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1.
Cell Microbiol ; 3(11): 731-44, 2001 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-11696033

RESUMO

After uptake by murine macrophages, Salmonella typhimurium is able to survive and replicate within specialized phagosomes called Salmonella-containing vacuoles (SCVs), which are segregated from the late endocytic pathway. The molecular basis of this process and the virulence factors required are not fully understood. In this study, we used confocal fluorescence microscopy to evaluate interactions between the endocytic pathway of the murine macrophage cell line RAW 264.7 and different S. typhimurium strains. The analysis was carried out using the fluid-phase marker Texas red-ovalbumin and antibodies against the lysosomal enzyme cathepsin D, the late endosomal lipid lysobisphosphatidic acid and the adaptor proteins AP-1 and AP-3. Less than 10% of wild-type SCVs were associated with these markers at 24 h after uptake by macrophages. A similar low level of association was observed for vacuoles containing mutant strains affected in the function of the Salmonella pathogenicity island (SPI)-2 type III secretion system or the virulence plasmid spv operon. However, at this time point, the proportion of vacuoles containing phoP-mutant bacteria that were associated with each of the markers ranged from 25% to 50%. These results show that the regulon controlled by the PhoP/Q two-component system makes a major contribution to trafficking of the SCV in macrophages. Segregation of SCVs from the endocytic pathway was also found to be dependent on bacterial proteins synthesized between 15 min and 4 h after uptake into macrophages. However, after this time, protein synthesis was not required to maintain the segregation of SCVs from late endosomes and lysosomes.


Assuntos
Proteínas de Bactérias/metabolismo , Macrófagos/microbiologia , Regulon/genética , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidade , Animais , Proteínas de Bactérias/genética , Catepsina D/metabolismo , Linhagem Celular , Endocitose , Regulação Bacteriana da Expressão Gênica , Lisossomos/fisiologia , Macrófagos/fisiologia , Camundongos , Microscopia Confocal , Fagossomos/microbiologia , Fagossomos/fisiologia , Infecções por Salmonella/microbiologia , Salmonella typhimurium/fisiologia , Virulência/genética
2.
EMBO J ; 19(13): 3235-49, 2000 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-10880437

RESUMO

A method based on the Competitive Index was used to identify Salmonella typhimurium virulence gene interactions during systemic infections of mice. Analysis of mixed infections involving single and double mutant strains showed that OmpR, the type III secretion system of Salmonella pathogenicity island 2 (SPI-2) and SifA [required for the formation in epithelial cells of lysosomal glycoprotein (lgp)-containing structures, termed Sifs] are all involved in the same virulence function. sifA gene expression was induced after Salmonella entry into host cells and was dependent on the SPI-2 regulator ssrA. A sifA(-) mutant strain had a replication defect in macrophages, similar to that of SPI-2 and ompR(-) mutant strains. Whereas wild-type and SPI-2 mutant strains reside in vacuoles that progressively acquire lgps and the vacuolar ATPase, the majority of sifA(-) bacteria lost their vacuolar membrane and were released into the host cell cytosol. We propose that the wild-type strain, through the action of SPI-2 effectors (including SpiC), diverts the Salmonella-containing vacuole from the endocytic pathway, and subsequent recruitment and maintenance of vacuolar ATPase/lgp-containing membranes that enclose replicating bacteria is mediated by translocation of SifA.


Assuntos
Genes Bacterianos , Salmonella typhimurium/patogenicidade , Vacúolos/ultraestrutura , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sequência de Bases , Linhagem Celular , Primers do DNA , Células HeLa , Humanos , Camundongos , Dados de Sequência Molecular , Ligação Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Salmonelose Animal/microbiologia , Salmonella typhimurium/genética , Salmonella typhimurium/ultraestrutura , Homologia de Sequência de Aminoácidos , Virulência/genética
3.
Mol Microbiol ; 32(4): 691-701, 1999 May.
Artigo em Inglês | MEDLINE | ID: mdl-10361274

