelium is a critical protective barrier. Its NAIP/NLRC4 inflammasome senses infection by Gram-negative bacteria, including Salmonella Typhimurium (S.Tm) and promotes expulsion of infected enterocytes. During the first ~12-24 h, this reduces mucosal S.Tm loads at the price of moderate enteropathy. It remained unknown how this NAIP/NLRC4-dependent tradeoff would develop during subsequent infection stages. In NAIP/NLRC4-deficient mice, S.Tm elicited severe enteropathy within 72 h, characterized by elevated mucosal TNF (>20 pg/mg) production from bone marrow-derived cells, reduced regeneration, excessive enterocyte loss, and a collapse of the epithelial barrier. TNF-depleting antibodies prevented this destructive pathology. In hosts proficient for epithelial NAIP/NLRC4, a heterogeneous enterocyte death response with both apoptotic and pyroptotic features kept S.Tm loads persistently in check, thereby preventing this dire outcome altogether. Our results demonstrate that immediate and selective removal of infected enterocytes, by locally acting epithelium-autonomous NAIP/NLRC4, is required to avoid a TNF-driven inflammatory hyper-reaction that otherwise destroys the epithelial barrier.
Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Enterócitos/imunologia , Inflamação/imunologia , Mucosa Intestinal/patologia , Proteína Inibidora de Apoptose Neuronal/metabolismo , Infecções por Salmonella/imunologia , Salmonella typhimurium/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Proteínas de Ligação ao Cálcio/genética , Células Cultivadas , Citotoxicidade Imunológica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Inibidora de Apoptose Neuronal/genética , Junções Íntimas/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Salmonella enterica serovar Typhimurium (S.Tm) infections of cultured cell lines have given rise to the ruffle model for epithelial cell invasion. According to this model, the Type-Three-Secretion-System-1 (TTSS-1) effectors SopB, SopE and SopE2 drive an explosive actin nucleation cascade, resulting in large lamellipodia- and filopodia-containing ruffles and cooperative S.Tm uptake. However, cell line experiments poorly recapitulate many of the cell and tissue features encountered in the host's gut mucosa. Here, we employed bacterial genetics and multiple imaging modalities to compare S.Tm invasion of cultured epithelial cell lines and the gut absorptive epithelium in vivo in mice. In contrast to the prevailing ruffle-model, we find that absorptive epithelial cell entry in the mouse gut occurs through "discreet-invasion". This distinct entry mode requires the conserved TTSS-1 effector SipA, involves modest elongation of local microvilli in the absence of expansive ruffles, and does not favor cooperative invasion. Discreet-invasion preferentially targets apicolateral hot spots at cell-cell junctions and shows strong dependence on local cell neighborhood. This proof-of-principle evidence challenges the current model for how S.Tm can enter gut absorptive epithelial cells in their intact in vivo context.
Assuntos
Aderência Bacteriana , Mucosa Intestinal/microbiologia , Infecções por Salmonella , Salmonella typhimurium , Sistemas de Secreção Tipo I/metabolismo , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cães , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Células HeLa , Humanos , Mucosa Intestinal/metabolismo , Mucosa Intestinal/patologia , Células Madin Darby de Rim Canino , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Infecções por Salmonella/genética , Infecções por Salmonella/metabolismo , Infecções por Salmonella/microbiologia , Infecções por Salmonella/patologia , Salmonella typhimurium/genética , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Sistemas de Secreção Tipo I/genéticaRESUMO
Initial enteropathogen growth in the microbiota-colonized gut is poorly understood. Salmonella Typhimurium is metabolically adaptable and can harvest energy by anaerobic respiration using microbiota-derived hydrogen (H2) as an electron donor and fumarate as an electron acceptor. As fumarate is scarce in the gut, the source of this electron acceptor is unclear. Here, transposon sequencing analysis along the colonization trajectory of S. Typhimurium implicates the C4-dicarboxylate antiporter DcuABC in early murine gut colonization. In competitive colonization assays, DcuABC and enzymes that convert the C4-dicarboxylates aspartate and malate into fumarate (AspA, FumABC), are required for fumarate/H2-dependent initial growth. Thus, S. Typhimurium obtains fumarate by DcuABC-mediated import and conversion of L-malate and L-aspartate. Fumarate reduction yields succinate, which is exported by DcuABC in exchange for L-aspartate and L-malate. This cycle allows S. Typhimurium to harvest energy by H2/fumarate respiration in the microbiota-colonized gut. This strategy may also be relevant for commensal E. coli diminishing the S. Typhimurium infection.
