RESUMEN
Nontyphoidal Salmonella (NTS) species cause self-limiting diarrhea and sometimes severe disease. Antibiotic treatment is considered only in severe cases and immune-compromised patients. The beneficial effects of antibiotic therapy and the consequences for adaptive immune responses are not well understood. We used a mouse model for Salmonella diarrhea to assess the effects of per os treatment with ciprofloxacin (15 mg/kg of body weight intragastrically 2 times/day, 5 days) or parenteral ceftriaxone (50 mg/kg intraperitoneally, 5 days), two common drugs used in human patients. The therapeutic and adverse effects were assessed with respect to generation of a protective adaptive immune response, fecal pathogen excretion, and the emergence of nonsymptomatic excreters. In the mouse model, both therapies reduced disease severity and reduced the level of fecal shedding. In line with clinical data, in most animals, a rebound of pathogen gut colonization/fecal shedding was observed 2 to 12 days after the end of the treatment. Yet, levels of pathogen shedding and frequency of appearance of nonsymptomatic excreters did not differ from those for untreated controls. Moreover, mice treated intraperitoneally with ceftriaxone developed an adaptive immunity protecting the mice from enteropathy in wild-type Salmonella enterica serovar Typhimurium challenge infections. In contrast, the mice treated intragastrically with ciprofloxacin were not protected. Thus, antibiotic treatment regimens can disrupt the adaptive immune response, but treatment regimens may be optimized in order to preserve the generation of protective immunity. It might be of interest to determine whether this also pertains to human patients. In this case, the mouse model might be a tool for further mechanistic studies.
Asunto(s)
Inmunidad Adaptativa/efectos de los fármacos , Ceftriaxona/administración & dosificación , Ciprofloxacina/administración & dosificación , Diarrea/tratamiento farmacológico , Infecciones por Salmonella/tratamiento farmacológico , Salmonella typhimurium/efectos de los fármacos , Administración Oral , Animales , Antibacterianos/administración & dosificación , Antibacterianos/uso terapéutico , Ceftriaxona/uso terapéutico , Ciprofloxacina/uso terapéutico , Recuento de Colonia Microbiana , Diarrea/inmunología , Diarrea/microbiología , Modelos Animales de Enfermedad , Esquema de Medicación , Heces/microbiología , Infusiones Parenterales , Ratones , Ratones Endogámicos C57BL , Infecciones por Salmonella/inmunología , Infecciones por Salmonella/microbiología , Salmonella typhimurium/inmunología , Índice de Severidad de la EnfermedadRESUMEN
The intestinal ecosystem is formed by a complex, yet highly characteristic microbial community. The parameters defining whether this community permits invasion of a new bacterial species are unclear. In particular, inhibition of enteropathogen infection by the gut microbiota ( = colonization resistance) is poorly understood. To analyze the mechanisms of microbiota-mediated protection from Salmonella enterica induced enterocolitis, we used a mouse infection model and large scale high-throughput pyrosequencing. In contrast to conventional mice (CON), mice with a gut microbiota of low complexity (LCM) were highly susceptible to S. enterica induced colonization and enterocolitis. Colonization resistance was partially restored in LCM-animals by co-housing with conventional mice for 21 days (LCM(con21)). 16S rRNA sequence analysis comparing LCM, LCM(con21) and CON gut microbiota revealed that gut microbiota complexity increased upon conventionalization and correlated with increased resistance to S. enterica infection. Comparative microbiota analysis of mice with varying degrees of colonization resistance allowed us to identify intestinal ecosystem characteristics associated with susceptibility to S. enterica infection. Moreover, this system enabled us to gain further insights into the general principles of gut ecosystem invasion by non-pathogenic, commensal bacteria. Mice harboring high commensal E. coli densities were more susceptible to S. enterica induced gut inflammation. Similarly, mice with high titers of Lactobacilli were more efficiently colonized by a commensal Lactobacillus reuteri(RR) strain after oral inoculation. Upon examination of 16S rRNA sequence data from 9 CON mice we found that closely related phylotypes generally display significantly correlated abundances (co-occurrence), more so than distantly related phylotypes. Thus, in essence, the presence of closely related species can increase the chance of invasion of newly incoming species into the gut ecosystem. We provide evidence that this principle might be of general validity for invasion of bacteria in preformed gut ecosystems. This might be of relevance for human enteropathogen infections as well as therapeutic use of probiotic commensal bacteria.