RESUMO

Presented here is the first evidence that Campylobacter jejuni secrete proteins upon co-cultivation with host cells and in INT 407 cell-conditioned medium. A C. jejuni gene designated ciaB for Campylobacter invasion antigen B was identified, using a differential screening technique, which is required for this secretion process and the efficient entry of this bacterium into a host cell. The C. jejuni ciaB gene encodes a protein of 610 amino acids with a calculated molecular mass of 73 154 Da. The deduced amino acid sequence of the CiaB protein shares similarity with type III secreted proteins associated with the invasion of host cells from other more extensively characterized bacterial pathogens. In vitro binding and internalization assays revealed that the binding of C. jejuni ciaB null mutants was indistinguishable from that of the parental isolate, whereas a significant reduction was noted in internalization. Confocal microscopic examination of C. jejuni-infected cells revealed that CiaB was translocated into the cytoplasm of the host cells. Culturing C. jejuni with INT 407 cells or in INT 407-conditioned medium resulted in the secretion of at least eight proteins, ranging in size from 12.8 to 108 kDa, into the culture medium. C. jejuni ciaB null mutants were deficient in the secretion of all eight proteins, indicating that CiaB is required for the secretion process. The identification of the C. jejuni ciaB gene represents a significant advance in understanding the molecular mechanism of C. jejuni internalization and the pathogenesis of C. jejuni-mediated enteritis.


Assuntos
Antígenos de Bactérias/genética , Campylobacter jejuni/genética , Genes Bacterianos , Sequência de Aminoácidos , Antígenos de Bactérias/química , Campylobacter jejuni/patogenicidade , Linhagem Celular , Clonagem Molecular , Técnicas de Cocultura , Meios de Cultivo Condicionados , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , Mutação , Plasmídeos , Proteínas Recombinantes/genética , Alinhamento de Sequência , Transdução de Sinais
4.
J Clin Microbiol ; 37(3): 510-7, 1999 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-9986804

RESUMO

Campylobacter jejuni and Campylobacter coli are common causes of gastroenteritis in humans. Infection with C. jejuni or C. coli is commonly acquired by eating undercooked chicken. The goal of this study was to develop specific detection assays for C. jejuni and C. coli isolates based on the cadF virulence gene and its product. The cadF gene from C. jejuni and C. coli encodes a 37-kDa outer membrane protein that promotes the binding of these pathogens to intestinal epithelial cells. A fragment of approximately 400 bp was amplified from 38 of 40 (95%) C. jejuni isolates and 5 of 6 (83.3%) C. coli isolates with primers designed to amplify an internal fragment of the cadF gene. PCR was then used to amplify Campylobacter DNA from store-bought chickens. A 400-bp band was amplified from 26 of the 27 chicken carcasses tested by the PCR-based assay. The CadF protein was detected in every C. jejuni and C. coli isolate tested, as judged by immunoblot analysis with a rabbit anti-C. jejuni 37-kDa serum. In addition, methanol-fixed samples of whole-cell C. jejuni and C. coli were detected with the rabbit anti-37-kDa serum by using an indirect-immunofluorescence microscopy assay. These findings indicate that the cadF gene and its product are conserved among C. jejuni and C. coli isolates and that a PCR assay based on the cadF gene may be useful for the detection of Campylobacter organisms in food products.


Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Campylobacter coli/patogenicidade , Campylobacter jejuni/patogenicidade , Proteínas de Transporte/genética , Carne/microbiologia , Animais , Sequência de Bases , Infecções por Campylobacter/etiologia , Infecções por Campylobacter/microbiologia , Campylobacter coli/classificação , Campylobacter coli/genética , Campylobacter jejuni/classificação , Campylobacter jejuni/genética , Galinhas/microbiologia , Gastroenterite/etiologia , Gastroenterite/microbiologia , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Polimorfismo Genético , Coelhos , Sensibilidade e Especificidade , Alinhamento de Sequência , Homologia de Sequência do Ácido Nucleico , Sorotipagem , Virulência/genética
5.
Adv Exp Med Biol ; 473: 215-24, 1999.
Artigo em Inglês | MEDLINE | ID: mdl-10659361

