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1.
Nihon Saikingaku Zasshi ; 75(2): 185-194, 2020.
Artigo em Japonês | MEDLINE | ID: mdl-33361654

RESUMO

Countless numbers of bacteria inhabit the intestinal tract. One of the important functions of gut microbiota is the "colonization resistance" against infection by pathogenic microorganisms. However, detailed mechanism of the colonization resistance of intestinal bacteria is still largely unknown. We tried to identify molecular and cellular mechanism of it and found that antigen presentation by dendritic cells is required for the induction of intestinal segmented filamentous bacteria (SFB)-induced T helper 17 (Th17) cells that contribute to the protection against infection by Citrobacter rodentium. We further identified that gut Th17 cells selectively recognize antigens derived from SFB. We also revealed that SFB induce α1,2-fucose, one of carbohydrate chains, expressed on the intestinal epithelial cells mediated by group 3 innate lymphoid cells. Epithelial α1,2-fucose protected against infection by pathogenic bacterium Salmonella typhimurium. Furthermore, it was found that intestinal bacteria inhibit colonization of the pathogenic fungus Candida albicans as well as pathogenic bacteria. From these studies, detailed mechanism of "colonization resistance" against pathogenic microorganisms by intestinal bacteria has been clarified.


Assuntos
Candida albicans/patogenicidade , Citrobacter rodentium/patogenicidade , Microbioma Gastrointestinal/imunologia , Microbioma Gastrointestinal/fisiologia , Interações entre Hospedeiro e Microrganismos/imunologia , Sistema Imunitário/imunologia , Mucosa Intestinal/microbiologia , Salmonella typhimurium/patogenicidade , Células Th17/imunologia , Animais , Apresentação do Antígeno , Antígenos de Bactérias/imunologia , Aderência Bacteriana/imunologia , Candida albicans/imunologia , Citrobacter rodentium/imunologia , Células Dendríticas/imunologia , Fucose/metabolismo , Humanos , Mucosa Intestinal/imunologia , Mucosa Intestinal/metabolismo , Camundongos , Salmonella typhimurium/imunologia
2.
PLoS Biol ; 18(9): e3000813, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32991574

RESUMO

Short-chain fatty acids (SCFAs) produced by gastrointestinal microbiota regulate immune responses, but host molecular mechanisms remain unknown. Unbiased screening using SCFA-conjugated affinity nanobeads identified apoptosis-associated speck-like protein (ASC), an adaptor protein of inflammasome complex, as a noncanonical SCFA receptor besides GPRs. SCFAs promoted inflammasome activation in macrophages by binding to its ASC PYRIN domain. Activated inflammasome suppressed survival of Salmonella enterica serovar Typhimurium (S. Typhimurium) in macrophages by pyroptosis and facilitated neutrophil recruitment to promote bacterial elimination and thus inhibit systemic dissemination in the host. Administration of SCFAs or dietary fibers, which are fermented to SCFAs by gut bacteria, significantly prolonged the survival of S. Typhimurium-infected mice through ASC-mediated inflammasome activation. SCFAs penetrated into the inflammatory region of the infected gut mucosa to protect against infection. This study provided evidence that SCFAs suppress Salmonella infection via inflammasome activation, shedding new light on the therapeutic activity of dietary fiber.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/metabolismo , Ácidos Graxos Voláteis/metabolismo , Inflamassomos/imunologia , Inflamassomos/metabolismo , Receptores Acoplados a Proteínas-G/metabolismo , Infecções por Salmonella/prevenção & controle , Animais , Proteínas Adaptadoras de Sinalização CARD/genética , Feminino , Microbioma Gastrointestinal/imunologia , Células HEK293 , Humanos , Imunidade Inata/fisiologia , Ativação de Macrófagos/genética , Ativação de Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica , Receptores Acoplados a Proteínas-G/genética , Infecções por Salmonella/genética , Infecções por Salmonella/imunologia , Infecções por Salmonella/metabolismo , Salmonella typhimurium/imunologia , Células U937
3.
PLoS Pathog ; 16(8): e1008763, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32834002