Asunto(s)
Bacterias/genética , Susceptibilidad a Enfermedades/microbiología , Enterocolitis/microbiología , Intestinos/microbiología , Infecciones por Salmonella/microbiología , Animales , Genes Bacterianos , Ratones , Filogenia , ARN Ribosómico 16S , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Salmonella enterica subspecies I serovar Typhimurium (S. Typhimurium) is a frequent cause of diarrhea worldwide. It employs 2 type III secretion systems (TTSS) to elicit mucosal inflammation via the TTSS-1-dependent 'classical' or the TTSS-2-dependent 'alternative' pathway. If TTSS-1 is defective (in invG or invC mutants), the pathogen is confined to the alternative pathway; transits the epithelium in a dendritic cell-dependent fashion, relocalizes from CD11c(+) dendritic cells to CD11c(-) cells, and elicits inflammation by day 3 post infection (p.i.). It has remained unclear whether other virulence factors may also contribute to this process. Here, we used the streptomycin mouse model to analyze whether spvB and spvC, virulence factors known to affect the pathogen-phagocyte interaction at systemic sites, might contribute to triggering colitis. By 12h p.i., spvBC mutants elicited wild-type levels of gut inflammation and mucosal cytokine induction via the classical pathway. However, spvBC mutants confined to the alternative pathway triggered reduced levels of gut inflammation by day 3 p.i. (S. tm(ΔinvGΔspvBC) vs. S. tm(ΔinvG)). Detailed analyses using spvB or spvC mutants (e.g. S. tm(ΔinvCΔspvB)) revealed that spvB was required for efficient lamina propria colonization and suggested that this was attributable to defective relocalization from dendritic- to CD11c(-) cells. This establishes a novel virulence phenotype for spvB in the alternative pathway of S. Typhimurium colitis.
Asunto(s)
ADP Ribosa Transferasas/metabolismo , Liasas de Carbono-Oxígeno/metabolismo , Colitis/microbiología , Colitis/patología , Salmonella typhimurium/patogenicidad , Factores de Virulencia/metabolismo , Animales , Carga Bacteriana , Colon/microbiología , Colon/patología , Citocinas/metabolismo , Histocitoquímica , Humanos , Inflamación/microbiología , Inflamación/patología , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Ratones , Ratones Endogámicos C57BL , Índice de Severidad de la Enfermedad , Factores de TiempoRESUMEN
Salmonella enterica subsp. I serovars Typhimurium and Enteritidis are major causes of enteric disease. The pathomechanism of enteric infection by serovar Typhimurium has been studied in detail. Serovar Typhimurium employs two pathways in parallel for triggering disease, i.e., the "classical" pathway, triggered by type III secretion system 1 (TTSS-1), and the "alternative" pathway, mediated by TTSS-2. It had remained unclear whether these two pathways would also explain the enteropathogenesis of strains from other serovars. We chose the isolate P125109 of the epidemic serovar Enteritidis PT4/6, generated isogenic mutants, and studied their virulence. Using in vitro and in vivo infection experiments, a dendritic cell depletion strategy, and MyD88(-/-) knockout mice, we found that P125109 employs both the "classical" and "alternative" pathways for triggering mucosal inflammation. The "classical" pathway was phenotypically similar in serovar Typhimurium strain SL1344 and in P125109. However, the kinetics of the "alternative" pathway differed significantly. Via TTSS-2, P125109 colonized the gut tissue more efficiently and triggered mucosal inflammation approximately 1 day faster than SL1344 did. In conclusion, our data demonstrate that different Salmonella spp. can differ in their capacity to trigger mucosal inflammation via the "alternative" pathway in vivo.
Asunto(s)
Colitis/microbiología , Infecciones por Salmonella/microbiología , Salmonella enteritidis/patogenicidad , Enfermedad Aguda , Animales , Colitis/inmunología , Células Dendríticas/fisiología , Células HeLa , Humanos , Ratones , Ratones Endogámicos C57BL , Factor 88 de Diferenciación Mieloide/fisiología , Infecciones por Salmonella/inmunología , Salmonella enteritidis/clasificación , Salmonella enteritidis/metabolismo , Salmonella typhimurium/patogenicidadRESUMEN
Intestinal inflammation induces alterations of the gut microbiota and promotes overgrowth of the enteric pathogen Salmonella enterica by largely unknown mechanisms. Here, we identified a host factor involved in this process. Specifically, the C-type lectin RegIIIß is strongly upregulated during mucosal infection and released into the gut lumen. In vitro, RegIIIß kills diverse commensal gut bacteria but not Salmonella enterica subspecies I serovar Typhimurium (S. Typhimurium). Protection of the pathogen was attributable to its specific cell envelope structure. Co-infection experiments with an avirulent S. Typhimurium mutant and a RegIIIß-sensitive commensal E. coli strain demonstrated that feeding of RegIIIß was sufficient for suppressing commensals in the absence of all other changes inflicted by mucosal disease. These data suggest that RegIIIß production by the host can promote S. Typhimurium infection by eliminating inhibitory gut microbiota.
