RESUMEN
Pili of pathogenic Neisseria are major virulence factors associated with adhesion, twitching motility, auto-aggregation, and DNA transformation. Pili of N. meningitidis are subject to several different post-translational modifications. Among these pilin modifications, the presence of phosphorylcholine (ChoP) and a glycan on the pilin protein are phase-variable (subject to high frequency, reversible on/off switching of expression). In this study we report the location of two ChoP modifications on the C-terminus of N. meningitidis pilin. We show that the surface accessibility of ChoP on pili is affected by phase variable changes to the structure of the pilin-linked glycan. We identify for the first time that the platelet activating factor receptor (PAFr) is a key, early event receptor for meningococcal adherence to human bronchial epithelial cells and tissue, and that synergy between the pilin-linked glycan and ChoP post-translational modifications is required for pili to optimally engage PAFr to mediate adherence to human airway cells.
Asunto(s)
Adhesión Bacteriana , Membrana Celular/metabolismo , Células Epiteliales/metabolismo , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Neisseria meningitidis/metabolismo , Procesamiento Proteico-Postraduccional , Mucosa Respiratoria/metabolismo , Linaje de la Célula , Membrana Celular/microbiología , Células Epiteliales/microbiología , Proteínas Fimbrias/genética , Fimbrias Bacterianas/genética , Humanos , Neisseria meningitidis/genética , Neisseria meningitidis/patogenicidad , Fosforilcolina/metabolismo , Mucosa Respiratoria/microbiologíaRESUMEN
Pilin is the major subunit of the essential virulence factor pili and is glycosylated at Ser63. In this study we investigated the gene pglI to determine whether it is involved in the biosynthesis of the pilin-linked glycan of Neisseria meningitidis strain C311#3. A N. meningitidis C311#3pglI mutant resulted in a change of apparent molecular weight in SDS-PAGE and altered binding of antisera, consistent with a role in the biosynthesis of the pilin-linked glycan. These data, in conjunction with homology with well-characterised acyltransferases suggests a specific role for pglI in the biosynthesis of the basal 2,4-diacetamido-2,4,6-trideoxyhexose residue of the pilin-linked glycan.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Fimbrias/metabolismo , Fimbrias Bacterianas/metabolismo , Neisseria meningitidis/metabolismo , Procesamiento Proteico-Postraduccional , Acetiltransferasas/genética , Proteínas Bacterianas/genética , Clonación Molecular , ADN Bacteriano/genética , Genes Bacterianos , Glicosilación , Humanos , Sueros Inmunes , Lipopolisacáridos/biosíntesis , Datos de Secuencia Molecular , Mutación , Neisseria meningitidis/genética , Neisseria meningitidis/patogenicidad , Fenotipo , Homología de Secuencia de Aminoácido , Trisacáridos/inmunología , VirulenciaRESUMEN
Pili of pathogenic Neisseria are major virulence factors associated with adhesion, cytotoxicity, twitching motility, autoaggregation, and DNA transformation. Pili are modified posttranslationally by the addition of phosphorylcholine. However, no genes involved in either the biosynthesis or the transfer of phosphorylcholine in Neisseria meningitidis have been identified. In this study, we identified five candidate open reading frames (ORFs) potentially involved in the biosynthesis or transfer of phosphorylcholine to pilin in N. meningitidis. Insertional mutants were constructed for each ORF in N. meningitidis strain C311#3 to determine their effect on phosphorylcholine expression. The effect of the mutant ORFs on the modification by phosphorylcholine was analyzed by Western analysis with phosphorylcholine-specific monoclonal antibody TEPC-15. Analysis of the mutants showed that ORF NMB0415, now defined as pptA (pilin phosphorylcholine transferase A), is involved in the addition of phosphorylcholine to pilin in N. meningitidis. Additionally, the phase variation (high frequency on-off switching of expression) of phosphorylcholine on pilin is due to changes in a homopolymeric guanosine tract in pptA.