RESUMO

The various sub-species of Salmonella enterica cause a range of disease in human hosts. The human-adapted Salmonella enterica serovar Typhi enters the gastrointestinal tract and invades systemic sites to cause enteric (typhoid) fever. In contrast, most non-typhoidal serovars of Salmonella are primarily restricted to gut tissues. Across Africa, invasive non-typhoidal Salmonella (iNTS) have emerged with an ability to spread beyond the gastrointestinal tract and cause systemic bloodstream infections with increased morbidity and mortality. To investigate this evolution in pathogenesis, we compared the genomes of African iNTS isolates with other Salmonella enterica serovar Typhimurium and identified several macA and macB gene variants unique to African iNTS. MacAB forms a tripartite efflux pump with TolC and is implicated in Salmonella pathogenesis. We show that macAB transcription is upregulated during macrophage infection and after antimicrobial peptide exposure, with macAB transcription being supported by the PhoP/Q two-component system. Constitutive expression of macAB improves survival of Salmonella in the presence of the antimicrobial peptide C18G. Furthermore, these macAB variants affect replication in macrophages and influence fitness during colonization of the murine gastrointestinal tract. Importantly, the infection outcome resulting from these macAB variants depends upon both the Salmonella Typhimurium genetic background and the host gene Nramp1, an important determinant of innate resistance to intracellular bacterial infection. The variations we have identified in the MacAB-TolC efflux pump in African iNTS may reflect evolution within human host populations that are compromised in their ability to clear intracellular Salmonella infections.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Bactérias/genética , Colite/patologia , Variação Genética , Macrófagos/imunologia , Salmonelose Animal/patologia , Salmonella typhimurium/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Proteínas de Bactérias/metabolismo , Linhagem da Célula , Colite/induzido quimicamente , Colite/imunologia , Colite/microbiologia , Análise Mutacional de DNA , Modelos Animais de Doenças , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Salmonelose Animal/imunologia , Salmonelose Animal/microbiologia , Replicação Viral
4.
PLoS Pathog ; 16(8): e1008766, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32857822

RESUMO

Pathogens commonly disrupt the intestinal epithelial barrier; however, how the epithelial immune system senses the loss of intestinal barrier as a danger signal to activate self-defense is unclear. Through an unbiased approach in the model nematode Caenorhabditis elegans, we found that the EGL-44/TEAD transcription factor and its transcriptional activator YAP-1/YAP (Yes-associated protein) were activated when the intestinal barrier was disrupted by infections with the pathogenic bacterium Pseudomonas aeruginosa PA14. Gene Ontology enrichment analysis of the genes containing the TEAD-binding sites revealed that "innate immune response" and "defense response to Gram-negative bacterium" were two top significantly overrepresented terms. Genetic inactivation of yap-1 and egl-44 significantly reduced the survival rate and promoted bacterial accumulation in worms after bacterial infections. Furthermore, we found that disturbance of the E-cadherin-based adherens junction triggered the nuclear translocation and activation of YAP-1/YAP in the gut of worms. Although YAP is a major downstream effector of the Hippo signaling, our study revealed that the activation of YAP-1/YAP was independent of the Hippo pathway during disruption of intestinal barrier. After screening 10 serine/threonine phosphatases, we identified that PP2A phosphatase was involved in the activation of YAP-1/YAP after intestinal barrier loss induced by bacterial infections. Additionally, our study demonstrated that the function of YAP was evolutionarily conserved in mice. Our study highlights how the intestinal epithelium recognizes the loss of the epithelial barrier as a danger signal to deploy defenses against pathogens, uncovering an immune surveillance program in the intestinal epithelium.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Permeabilidade da Membrana Celular , Células Epiteliais/imunologia , Microbioma Gastrointestinal/imunologia , Salmonelose Animal/imunologia , Salmonella typhimurium/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Camundongos , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Transdução de Sinais
5.
Nat Commun ; 11(1): 3547, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669546

RESUMO

Neutrophils provide first line of host defense against bacterial infections utilizing glycolysis for their effector functions. How glycolysis and its major byproduct lactate are triggered in bone marrow (BM) neutrophils and their contribution to neutrophil mobilization in acute inflammation is not clear. Here we report that bacterial lipopolysaccharides (LPS) or Salmonella Typhimurium triggers lactate release by increasing glycolysis, NADPH-oxidase-mediated reactive oxygen species and HIF-1α levels in BM neutrophils. Increased release of BM lactate preferentially promotes neutrophil mobilization by reducing endothelial VE-Cadherin expression, increasing BM vascular permeability via endothelial lactate-receptor GPR81 signaling. GPR81-/- mice mobilize reduced levels of neutrophils in response to LPS, unless rescued by VE-Cadherin disrupting antibodies. Lactate administration also induces release of the BM neutrophil mobilizers G-CSF, CXCL1 and CXCL2, indicating that this metabolite drives neutrophil mobilization via multiple pathways. Our study reveals a metabolic crosstalk between lactate-producing neutrophils and BM endothelium, which controls neutrophil mobilization under bacterial infection.


