RESUMEN
BACKGROUND & AIMS: The diarrheagenic pathogen, enteropathogenic Escherichia coli (EPEC), uses a type III secretion system to deliver effector molecules into intestinal epithelial cells (IECs). While exploring the basis for the lateral membrane separation of EPEC-infected IECs, we observed infection-induced loss of the desmosomal cadherin desmoglein-2 (DSG2). We sought to identify the molecule(s) involved in, and delineate the mechanisms and consequences of, EPEC-induced DSG2 loss. METHODS: DSG2 abundance and localization was monitored via immunoblotting and immunofluorescence, respectively. Junctional perturbations were visualized by electron microscopy, and cell-cell adhesion was assessed using dispase assays. EspH alanine-scan mutants as well as pharmacologic agents were used to evaluate impacts on desmosomal alterations. EPEC-mediated DSG2 loss, and its impact on bacterial colonization in vivo, was assessed using a murine model. RESULTS: The secreted virulence protein EspH mediates EPEC-induced DSG2 degradation, and contributes to desmosomal perturbation, loss of cell junction integrity, and barrier disruption in infected IECs. EspH sequesters Rho guanine nucleotide exchange factors and inhibits Rho guanosine triphosphatase signaling; EspH mutants impaired for Rho guanine nucleotide exchange factor interaction failed to inhibit RhoA or deplete DSG2. Cytotoxic necrotizing factor 1, which locks Rho guanosine triphosphatase in the active state, jasplakinolide, a molecule that promotes actin polymerization, and the lysosomal inhibitor bafilomycin A, respectively, rescued infected cells from EPEC-induced DSG2 loss. Wild-type EPEC, but not an espH-deficient strain, colonizes mouse intestines robustly, widens paracellular junctions, and induces DSG2 re-localization in vivo. CONCLUSIONS: Our studies define the mechanism and consequences of EPEC-induced desmosomal alterations in IECs. These perturbations contribute to the colonization and virulence of EPEC, and likely related pathogens.
RESUMEN
Members of the genus Neisseria have been isolated from or detected in a wide range of animals, from non-human primates and felids to a rodent, the guinea pig. By means of selective culture, biochemical testing, Gram staining and PCR screening for the Neisseria-specific internal transcribed spacer region of the rRNA operon, we isolated four strains of the genus Neisseria from the oral cavity of the wild house mouse, Mus musculus subsp. domesticus. The isolates are highly related and form a separate clade in the genus, as judged by tree analyses using either multi-locus sequence typing of ribosomal genes or core genes. One isolate, provisionally named Neisseria musculi sp. nov. (type strain AP2031T=DSM 101846T=CCUG 68283T=LMG 29261T), was studied further. Strain AP2031T/N. musculi grew well in vitro. It was naturally competent, taking up DNA in a DNA uptake sequence and pilT-dependent manner, and was amenable to genetic manipulation. These and other genomic attributes of N. musculi sp. nov. make it an ideal candidate for use in developing a mouse model for studying Neisseria-host interactions.
Asunto(s)
Ratones/microbiología , Neisseria/clasificación , Filogenia , Animales , Técnicas de Tipificación Bacteriana , Composición de Base , ADN Bacteriano/genética , Genes Bacterianos , Boca/microbiología , Tipificación de Secuencias Multilocus , Neisseria/genética , Neisseria/aislamiento & purificación , América del Norte , Hibridación de Ácido Nucleico , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND/AIMS: Sex differences in gene expression program have not been effectively explored at the transcriptome level. We aimed to develop a method for the analysis of transcriptome data to identify sex differences and sex-dimorphic responses to experimental conditions in mice. METHODS: Profiling of the small intestine transcriptome of chow-fed C57BL/6J (wild-type, WT) and Fabp2â»/â» mice was carried out by microarray analysis. Sex-specific and androgynous effects of Fabp2 gene ablation were examined using FlexArray V1.6 by comparing WT to Fabp2â»/â» mice. The data generated were exported into a single spreadsheet, collated and transformed to identify the differentially expressed genes for pathway analysis. RESULTS: The method revealed enrichment of 17 sex-dimorphic pathways in the small intestine of WT mice compared to only 4 in Fabp2â»/â» mice. Comparison of the effects of Fabp2 loss in individual sexes revealed a male-specific upregulation of 5 pathways involved in the production of unsaturated fatty acids, and a female-specific downregulation of pathways involved in xenobiotic metabolism. CONCLUSIONS: Our approach detected the common as well as sex-differential pathways that are modified due to the loss of Fabp2. These findings suggest that the pathways involved in nutrient and xenobiotic metabolism in the intestine are regulated by sex-specific mechanisms.
Asunto(s)
Proteínas de Unión a Ácidos Grasos/genética , Mucosa Intestinal/metabolismo , Caracteres Sexuales , Transcriptoma , Animales , Regulación hacia Abajo , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Regulación hacia ArribaRESUMEN
We identified and characterized 14 novel short-tandem-repeats (STRs) on the Y chromosome and typed them in two samples, a globally diverse panel of 73 cell lines, and 148 individuals from a European-American population. These Y-STRs include eight tetranucleotide repeats (DYS449, DYS453, DYS454, DYS455, DYS456, DYS458, DYS459, and DYS464), five pentanucleotide repeats (DYS446, DYS447, DYS450, DYS452, and DYS463), and one hexanucleotide repeat (DYS448). Sequence data were obtained to designate a repeat number nomenclature. The gene diversities of an additional 22 Y-STRs, including the most commonly used in forensic databases, were directly compared in the cell line DNAs. Six of the 10 most polymorphic markers include the newly identified Y-STRs. Furthermore, these novel Y-STRs greatly improved the resolution of paternal lineages, above the level obtained with commonly used Y-STRs, in the European-American population.