RESUMO

Clinical and in vitro experimental data suggest that invasion of intestinal epithelial cells is an essential step in the pathogenesis of Campylobacter jejuni-mediated enteritis. However, the molecular mechanism of C. jejuni internalization remains poorly defined. The goal of this study was to identify a gene that encodes a protein required for the internalization of C. jejuni into host cells. A C. jejuni gene, designated ciaB, was identified upon immunoscreening C. jejuni genomic DNA-phage libraries with an antiserum generated against C. jejuni co-cultivated with INT 407 cells. The C. jejuni ciaB gene encodes a protein of 610 amino acids with a calculated molecular mass of 73,154 Da. The deduced amino acid sequence of the CiaB protein shares similarity with type III secreted proteins, associated with invasion of host cells, from other more extensively characterized bacterial pathogens. In vitro binding and internalization assays revealed that the binding of C. jejuni ciaB null mutants was indistinguishable from that of the parental isolate, whereas a significant reduction was noted in internalization. Immunoblot analysis using an anti-CiaB specific antibody revealed that CiaB is secreted into the supernatant fluids upon co-cultivation of C. jejuni with INT 407 cell conditioned medium. Metabolic labeling experiments revealed that at least eight C. jejuni proteins, ranging in size from 12.8 to 108 kDa, are secreted into the culture medium. C. jejuni ciaB null mutants were deficient in the secretion of all proteins, indicating that CiaB is required for the secretion process. Identification of the C. jejuni ciaB gene represents a significant advance in understanding the molecular mechanism of C. jejuni internalization.


Assuntos
Antígenos de Bactérias/fisiologia , Proteínas de Bactérias/fisiologia , Campylobacter jejuni/imunologia , Fagocitose , Animais , Antígenos de Bactérias/genética , Proteínas de Bactérias/genética , Células Cultivadas , Clonagem Molecular , Coelhos
6.
Mol Microbiol ; 24(5): 953-63, 1997 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-9220003

RESUMO

Campylobacter jejuni, a Gram-negative bacterium, is a common cause of gastrointestinal disease. By analogy with other enteric pathogens such as Salmonella and Shigella, the ability of C. jejuni to bind to host cells is thought to be essential in the pathogenesis of enteritis. Scanning electron microscopy of infected INT407 cells suggested that C. jejuni bound to a component of the extracellular matrix. Binding assays using immobilized extracellular matrix proteins and soluble fibronectin showed specific and saturable binding of fibronectin to C. jejuni. Ligand immunoblot assays using 125I-labelled fibronectin revealed specific binding to an outer membrane protein with an apparent molecular mass of 37 kDa. A rabbit antiserum, raised against the gel-purified protein, reacted with a 37 kDa protein in all C. jejuni isolates (n = 15) as tested by immunoblot analysis. Antibodies present in convalescent serum from C. jejuni-infected individuals also recognized a 37 kDa protein. The gene encoding the immunoreactive 37kDa protein was cloned and sequenced. Sequencing of overlapping DNA fragments revealed an open reading frame (ORF) that encodes a protein of 326 amino acids with a calculated molecular mass of 36872Da. The deduced amino acid sequence of the ORF exhibited 52% similarity and 28% identity to the root adhesin protein from Pseudomonas fluorescens. Isogenic C. jejuni mutants which lack the 37 kDa outer membrane protein, which we have termed CadF, displayed significantly reduced binding to fibronectin. Biotinylated fibronectin bound to a protein with an apparent molecular mass of 37 kDa in the outer membrane protein extracts from wild-type C. jejuni as judged by ligand-binding blots. These results indicate that the binding of C. jejuni to fibronectin is mediated by the 37 kDa outer membrane protein which is conserved among C. jejuni isolates.