Assuntos
Células da Medula Óssea/imunologia , Ácido Láctico/metabolismo , Neutrófilos/imunologia , Receptores Acoplados a Proteínas-G/metabolismo , Infecções por Salmonella/imunologia , Animais , Medula Óssea/irrigação sanguínea , Células da Medula Óssea/metabolismo , Modelos Animais de Doenças , Endotélio Vascular/metabolismo , Feminino , Humanos , Lipopolissacarídeos/imunologia , Masculino , Camundongos , Camundongos Knockout , Neutrófilos/metabolismo , Receptores Acoplados a Proteínas-G/genética , Infecções por Salmonella/microbiologia , Salmonella typhimurium/imunologia , Transdução de Sinais/imunologia
6.
BMC Infect Dis ; 20(1): 422, 2020 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-32552753

RESUMO

BACKGROUND: In Kenya, typhoid fever and invasive non-typhoidal salmonellosis present a huge burden of disease, especially in poor-resource settings where clean water supply and sanitation conditions are inadequate. The epidemiology of both diseases is poorly understood in terms of severity and risk factors. The aim of the study was to determine the disease burden and spatial distribution of salmonellosis, as well as socioeconomic and environmental risk factors for these infections, in a large informal settlement near the city of Nairobi, from 2013 to 2017. METHODS: Initially, a house-to-house baseline census of 150,000 population in Mukuru informal settlement was carried out and relevant socioeconomic, demographic, and healthcare utilization information was collected using structured questionnaires. Salmonella bacteria were cultured from the blood and faeces of children < 16 years of age who reported at three outpatient facilities with fever alone or fever and diarrhea. Tests of association between specific Salmonella serotypes and risk factors were conducted using Pearson Chi-Square (χ2) test. RESULTS: A total of 16,236 children were recruited into the study. The prevalence of bloodstream infections by Non-Typhoidal Salmonella (NTS), consisting of Salmonella Typhimurium/ Enteriditis, was 1.3%; Salmonella Typhi was 1.4%, and this was highest among children < 16 years of age. Occurrence of Salmonella Typhimurium/ Enteriditis was not significantly associated with rearing any domestic animals. Rearing chicken was significantly associated with high prevalence of S. Typhi (2.1%; p = 0.011). The proportion of children infected with Salmonella Typhimurium/ Enteriditis was significantly higher in households that used water pots as water storage containers compared to using water directly from the tap (0.6%). Use of pit latrines and open defecation were significant risk factors for S. Typhi infection (1.6%; p = 0.048). The proportion of Salmonella Typhimurium/ Enteriditis among children eating street food 4 or more times per week was higher compared to 1 to 2 times/week on average (1.1%; p = 0.032). CONCLUSION: Typhoidal and NTS are important causes of illness in children in Mukuru informal settlement, especially among children less than 16 years of age. Improving Water, Sanitation and Hygiene (WASH) including boiling water, breastfeeding, hand washing practices, and avoiding animal contact in domestic settings could contribute to reducing the risk of transmission of Salmonella disease from contaminated environments.


Assuntos
Infecções por Salmonella/epidemiologia , Salmonella typhi/imunologia , Salmonella typhimurium/imunologia , Febre Tifoide/epidemiologia , Adolescente , Animais , Galinhas , Criança , Pré-Escolar , Características da Família , Fezes/microbiologia , Feminino , Humanos , Higiene , Lactente , Recém-Nascido , Quênia/epidemiologia , Masculino , Áreas de Pobreza , Prevalência , Fatores de Risco , Infecções por Salmonella/microbiologia , Salmonella typhi/isolamento & purificação , Salmonella typhimurium/isolamento & purificação , Saneamento , Testes Sorológicos , Febre Tifoide/microbiologia
7.
PLoS Negl Trop Dis ; 14(5): e0008326, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32463817

RESUMO

Salmonella and Shigella species are food- and water-borne pathogens that are responsible for enteric infections in both humans and animals and are still the major cause of morbidity and mortality in the emerging countries. The existence of multiple Salmonella and Shigella serotypes as well as the emergence of strains resistant to antibiotics require the development of broadly protective therapies. Those bacteria utilize a Type III Secretion System (T3SS), necessary for their pathogenicity. The structural proteins composing the T3SS are common to all virulent Salmonella and Shigella spp., particularly the needle-tip proteins SipD (Salmonella) and IpaD (Shigella). We investigated the immunogenicity and protective efficacy of SipD and IpaD administered by intranasal and intragastric routes, in a mouse model of Salmonella enterica serotype Typhimurium (S. Typhimurium) intestinal challenge. Robust IgG (in all immunization routes) and IgA (in intranasal and oral immunization routes) antibody responses were induced against both proteins. Mice immunized with SipD or IpaD were protected against lethal intestinal challenge with S. Typhimurium or Shigella flexneri (100 Lethal Dose 50%). We have shown that SipD and IpaD are able to induce a cross-protection in a murine model of infection by Salmonella and Shigella. We provide the first demonstration that Salmonella and Shigella T3SS SipD and IpaD are promising antigens for the development of a cross-protective Salmonella-Shigella vaccine. These results open the way to the development of cross-protective therapeutic molecules.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas de Bactérias/imunologia , Proteção Cruzada , Disenteria Bacilar/prevenção & controle , Proteínas de Membrana/imunologia , Infecções por Salmonella/prevenção & controle , Vacinas contra Salmonella/imunologia , Vacinas contra Shigella/imunologia , Administração Intranasal , Administração Oral , Animais , Anticorpos Antibacterianos/análise , Modelos Animais de Doenças , Feminino , Imunoglobulina A/análise , Imunoglobulina G/análise , Camundongos Endogâmicos BALB C , Vacinas contra Salmonella/administração & dosagem , Salmonella typhimurium/imunologia , Vacinas contra Shigella/administração & dosagem , Shigella flexneri/imunologia , Análise de Sobrevida
8.
PLoS Pathog ; 16(4): e1008360, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32330185

