RESUMO
Yersinia pestis is a Gram-negative bacterium that causes plague. Currently, plague is considered a re-emerging infectious disease and Y. pestis a potential bioterrorism agent. Autotransporters (ATs) are virulence proteins translocated by a variety of pathogenic Gram-negative bacteria across the cell envelope to the cell surface or extracellular environment. In this study, we screened the genome of Yersinia pestis KIM for AT genes whose expression might be relevant for the pathogenicity of this plague-causing organism. By in silico analyses, we identified ten putative AT genes in the genomic sequence of Y. pestis KIM; two of these genes are located within known pathogenicity islands. The expression of all ten putative AT genes in Y. pestis KIM was confirmed by RT-PCR. Five genes, designated yapA, yapC, yapG, yapK and yapN, were subsequently cloned and expressed in Escherichia coli K12 for protein secretion studies. Two forms of the YapA protein (130 kDa and 115 kDa) were found secreted into the culture medium. Protease cleavage at the C terminus of YapA released the protein from the cell surface. Outer membrane localization of YapC (65 kDa), YapG (100 kDa), YapK (130 kDa), and YapN (60 kDa) was established by cell fractionation, and cell surface localization of YapC and YapN was demonstrated by protease accessibility experiments. In functional studies, YapN and YapK showed hemagglutination activity and YapC exhibited autoagglutination activity. Data reported here represent the first study on Y. pestis ATs.
Assuntos
Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Fatores de Virulência/metabolismo , Yersinia pestis/genética , Yersinia pestis/patogenicidade , Aderência Bacteriana , Proteínas de Bactérias/química , Clonagem Molecular , Biologia Computacional , Escherichia coli , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos/genética , Hemaglutinação , Filogenia , Porinas/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Homologia de Sequência de Aminoácidos , Fatores de Virulência/química , Fatores de Virulência/genética , Yersinia pestis/classificação , Yersinia pestis/metabolismoRESUMO
Bacteria secrete a wide variety of proteins, many of which play important roles in virulence. In gram-negative bacteria, these proteins must cross the cytoplasmic or inner membrane, periplasm, and outer membrane to reach the cell surface. Gram-negative bacteria have evolved multiple pathways to allow protein secretion across their complex envelope. ATP is not available in the periplasm and many of these secretion pathways encode components that harness energy available at the inner membrane to drive secretion across the outer membrane. In contrast, the autotransporter, two-partner secretion and chaperone/usher pathways are comparatively simple systems that allow secretion across the outer membrane without the need for input of energy from the inner membrane. This review will present overviews of these 'self-sufficient' pathways, focusing on recent advances and secretion mechanisms. Similarities among the pathways and with other protein translocation mechanisms will be highlighted.