Assuntos
Adesinas Bacterianas , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Campylobacter jejuni/genética , Proteínas de Transporte/genética , Sequência de Aminoácidos , Animais , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/metabolismo , Sequência de Bases , Campylobacter jejuni/metabolismo , Proteínas de Transporte/metabolismo , Linhagem Celular , Clonagem Molecular , DNA Bacteriano , Matriz Extracelular/metabolismo , Genes Bacterianos , Humanos , Dados de Sequência Molecular , Mutagênese Insercional , Fases de Leitura Aberta , Coelhos , Homologia de Sequência de Aminoácidos
7.
Gene ; 185(1): 63-7, 1997 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-9034314

RESUMO

The final step of L-cysteine biosynthesis in Escherichia coli and Salmonella typhimurium consists of the formation of L-cysteine from O-acetylserine and sulfide. This reaction can be catalyzed by two enzymes, O-acetylserine sulfhydrylase A and O-acetylserine sulfhydrylase B, the former of which has been more rigorously characterized. In contrast to O-acetylserine sulfhydrylase A, O-acetylserine sulfhydrylase B is preferentially used for cysteine biosynthesis during anaerobic growth and is able to utilize thiosulfate as a substrate. Campylobacter jejuni is a micro-aerophilic, Gram-negative bacterium, and a member of the epsilon subdivision of eubacteria. We have cloned, sequenced, and expressed a gene from C. jejuni that encodes a protein of 299 aa with a calculated molecular mass of 32,367 Da. Complementation analysis of an E. coli cysteine auxotroph with the pMEK34-14 recombinant plasmid containing a 1.2-kb insert of chromosomal DNA from C. jejuni revealed that transformants were capable of growth in medium containing either sulfide or thiosulfate as sole sulfur sources. These data indicate that the cloned C. jejuni gene is a functional homolog of the cysM gene that codes for O-acetylserine sulfhydrylase B in E. coli and S. typhimurium.


Assuntos
Campylobacter jejuni/enzimologia , Campylobacter jejuni/genética , Cisteína Sintase/genética , Sequência de Aminoácidos , Sequência de Bases , Southern Blotting , Clonagem Molecular , Cisteína/metabolismo , DNA Bacteriano/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Teste de Complementação Genética , Dados de Sequência Molecular , Mutagênese Insercional , Plasmídeos , Recombinação Genética , Salmonella typhimurium/genética , Sulfetos/metabolismo , Tiossulfatos/metabolismo
9.
Infect Immun ; 64(9): 3537-43, 1996 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-8751896

RESUMO

Campylobacter jejuni, a gram-negative, microaerophilic, spiral bacterium, is a common cause of human gastrointestinal disease. Although investigators commonly use C. jejuni glycine-hydrochloride extracts in assays to determine the products that promote the binding of the organism to eukaryotic cells, the proteins contained within these extracts remain ill defined. Characterization of these proteins will provide a better understanding of C. jejuni gene regulation and organization. An antiserum was raised against a C. jejuni 29-kDa gel-purified protein detected in glycine-hydrochloride extracts. This antiserum was used to screen an expression library of C. jejuni. A reactive clone that contained an open reading frame of 256 amino acids was identified. The cloned gene was transcribed and translated, and the product was exported to the periplasmic space in Escherichia coli XL1-Blue. The translated C. jejuni product, designated P29, exhibited significant similarity to the histidine and lysine-arginine-ornithine periplasmic binding proteins (HisJ and LAO, respectively) of Salmonella typhimurium. The C. jejuni gene encoding the P29 protein complemented an S. typhimurium HisJ mutant but not a LAO mutant when provided in trans. These data suggest that the C. jejuni gene encoding the P29 protein is a homolog of the S. typhimurium hisJ gene.


Assuntos
Proteínas de Bactérias/genética , Campylobacter jejuni/genética , Proteínas de Transporte/genética , Genes Bacterianos , Proteínas Periplásmicas de Ligação , Sequência de Aminoácidos , Aderência Bacteriana , Sequência de Bases , Campylobacter jejuni/patogenicidade , Proteínas de Transporte/química , Primers do DNA/química , Dados de Sequência Molecular , Peso Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos
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