RESUMO

Intestinal epithelial cells (IECs) are at the forefront of host-pathogen interactions, coordinating a cascade of immune responses to protect against pathogens. Here we show that IEC-intrinsic vitamin A signaling restricts pathogen invasion early in the infection and subsequently activates immune cells to promote pathogen clearance. Mice blocked for retinoic acid receptor (RAR) signaling selectively in IECs (stopΔIEC) showed higher Salmonella burden in colonic tissues early in the infection that associated with higher luminal and systemic loads of the pathogen at later stages. Higher pathogen burden in stopΔIEC mice correlated with attenuated mucosal interferon gamma (IFNγ) production by underlying immune cells. We found that, at homeostasis, the intestinal epithelium of stopΔIEC mice produced significantly lower amounts of interleukin 18 (IL-18), a potent inducer of IFNγ. Regulation of IL-18 by vitamin A was also observed in a dietary model of vitamin A supplementation. IL-18 reconstitution in stopΔIEC mice restored resistance to Salmonella by promoting epithelial cell shedding to eliminate infected cells and limit pathogen invasion early in infection. Further, IL-18 augmented IFNγ production by underlying immune cells to restrict pathogen burden and systemic spread. Our work uncovers a critical role for vitamin A in coordinating a biphasic immune response to Salmonella infection by regulating IL-18 production by IECs.


Assuntos
Microbioma Gastrointestinal , Interleucina-18/metabolismo , Mucosa Intestinal/imunologia , Proteínas Associadas aos Microtúbulos/fisiologia , Infecções por Salmonella/prevenção & controle , Salmonella typhimurium/imunologia , Vitamina A/metabolismo , Animais , Interações Hospedeiro-Patógeno , Interferon gama/metabolismo , Mucosa Intestinal/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores do Ácido Retinoico/metabolismo , Infecções por Salmonella/imunologia , Infecções por Salmonella/microbiologia , Infecções por Salmonella/patologia , Transdução de Sinais
9.
Vet Res Commun ; 44(2): 51-59, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32297137

RESUMO

Inflammasome, a cytosolic multi-protein complex, assembly is a response to sensing intracellular pathogenic and endogenic danger signals followed by caspase-1 activation, which maturates precursor cytokines such as interleukin (IL)-1ß. Most inflammasome research has been undertaken in humans and rodents, and inflammasomes in veterinary species have not been well-characterized. In this study, we observed the effects of well-known inflammasome activators on equine peripheral blood monocytes (PBMCs). The NLRP3 inflammasome triggers include ATP, nigericin, aluminum crystals, and monosodium urate crystals, and NLRP3 activation induces IL-1ß secretion in a dose-dependent manner. Activators of NLRC4 and AIM2 inflammasomes include cytosolic flagellin and dsDNA, and their activation induces IL-1ß secretion. The bacterial inflammasome triggers Salmonella Typhimurium and Listeria monocytogenes also induce IL-ß releases. To elucidate the role of potassium efflux as an upstream signal of NLRP3 inflammasome activation, equine PBMCs were treated with blockers of potassium efflux in the presence of NLRP3 triggers. As a result, the IL-1ß secretion stemming from equine NLRP3 inflammasome activation was not completely attenuated by the inhibition of potassium efflux. Taken together, the results indicate that equine PBMCs normally secrete IL-1ß in response to well-known inflammasome activators, although equine NLRP3 inflammasome activation might not be dependent on potassium efflux.


Assuntos
Cavalos/imunologia , Inflamassomos/imunologia , Monócitos/imunologia , Trifosfato de Adenosina/farmacologia , Alumínio/farmacologia , Animais , DNA/farmacologia , Flagelina/farmacologia , Inflamassomos/efeitos dos fármacos , Interleucina-1beta/imunologia , Listeria monocytogenes/imunologia , Nigericina/farmacologia , Salmonella typhimurium/imunologia , Ácido Úrico/farmacologia
10.
PLoS Pathog ; 16(4): e1008498, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32282854

RESUMO

We investigated the role of the inflammasome effector caspases-1 and -11 during Salmonella enterica serovar Typhimurium infection of murine intestinal epithelial cells (IECs). Salmonella burdens were significantly greater in the intestines of caspase-1/11 deficient (Casp1/11-/-), Casp1-/- and Casp11-/- mice, as compared to wildtype mice. To determine if this reflected IEC-intrinsic inflammasomes, enteroid monolayers were derived and infected with Salmonella. Casp11-/- and wildtype monolayers responded similarly, whereas Casp1-/- and Casp1/11-/- monolayers carried significantly increased intracellular burdens, concomitant with marked decreases in IEC shedding and death. Pretreatment with IFN-γ to mimic inflammation increased caspase-11 levels and IEC death, and reduced Salmonella burdens in Casp1-/- monolayers, while high intracellular burdens and limited cell shedding persisted in Casp1/11-/- monolayers. Thus caspase-1 regulates inflammasome responses in IECs at baseline, while proinflammatory activation of IECs reveals a compensatory role for caspase-11. These results demonstrate the importance of IEC-intrinsic canonical and non-canonical inflammasomes in host defense against Salmonella.


