RESUMEN
The life cycle of the opportunistic fungal pathogen Pneumocystis murina consists of a trophic stage and an ascus-like cystic stage. Infection with the cyst stage induces proinflammatory immune responses, while trophic forms suppress the cytokine response to multiple pathogen-associated molecular patterns (PAMPs), including ß-glucan. A targeted gene expression assay was used to evaluate the dendritic cell response following stimulation with trophic forms alone, with a normal mixture of trophic forms and cysts, or with ß-glucan. We demonstrate that stimulation with trophic forms downregulated the expression of multiple genes normally associated with the response to infection, including genes encoding transcription factors. Trophic forms also suppressed the expression of genes related to antigen processing and presentation, including the gene encoding the major histocompatibility complex (MHC) class II transactivator, CIITA. Stimulation of dendritic cells with trophic forms, but not a mixture of trophic forms and cysts, reduced the expression of MHC class II and the costimulatory molecule CD40 on the surface of the cells. These defects in the expression of MHC class II and costimulatory molecules corresponded with a reduced capacity for trophic form-loaded dendritic cells to stimulate CD4+ T cell proliferation and polarization. These data are consistent with the delayed innate and adaptive responses previously observed in immunocompetent mice inoculated with trophic forms compared to responses in mice inoculated with a mixture of trophic forms and cysts. We propose that trophic forms broadly inhibit the ability of dendritic cells to fulfill their role as antigen-presenting cells.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Células Dendríticas/inmunología , Células Dendríticas/microbiología , Pneumocystis/crecimiento & desarrollo , Pneumocystis/inmunología , Animales , Presentación de Antígeno , Células Dendríticas/metabolismo , Células Dendríticas/patología , Regulación de la Expresión Génica , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Activación de Linfocitos , Ratones , Moléculas de Patrón Molecular Asociado a Patógenos/inmunología , Neumonía por Pneumocystis/inmunología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , beta-Glucanos/inmunología , beta-Glucanos/metabolismoRESUMEN
The DNA damage response triggered by bacterial cytolethal distending toxins (CDTs) is associated with activation of the actin-regulating protein RhoA and phosphorylation of the downstream-regulated mitogen-activated protein kinase (MAPK) p38, which promotes the survival of intoxicated (i.e. cells exposed to a bacterial toxin) cells. To identify the effectors of this CDT-induced survival response, we screened a library of 4492 Saccharomyces cerevisiae mutants that carry deletions in nonessential genes for reduced growth following inducible expression of CdtB. We identified 78 genes whose deletion confers hypersensitivity to toxin. Bioinformatics analysis revealed that DNA repair and endocytosis were the two most overrepresented signaling pathways. Among the human orthologs present in our data set, FEN1 and TSG101 regulate DNA repair and endocytosis, respectively, and also share common interacting partners with RhoA. We further demonstrate that FEN1, but not TSG101, regulates cell survival, MAPK p38 phosphorylation, RhoA activation and actin cytoskeleton reorganization in response to DNA damage. Our data reveal a previously unrecognized crosstalk between DNA damage and cytoskeleton dynamics in the regulation of cell survival, and might provide new insights on the role of chronic bacteria infection in carcinogenesis.
Asunto(s)
Toxinas Bacterianas/metabolismo , Supervivencia Celular , Citoesqueleto/metabolismo , Endonucleasas de ADN Solapado/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Actinas/metabolismo , Toxinas Bacterianas/genética , Supervivencia Celular/genética , Biología Computacional , Citoesqueleto/ultraestructura , Daño del ADN , Reparación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endocitosis/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/genética , Complejos de Clasificación Endosomal Requeridos para el Transporte/metabolismo , Endonucleasas de ADN Solapado/genética , Células HeLa , Humanos , Saccharomyces cerevisiae/genética , Eliminación de Secuencia/genética , Transducción de Señal/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transgenes/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Synthesis of the siderophore yersiniabactin (Ybt) proceeds by a mixed nonribosomal peptide synthetase/polyketide synthase mechanism. Transcription of ybt genes encoding biosynthetic and transport functions is repressed under excess iron conditions by Fur, but is also activated by Ybt via the transcriptional regulator YbtA. While mutations in most biosynthetic genes and ybtA negate transcription activation from the regulated promoters, three biosynthetic mutations do not reduce this transcriptional activation. Here we show that two of these mutants, one lacking the putative type II thioesterase (TE) YbtT and the other with a mutation in the TE domain of HMWP1, produce reduced levels of authentic Ybt that are capable of signalling activity. Alanine substitutions in two residues of YbtT that are essential for catalytic activity in other type II TEs reduced the ability of Yersinia pestis to grow under iron-chelated conditions. The third mutant, which lacks the salicylate synthase YbtS, did not make authentic Ybt but did produce a signalling molecule. Finally, a Delta pgm strain of Y. pestis, which lacks essential Ybt biosynthetic genes, also produced a signalling molecule that can activate transcription of ybt genes. The non-Ybt signal molecules from these two mutants are likely separate compounds. While these compounds are not biologically relevant to normal Ybt regulation, a comparison of the structures of Ybt and other signalling molecules will help in determining the chemical structures recognized as a Ybt signal.