Asunto(s)
Mucosa Intestinal/metabolismo , Mucosa Intestinal/microbiología , Metagenoma , Viabilidad Microbiana , Proteínas/metabolismo , Salmonella typhimurium/metabolismo , Animales , Antígenos Bacterianos/inmunología , Recuento de Colonia Microbiana , Escherichia coli/crecimiento & desarrollo , Ratones , Modelos Biológicos , Proteínas Asociadas a Pancreatitis , Infecciones por Salmonella/microbiología , Salmonella typhimurium/inmunologíaRESUMEN
The innate immune system is of vital importance for protection against infectious pathogens. Inflammasome mediated caspase-1 activation and subsequent release of pro-inflammatory cytokines like IL-1beta and IL-18 is an important arm of the innate immune system. Salmonella enterica subspecies 1 serovar Typhimurium (S. Typhimurium, SL1344) is an enteropathogenic bacterium causing diarrheal diseases. Different reports have shown that in macrophages, S. Typhimurium may activate caspase-1 by at least three different types of stimuli: flagellin, the type III secretion system 1 (T1) and the T1 effector protein SopE. However, the relative importance and interdependence of the different factors in caspase-1 activation is still a matter of debate. Here, we have analyzed their relative contributions to caspase-1 activation in LPS-pretreated RAW264.7 macrophages. Using flagellar mutants (fliGHI, flgK) and centrifugation to mediate pathogen-host cell contact, we show that flagellins account for a small part of the caspase-1 activation in RAW264.7 cells. In addition, functional flagella are of key importance for motility and host cell attachment which is a prerequisite for mediating caspase-1 activation via these three stimuli. Using site directed mutants lacking several T1 effector proteins and flagellin expression, we found that SopE elicits caspase-1 activation even when flagellins are absent. In contrast, disruption of essential genes of the T1 protein injection system (invG, sipB) completely abolished caspase-1 activation. However, a robust level of caspase-1 activation is retained by the T1 system (or unidentified T1 effectors) in the absence of flagellin and SopE. T1-mediated inflammasome activation is in line with recent work by others and suggests that the T1 system itself may represent the basic caspase-1 activating stimulus in RAW264.7 macrophages which is further enhanced independently by SopE and/or flagellin.
Asunto(s)
Proteínas Bacterianas/farmacología , Caspasa 1/metabolismo , Flagelina , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Salmonella typhimurium , Animales , Adhesión Bacteriana , Proteínas Bacterianas/metabolismo , Biocatálisis , Línea Celular , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Macrófagos/enzimología , Ratones , Salmonella typhimurium/fisiologíaRESUMEN
Salmonella Typhimurium causes bacterial enterocolitis. The type III secretion system (TTSS)-1 is a key virulence determinant of S. Typhimurium mediating host cell invasion and acute enterocolitis. The TTSS-1 effector protein SipA is transported into host cells, accumulates in characteristic foci at the bacteria-host cell interface, manipulates signalling and affects virulence. Two functional domains of SipA have previously been characterized: The N-terminal SipA region (amino acids 1-105) mediates TTSS-1 transport and the C-terminal SipA 'actin-binding' domain (ABD; amino acids 446-685) manipulates F-actin assembly. Little is known about the central region of SipA. In a deletion analysis we found that the central SipA region harbours two distinct functional domains, F1 and F2. They are involved in SipA focus formation and host manipulation. The F1 domain (amino acids 170-271) drives SipA focus formation and domain F2 (amino acids 280-394) enhances this process by mediating SipA-SipA interactions. SipA variants lacking the F1-, the F2- or the actin binding domain were attenuated in virulence assays, namely host cell invasion and/or virulence in a mouse model for enterocolitis. Our results show that the newly identified SipA domains have distinct functions. Nevertheless, cooperation between the SipA domains F1, F2 and ABD is required to promote Salmonella virulence.