Assuntos
Caspase 1/imunologia , Caspases Iniciadoras/imunologia , Inflamassomos/imunologia , Intestinos/enzimologia , Intestinos/imunologia , Infecções por Salmonella/enzimologia , Salmonella typhimurium/imunologia , Animais , Células Epiteliais/enzimologia , Células Epiteliais/imunologia , Células Epiteliais/microbiologia , Feminino , Imunidade nas Mucosas , Inflamassomos/metabolismo , Interferon gama/imunologia , Mucosa Intestinal/enzimologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/microbiologia , Intestinos/microbiologia , Lipopolissacarídeos , Macrófagos/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Infecções por Salmonella/imunologia , Salmonella typhimurium/patogenicidade
11.
Nat Commun ; 11(1): 1007, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32081907

RESUMO

Bacterial biofilms, especially those associated with implanted medical devices, are difficult to eradicate. Curli amyloid fibers are important components of the biofilms formed by the Enterobacteriaceae family. Here, we show that a human monoclonal antibody with pan-amyloid-binding activity (mAb 3H3) can disrupt biofilms formed by Salmonella enterica serovar Typhimurium in vitro and in vivo. The antibody disrupts the biofilm structure, enhancing biofilm eradication by antibiotics and immune cells. In mice, 3H3 injections allow antibiotic-mediated clearance of catheter-associated S. Typhimurium biofilms. Thus, monoclonal antibodies that bind a pan-amyloid epitope have potential to prevent or eradicate bacterial biofilms.


Assuntos
Amiloide/imunologia , Proteínas de Bactérias/imunologia , Biofilmes/crescimento & desenvolvimento , Salmonella typhimurium/imunologia , Salmonella typhimurium/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Infecções Relacionadas a Cateter/prevenção & controle , Epitopos/imunologia , Humanos , Macrófagos/imunologia , Camundongos , Infecções por Salmonella/prevenção & controle
12.
mBio ; 11(1)2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32071273

RESUMO

A major obstacle in infection biology is the limited ability to recapitulate human disease trajectories in traditional cell culture and animal models, which impedes the translation of basic research into clinics. Here, we introduce a three-dimensional (3D) intestinal tissue model to study human enteric infections at a level of detail that is not achieved by conventional two-dimensional monocultures. Our model comprises epithelial and endothelial layers, a primary intestinal collagen scaffold, and immune cells. Upon Salmonella infection, the model mimics human gastroenteritis, in that it restricts the pathogen to the epithelial compartment, an advantage over existing mouse models. Application of dual transcriptome sequencing to the Salmonella-infected model revealed the communication of epithelial, endothelial, monocytic, and natural killer cells among each other and with the pathogen. Our results suggest that Salmonella uses its type III secretion systems to manipulate STAT3-dependent inflammatory responses locally in the epithelium without accompanying alterations in the endothelial compartment. Our approach promises to reveal further human-specific infection strategies employed by Salmonella and other pathogens.IMPORTANCE Infection research routinely employs in vitro cell cultures or in vivo mouse models as surrogates of human hosts. Differences between murine and human immunity and the low level of complexity of traditional cell cultures, however, highlight the demand for alternative models that combine the in vivo-like properties of the human system with straightforward experimental perturbation. Here, we introduce a 3D tissue model comprising multiple cell types of the human intestinal barrier, a primary site of pathogen attack. During infection with the foodborne pathogen Salmonella enterica serovar Typhimurium, our model recapitulates human disease aspects, including pathogen restriction to the epithelial compartment, thereby deviating from the systemic infection in mice. Combination of our model with state-of-the-art genetics revealed Salmonella-mediated local manipulations of human immune responses, likely contributing to the establishment of the pathogen's infection niche. We propose the adoption of similar 3D tissue models to infection biology, to advance our understanding of molecular infection strategies employed by bacterial pathogens in their human host.