Asunto(s)
Proteínas Bacterianas/genética , Fenoles/metabolismo , Sideróforos/biosíntesis , Tiazoles/metabolismo , Activación Transcripcional , Yersinia pestis/genética , Yersinia pestis/metabolismo , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión GénicaRESUMEN
Cytolethal distending toxin (CDT) is a multisubunit protein found in various gram-negative bacterial pathogens of humans which is thought to cause cell death by direct DNA damage of host cells. We sought to determine if a cellular response to DNA damage could be detected by exogenous addition of the holotoxin. Exogenous addition of the Campylobacter jejuni 81-176 CDT to primary human fibroblasts resulted in formation of Rad50 foci, which are formed around double-stranded-DNA breaks. Moreover, such foci are formed in both proliferating and nonproliferating cells that are treated with C. jejuni CDT. Fibroblasts that were intoxicated and later stimulated to proliferate failed to divide and remained arrested in the G(1) phase of the cell cycle.
Asunto(s)
Toxinas Bacterianas/farmacología , Campylobacter jejuni/metabolismo , Enzimas Reparadoras del ADN , Reparación del ADN/efectos de los fármacos , Ácido Anhídrido Hidrolasas , Ciclo Celular/efectos de los fármacos , División Celular/efectos de los fármacos , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Fibroblastos , HumanosRESUMEN
Campylobacter jejuni produces a toxin, called cytolethal distending toxin (CDT), which causes direct DNA damage leading to invocation of DNA damage checkpoint pathways. The affected cells arrest in G(1) or G(2) and eventually die. CDT consists of three protein subunits, CdtA, CdtB, and CdtC, with CdtB recently identified as a nuclease. However, little is known about the functions of CdtA or CdtC. In this work, enzyme-linked immunosorbent assay-based experiments were used to show, for the first time, that both CdtA and CdtC bound with specificity to the surface of HeLa cells, whereas CdtB did not. Varying the order of the addition of subunits for reconstitution of the holotoxin had no effect on activity. In addition, mutants containing deletions of conserved regions of CdtA and CdtC were able to bind to the surface of HeLa cells but were not able to participate in holotoxin assembly. Finally, both Cdt mutant subunits were able to effectively compete with CDT holotoxin in the HeLa cell binding assay.
Asunto(s)
Toxinas Bacterianas/toxicidad , Campylobacter jejuni/patogenicidad , Secuencia de Aminoácidos , Toxinas Bacterianas/química , Toxinas Bacterianas/genética , Secuencia de Bases , Unión Competitiva , Biotina , Campylobacter jejuni/genética , Campylobacter jejuni/metabolismo , Campylobacter jejuni/fisiología , ADN Bacteriano/genética , Genes Bacterianos , Células HeLa , Humanos , Datos de Secuencia Molecular , Mutación , Unión Proteica , Subunidades de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/toxicidadRESUMEN
A collection of 20 Escherichia coli strains that produce cytolethal distending toxin (CDT) were analyzed for their virulence-associated genes. All of these strains were serotyped, and multiplex PCR analysis was used to ascertain the presence of genes encoding other virulence factors, including Shiga toxin, intimin, enterohemolysin, cytotoxic necrotizing factor type 1 (CNF1) and CNF2, heat-stable toxin, and heat-labile toxin. These CDT-producing strains possessed various combinations of known virulence genes, some of which have not been noted before. Partial cdtB sequences were obtained from 10 of these strains, and their predicted CdtB sequences were compared to known E. coli CdtB sequences; some of the sequences were identical to known CdtB sequences, but two were not. PCR primers based on sequence differences between the known cdt sequences were tested for their ability to detect CDT producers and to determine CDT type. Correlations between the type of CDT produced, the presence of other virulence properties, and overall strain relatedness revealed that the CDT producers studied here can be divided into three general groups, with distinct differences in CDT type and in their complement of virulence-associated genes.