RESUMO
The opc gene is widespread in epidemic and endemic Neisseria meningitidis, but most strains of certain epidemic clones (ET-37 complex, Cluster A4) and a few random endemic isolates lack an opc gene. Four percent of the 1148 bp that contain opc plus the surrounding intergenic region was polymorphic (18 alleles), and many of the alleles contained a 230 bp insertion at a fixed location in the intergenic region. The presence or absence of the insertion reflects site-specific recombination. The alleles are stably inherited within clonal groupings for up to at least 50 years, with rare cases of horizontal genetic exchange. Most statistical methods indicated significant intragenic recombination events within this dataset.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Genes Bacterianos , Neisseria meningitidis/genética , Polimorfismo Genético , Recombinação Genética , Alelos , Sequência de Bases , Transferência Genética Horizontal , Humanos , Infecções Meningocócicas/microbiologia , Dados de Sequência Molecular , Neisseria meningitidis/classificação , Filogenia , Sinais Direcionadores de Proteínas/genética , Sorotipagem , Especificidade da EspécieRESUMO
Opa proteins of Neisseria meningitidis exhibit translational phase variation via addition or deletion of repetitive coding repeat units within the DNA encoding the protein leader sequence. In contrast, Opc phase variation is the result of transcriptional regulation. Transcription starts 13 nucleotides after the -10 region of an unusual promoter sequence containing a variable number of contiguous cytidine residues and lacking a -35 region. Efficient expression of Opc occurred in strains with 12 to 13 cytidine residues, intermediate expression in strains with 11 or 14 residues and no expression with < or = 10 or > or = 15 residues. This unusual regulation may have evolved because the Opc protein enables meningococcal invasion and is immunogenic.
Assuntos
Proteínas da Membrana Bacteriana Externa/biossíntese , Regulação Bacteriana da Expressão Gênica , Neisseria meningitidis/genética , Regiões Promotoras Genéticas , Proteínas da Membrana Bacteriana Externa/genética , Sequência de Bases , Dados de Sequência Molecular , Neisseria meningitidis/metabolismo , Poli C , Transcrição GênicaRESUMO
Whereas capsulate strains of Neisseria meningitidis are dependent on pili for adhesion to human endothelial and epithelial cells, strains which lacked assembled pili and were partially capsule-deficient adhered to and invaded human endothelial and epithelial cells if they expressed the Opc protein. Bacteria expressing low or undetectable levels of Opc protein failed to adhere to or invade eukaryotic cells. In addition, the presence of OpaAC751 protein on the surface of bacteria did not increase bacterial interactions with host cells. Association of Opc-expressing bacteria was inhibited by antibodies against Opc. Invasion was dependent on the host-cell cytoskeletal activity and was inhibited by cytochalasin D. In some cells, infected at the apical surface, bacteria emerging from basal surface were detected by electron microscopy. Opc is found in diverse meningococci and may represent a common virulence factor which facilitates adherence and invasion by these bacteria.
Assuntos
Aderência Bacteriana , Proteínas da Membrana Bacteriana Externa/metabolismo , Endotélio Vascular/microbiologia , Epitélio/microbiologia , Neisseria meningitidis/patogenicidade , Animais , Cápsulas Bacterianas/fisiologia , Proteínas da Membrana Bacteriana Externa/biossíntese , Linhagem Celular , Citocalasina D/farmacologia , Proteínas de Fímbrias , Humanos , Microscopia Eletrônica , Microscopia Eletrônica de Varredura , Células Tumorais Cultivadas , VirulênciaRESUMO
A genomic library was constructed in a lambda gt11 vector using chromosomal DNA from a meningococcal serogroup A strain and plaques expressing the class 5C protein were recognized by screening with specific monoclonal antibodies. The opc insert was subcloned into a multicopy plasmid which induced expression of that protein in Escherichia coli as a surface-exposed major outer membrane protein. The nucleotide sequence of opc is typical of an outer membrane protein with a promoter and terminator region, a leader peptide which is cleaved during expression and a complete open reading frame. Unlike other meningococcal class 5 proteins or gonococcal P.II proteins, the sequence did not contain any pentanucleotide repeats and the sequence showed little homology to these other functionally related proteins. However, the predicted amino acid sequence of the mature protein for opc showed 27% similarity to that for a second opa gene cloned from the same meningococcal strain. This is the first report of cloning and expression of a functional meningococcal gene encoding a class 5 outer membrane protein in E. coli.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Neisseria meningitidis/genética , Sequência de Aminoácidos , Anticorpos Monoclonais , Antígenos de Bactérias/química , Antígenos de Bactérias/genética , Antígenos de Superfície/química , Antígenos de Superfície/genética , Sequência de Bases , Southern Blotting , Western Blotting , Clonagem Molecular , DNA Bacteriano/genética , Eletroforese em Gel de Poliacrilamida , Expressão Gênica , Genes Bacterianos , Dados de Sequência Molecular , Neisseria meningitidis/química , Neisseria meningitidis/imunologia , Biossíntese de Proteínas , Sequências Repetitivas de Ácido NucleicoRESUMO
Extrachromosomal circular DNA molecules consisting of IS1-cat repeats, (IS1-cat)n, were isolated from an E. coli strain harboring nearly 30 copies of tandemly amplified transposon Tn9 located on the chromosome. The DNA 'circles' were characterized by restriction analysis followed by Southern blotting and electron microscopic examination. Their size varied from approximately 5.5 kb to 53 kb.