Assuntos
Técnicas de Cocultura/métodos , Interações Hospedeiro-Patógeno/fisiologia , Intestinos/microbiologia , Infecções por Salmonella/microbiologia , Animais , Sistemas CRISPR-Cas , Células CACO-2 , Modelos Animais de Doenças , Células Epiteliais/microbiologia , Epitélio/microbiologia , Gastroenterite/microbiologia , Regulação Bacteriana da Expressão Gênica , Humanos , Células Matadoras Naturais , Camundongos , Fator de Transcrição STAT3/metabolismo , Infecções por Salmonella/imunologia , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Transcriptoma , Sistemas de Secreção Tipo III
13.
Nat Commun ; 11(1): 851, 2020 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-32051408

RESUMO

Lipopolysaccharide (LPS) O-antigen (O-Ag) is known to limit antibody binding to surface antigens, although the relationship between antibody, O-Ag and other outer-membrane antigens is poorly understood. Here we report, immunization with the trimeric porin OmpD from Salmonella Typhimurium (STmOmpD) protects against infection. Atomistic molecular dynamics simulations indicate this is because OmpD trimers generate footprints within the O-Ag layer sufficiently sized for a single IgG Fab to access. While STmOmpD differs from its orthologue in S. Enteritidis (SEn) by a single amino-acid residue, immunization with STmOmpD confers minimal protection to SEn. This is due to the OmpD-O-Ag interplay restricting IgG binding, with the pairing of OmpD with its native O-Ag being essential for optimal protection after immunization. Thus, both the chemical and physical structure of O-Ag are key for the presentation of specific epitopes within proteinaceous surface-antigens. This enhances combinatorial antigenic diversity in Gram-negative bacteria, while reducing associated fitness costs.


Assuntos
Anticorpos Antibacterianos/imunologia , Proteínas da Membrana Bacteriana Externa/química , Proteínas da Membrana Bacteriana Externa/imunologia , Imunização , Antígenos O/imunologia , Salmonella typhimurium/imunologia , Animais , Anticorpos Antibacterianos/sangue , Formação de Anticorpos , Especificidade de Anticorpos , Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteção Cruzada , Modelos Animais de Doenças , Epitopos/química , Epitopos/imunologia , Imunoglobulina G/sangue , Camundongos , Modelos Moleculares , Antígenos O/química , Antígenos O/genética , Porinas/química , Porinas/genética , Porinas/imunologia , Conformação Proteica , Salmonelose Animal/imunologia , Salmonelose Animal/prevenção & controle , Análise de Sequência de Proteína
14.
Cell ; 180(1): 50-63.e12, 2020 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-31923399

RESUMO

Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.


Assuntos
Imunidade nas Mucosas/imunologia , Interleucina-18/imunologia , Mucosa Intestinal/imunologia , Animais , Citocinas/imunologia , Sistema Nervoso Entérico/imunologia , Sistema Nervoso Entérico/metabolismo , Células Epiteliais/imunologia , Feminino , Células Caliciformes/imunologia , Interleucina-18/biossíntese , Mucosa Intestinal/metabolismo , Intestino Delgado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/imunologia , Ratos , Ratos Sprague-Dawley , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Transdução de Sinais/imunologia
15.
Vet Microbiol ; 240: 108505, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31902485

RESUMO

Salmonella enterica Subsp enterica serovar Typhimurium (S. Typhimurium, ST) is one of the most important serovars of the genus Salmonella in human and animals. Because of its intracellular tropism, monocytes/macrophages are pivotal in killing of Salmonella serovars; they are also responsible for transporting of ST to extra-intestinal organs. To investigate the effect of the ST on the functions of avian innate immune cells, almost homogeneous enriched monocytes (EMo) were isolated from peripheral blood mononuclear cells of 2-3 weeks-old of healthy broilers. The EMo were then divided in three groups: control (media only), treatments (challenged with ST clinical isolates) and [doxorubicin (Dox), specifically as positive control for EMo apoptosis] groups. Cellular-molecular damage caused by ST in EMo was assessed with bioluminescence (for caspase-3, 7, and 9 activities and intracellular ATP content), chemiluminescence (for pro/anti-oxidant capacities) and flow cytometry (for apoptosis/necrosis). Further, phagocytosis capacity of post-ST challenged EMo was assessed using a flow cytometry-based internalisation of FITC-loaded polystyrene microparticles. Like the effects of Dox, in post-ST challenged EMo much higher caspase-3, 7 and 9 activities and ATP depletion along with decreased phagocytosis capacity and anti-oxidant load were observed. The results herein indicate that ST weakens EMo particularly through caspases activation/apoptosis. These findings can open a new window on the molecular aspects of Salmonella-macrophage interactions and immunopathology/pathogenicity of salmonellosis in animals especially avian species.