Assuntos
Ácido Aspártico/metabolismo , Fumaratos/metabolismo , Microbioma Gastrointestinal/fisiologia , Malatos/metabolismo , Salmonella/metabolismo , Administração Oral , Animais , Ácido Aspártico/administração & dosagem , Proteínas de Bactérias/metabolismo , Ciclo do Ácido Cítrico , Modelos Animais de Doenças , Escherichia coli/metabolismo , Fezes/microbiologia , Feminino , Microbioma Gastrointestinal/genética , Intestinos/microbiologia , Malatos/administração & dosagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mutagênese , RNA Ribossômico 16S/genética , Salmonella/genética , Salmonella/crescimento & desenvolvimento , Salmonella typhimurium , Análise de Sequência de DNA , Ácido SuccínicoRESUMO
Inflammasomes can prevent systemic dissemination of enteropathogenic bacteria. As adapted pathogens including Salmonella Typhimurium (S. Tm) have evolved evasion strategies, it has remained unclear when and where inflammasomes restrict their dissemination. Bacterial population dynamics establish that the NAIP/NLRC4 inflammasome specifically restricts S. Tm migration from the gut to draining lymph nodes. This is solely attributable to NAIP/NLRC4 within intestinal epithelial cells (IECs), while S. Tm evades restriction by phagocyte NAIP/NLRC4. NLRP3 and Caspase-11 also fail to restrict S. Tm mucosa traversal, migration to lymph nodes, and systemic pathogen growth. The ability of IECs (not phagocytes) to mount a NAIP/NLRC4 defense in vivo is explained by particularly high NAIP/NLRC4 expression in IECs and the necessity for epithelium-invading S. Tm to express the NAIP1-6 ligands-flagella and type-III-secretion-system-1. Imaging reveals both ligands to be promptly downregulated following IEC-traversal. These results highlight the importance of intestinal epithelial NAIP/NLRC4 in blocking bacterial dissemination in vivo, and explain why this constitutes a uniquely evasion-proof defense against the adapted enteropathogen S. Tm.
Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Proteína Inibidora de Apoptose Neuronal/metabolismo , Moléculas com Motivos Associados a Patógenos , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Animais , Caspases/metabolismo , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Tecido Linfoide/imunologia , Tecido Linfoide/metabolismo , Camundongos , Camundongos Knockout , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Especificidade de Órgãos/imunologia , Fagócitos/imunologia , Fagócitos/metabolismo , Infecções por Salmonella/metabolismoRESUMO
The emergence of antibiotic-resistant bacteria through mutations or the acquisition of genetic material such as resistance plasmids represents a major public health issue1,2. Persisters are subpopulations of bacteria that survive antibiotics by reversibly adapting their physiology3-10, and can promote the emergence of antibiotic-resistant mutants11. We investigated whether persisters can also promote the spread of resistance plasmids. In contrast to mutations, the transfer of resistance plasmids requires the co-occurrence of both a donor and a recipient bacterial strain. For our experiments, we chose the facultative intracellular entero-pathogen Salmonella enterica serovar Typhimurium (S. Typhimurium) and Escherichia coli, a common member of the microbiota12. S. Typhimurium forms persisters that survive antibiotic therapy in several host tissues. Here we show that tissue-associated S. Typhimurium persisters represent long-lived reservoirs of plasmid donors or recipients. The formation of reservoirs of S. Typhimurium persisters requires Salmonella pathogenicity island (SPI)-1 and/or SPI-2 in gut-associated tissues, or SPI-2 at systemic sites. The re-seeding of these persister bacteria into the gut lumen enables the co-occurrence of donors with gut-resident recipients, and thereby favours plasmid transfer between various strains of Enterobacteriaceae. We observe up to 99% transconjugants within two to three days of re-seeding. Mathematical modelling shows that rare re-seeding events may suffice for a high frequency of conjugation. Vaccination reduces the formation of reservoirs of persisters after oral infection with S. Typhimurium, as well as subsequent plasmid transfer. We conclude that-even without selection for plasmid-encoded resistance genes-small reservoirs of pathogen persisters can foster the spread of promiscuous resistance plasmids in the gut.
Assuntos
Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Microbioma Gastrointestinal/genética , Transferência Genética Horizontal , Mucosa Intestinal/microbiologia , Plasmídeos/genética , Salmonella typhimurium/genética , Animais , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Fezes/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Camundongos , Modelos Teóricos , Salmonella typhimurium/efeitos dos fármacos , VacinaçãoRESUMO
Mucus separates gut-luminal microbes from the tissue. It is unclear how pathogens like Salmonella Typhimurium (S.Tm) can overcome this obstacle. Using live microscopy, we monitored S.Tm interactions with native murine gut explants and studied how mucus affects the infection. A dense inner mucus layer covers the distal colon tissue, limiting direct tissue access. S.Tm performs near-surface swimming on this mucus layer, which allows probing for colon mucus heterogeneities, but can also entrap the bacterium in the dense inner colon mucus layer. In the cecum, dense mucus fills only the bottom of the intestinal crypts, leaving the epithelium between crypts unshielded and prone to access by motile and non-motile bacteria alike. This explains why the cecum is highly infection permissive and represents the primary site of S.Tm enterocolitis in the streptomycin mouse model. Our findings highlight the importance of mucus in intestinal defense and homeostasis.