Assuntos
Trifosfato de Adenosina/análise , Leucócitos Mononucleares/microbiologia , Leucócitos Mononucleares/patologia , Fagocitose , Piroptose , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Animais , Caspases/análise , Morte Celular , Galinhas , Doxorrubicina/administração & dosagem , Citometria de Fluxo , Interações entre Hospedeiro e Microrganismos/imunologia , Leucócitos Mononucleares/imunologia , Medições Luminescentes , Macrófagos/imunologia , Macrófagos/microbiologia , Salmonelose Animal
16.
Nat Commun ; 11(1): 234, 2020 01 13.
Artigo em Inglês | MEDLINE | ID: mdl-31932605

RESUMO

Microfold cells (M cells) are responsible for antigen uptake to initiate immune responses in the gut-associated lymphoid tissue (GALT). Receptor activator of nuclear factor-κB ligand (RANKL) is essential for M cell differentiation. Follicle-associated epithelium (FAE) covers the GALT and is continuously exposed to RANKL from stromal cells underneath the FAE, yet only a subset of FAE cells undergoes differentiation into M cells. Here, we show that M cells express osteoprotegerin (OPG), a soluble inhibitor of RANKL, which suppresses the differentiation of adjacent FAE cells into M cells. Notably, OPG deficiency increases M cell number in the GALT and enhances commensal bacterium-specific immunoglobulin production, resulting in the amelioration of disease symptoms in mice with experimental colitis. By contrast, OPG-deficient mice are highly susceptible to Salmonella infection. Thus, OPG-dependent self-regulation of M cell differentiation is essential for the balance between the infectious risk and the ability to perform immunosurveillance at the mucosal surface.


Assuntos
Mucosa Intestinal/citologia , Mucosa Intestinal/imunologia , Osteoprotegerina/metabolismo , Animais , Anticorpos Antibacterianos/imunologia , Ceco/citologia , Ceco/imunologia , Ceco/metabolismo , Ceco/microbiologia , Diferenciação Celular , Colite/induzido quimicamente , Colite/imunologia , Colite/patologia , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Microbioma Gastrointestinal/imunologia , Homeostase , Imunidade nas Mucosas , Imunoglobulina G/imunologia , Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiologia , Tecido Linfoide/citologia , Tecido Linfoide/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Osteoprotegerina/genética , Ligante RANK/metabolismo , Receptor Ativador de Fator Nuclear kappa-B/metabolismo , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Salmonella typhimurium/patogenicidade , Transdução de Sinais
17.
Appl Microbiol Biotechnol ; 104(6): 2651-2661, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31997109

RESUMO

In the present study, we have generated a murine monoclonal antibody (mAb) named Sal-06 by using the crude outer membrane protein preparation of Salmonella enteric subsp. enterica serovar Typhimurium ATCC 14028 strain as antigen. Sal-06mAb belonging to IgG1 isotype demonstrated broad cross-reactivity to standard and isolated strains of genus Salmonella and others such as Escherichia coli, Klebsiella pneumonia, and Proteus mirabilis. Cross-reactivity across several bacterial genera indicated that the epitopes reactive to Sal-06mAb are conserved among these members. Neutralizing effects of Sal-06mAb on Salmonella growth and survival was evaluated in vitro using bacteriostatic and bactericidal activity with and without complement and bacterial invasion inhibition assay. Sal-06mAb demonstrated a bacteriostatic effect on the growth of S. typhimurium ATCC 14028 strain which is both time and concentration (of mAb) dependent. It was also found that the bacterial growth inhibition was complement independent. When the bacterial cells were preincubated with Sal-06mAb, it reduced the adherence and invasion of bacterial cells into A549 epithelial cell line. This was confirmed by CFU count analysis, phase contrast, and fluorescence microscopy. Scanning electron microscope (SEM) imaging confirmed the antimicrobial effects of Sal-06mAb on S. typhimurium ATCC 14028. The development of broadly reactive and cross protective Sal-06mAb opens new possibilities for immunotherapy of sepsis caused by Gram-negative Enterobacteriaceae members.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Proteínas da Membrana Bacteriana Externa/imunologia , Salmonella typhimurium/imunologia , Células A549 , Animais , Antígenos de Bactérias/imunologia , Aderência Bacteriana , Proteínas do Sistema Complemento , Reações Cruzadas , Enterobacteriaceae/imunologia , Escherichia coli/imunologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C
18.
Cancer Lett ; 469: 102-110, 2020 01 28.
Artigo em Inglês | MEDLINE | ID: mdl-31666180

RESUMO

Conventional chemotherapies have some limitations, including the lack of selectivity, high toxicity to normal tissues, multidrug resistance, and tumor relapse. Recently, great progress was made in immunotherapies for anticancer research, with bacteria-mediated cancer therapy one of the most promising approaches among them. Attenuated Salmonella have very specific targeting to various solid cancers, making them ideal vectors for the delivery and expression of immunostimulators. They have native bacterial immunogenicity and induce strong anticancer immunity in vivo. In this review, the recent advances in Salmonella-mediated cancer immunotherapies and the related mechanisms of Salmonella-based cancer therapies are summarized.


Assuntos
Antígenos de Neoplasias/genética , Vacinas Anticâncer/imunologia , Imunoterapia/métodos , Neoplasias/terapia , Salmonella typhimurium/imunologia , Animais , Antígenos de Neoplasias/imunologia , Vacinas Anticâncer/administração & dosagem , Vacinas Anticâncer/genética , Ensaios Clínicos Fase I como Assunto , Engenharia Genética , Humanos , Imunogenicidade da Vacina , Neoplasias/imunologia , Salmonella typhimurium/genética , Resultado do Tratamento , Microambiente Tumoral/imunologia , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/genética , Vacinas Atenuadas/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
19.
Microb Pathog ; 138: 103857, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31705999

RESUMO

In the present study, the importance of sodium bicarbonate antacid as an agent for an orally delivered attenuated Salmonella strain secreting Brucella antigens Cu-Zn superoxide dismutase (SodC) and outer membrane protein 19 (Omp19) as a live vaccine candidate against Brucella infection was investigated. First, Brucella antigens SodC and Omp19 were cloned into a prokaryotic constitutive expression vector, pJHL65. Then secretion of proteins was verified after transformation into an attenuated Salmonella typhimurium (ST) strain, JOL1800 (Δlon, ΔcpxR, Δasd, ΔrfaL), using western blot analysis. Mice were orally inoculated with phosphate-buffered saline (PBS) or with a co-mixture Salmonella secreting each antigens at a 1:1 ratio, each containing 1 × 108 CFU/mouse with and without sodium bicarbonate treatment. For antacid treatment, 1.3% w/v sodium bicarbonate was orally administered 30 min before and immediately after immunization with the Salmonella formulation. Humoral and cell-mediated immune responses were evaluated to investigate the efficacy of sodium bicarbonate in an oral formulation. The results indicated that addition of sodium bicarbonate to the vaccine significantly increased (P < 0.05) levels of anti-Brucella-specific systemic IgG responses, lymphocyte proliferation, and CD4+ T cell responses, indicating induction of a mixed Th1-Th2 response. Immunohistochemical assays and bacterial enumeration in intestinal samples also indicated that administration of sodium bicarbonate enhanced colonization of Salmonella. These results indicate that ingestion of the Salmonella formulation with sodium bicarbonate can enhance colonization of Salmonella and induce a significant protective immune response against Brucella compared with a formulation without sodium bicarbonate. Thus, incorporation of sodium bicarbonate as an antacid buffer is highly recommended for this oral live vaccine.


Assuntos
Vacina contra Brucelose , Bicarbonato de Sódio , Vacinas Atenuadas , Administração Oral , Animais , Antígenos de Bactérias/imunologia , Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/biossíntese , Vacinas Bacterianas/química , Vacina contra Brucelose/administração & dosagem , Vacina contra Brucelose/biossíntese , Vacina contra Brucelose/química , Imunidade Celular , Imunidade Humoral , Intestinos/imunologia , Intestinos/microbiologia , Camundongos , Microrganismos Geneticamente Modificados , Salmonella typhimurium/genética , Salmonella typhimurium/imunologia , Bicarbonato de Sódio/administração & dosagem , Transformação Bacteriana , Vacinação/métodos , Vacinas Atenuadas/administração & dosagem , Vacinas Atenuadas/biossíntese , Vacinas Atenuadas/química
20.
Cell Host Microbe ; 27(1): 41-53.e6, 2020 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-31862381

RESUMO

Many Gram-negative bacterial pathogens antagonize anti-bacterial immunity through translocated effector proteins that inhibit pro-inflammatory signaling. In addition, the intracellular pathogen Salmonella enterica serovar Typhimurium initiates an anti-inflammatory transcriptional response in macrophages through its effector protein SteE. However, the target(s) and molecular mechanism of SteE remain unknown. Here, we demonstrate that SteE converts both the amino acid and substrate specificity of the host pleiotropic serine/threonine kinase GSK3. SteE itself is a substrate of GSK3, and phosphorylation of SteE is required for its activity. Remarkably, phosphorylated SteE then forces GSK3 to phosphorylate the non-canonical substrate signal transducer and activator of transcription 3 (STAT3) on tyrosine-705. This results in STAT3 activation, which along with GSK3 is required for SteE-mediated upregulation of the anti-inflammatory M2 macrophage marker interleukin-4Rα (IL-4Rα). Overall, the conversion of GSK3 to a tyrosine-directed kinase represents a tightly regulated event that enables a bacterial virulence protein to reprogram innate immune signaling and establish an anti-inflammatory environment.


Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Macrófagos/microbiologia , Proteínas Serina-Treonina Quinases/metabolismo , Fator de Transcrição STAT3/metabolismo , Salmonella typhimurium , Animais , Proteínas de Bactérias/metabolismo , Células HEK293 , Células HeLa , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Interleucina-4/metabolismo , Ativação de Macrófagos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Tirosina Quinases/metabolismo , Salmonella typhimurium/imunologia , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Virulência/imunologia
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