Power P M, Roddam L F, Rutter K, Fitzpatrick S Z, Srikhanta Y N, Jennings M P
Department of Microbiology and Parasitology, The University of Queensland, Brisbane, Queensland, Australia.
Mol Microbiol. 2003 Aug;49(3):833-47. doi: 10.1046/j.1365-2958.2003.03602.x.
Pili of Neisseria meningitidis are a key virulence factor, being the major adhesin of this capsulate organism and contributing to specificity for the human host. Pili are post-translationally modified by addition of either an O-linked trisaccharide, Gal (beta1-4) Gal (alpha1-3) 2,4-diacetamido-2,4,6-trideoxyhexose or an O-linked disaccharide Gal (alpha1,3) GlcNAc. The role of these structures in meningococcal pathogenesis has not been resolved. In previous studies we identified two separate genetic loci, pglA and pglBCD, involved in pilin glycosylation. Putative functions have been allocated to these genes; however, there are not enough genes to account for the complete biosynthesis of the described structures, suggesting additional genes remain to be identified. In addition, it is not known why some strains express the trisaccharide structure and some the disaccharide structure. In order to find additional genes involved in the biosynthesis of these structures, we used the recently published group A strain Z2491 and group B strain MC58 Neisseria meningitidis genomes and the unfinished Neisseria meningitidis group C strain FAM18 and Neisseria gonorrhoeae strain FA1090 genomes to identify novel genes involved in pilin glycosylation, based on homology to known oligosaccharide biosynthetic genes. We identified a new gene involved in pilin glycosylation designated pglE and examined four additional genes pglB/B2, pglF, pglG and pglH. A strain survey revealed that pglE and pglF were present in each strain examined. The pglG, pglH and pglB2 polymorphisms were not found in strain C311 musical sharp 3 but were present in a large number of clinical isolates. Insertional mutations were constructed in pglE and pglF in N. meningitidis strain C311 musical sharp 3, a strain with well-defined lipopolysaccharide (LPS) and pilin-linked glycan structures. Increased gel migration of the pilin subunit molecules of pglE and pglF mutants was observed by Western analysis, indicating truncation of the trisaccharide structure. Antisera specific for the C311 musical sharp 3 trisaccharide failed to react with pilin from these pglE and pglF mutants. GC-MS analysis of the sugar composition of the pglE mutant showed a reduction in galactose compared with C311 musical sharp 3 wild type. Analysis of amino acid sequence homologies has suggested specific roles for pglE and pglF in the biosynthesis of the trisaccharide structure. Further, we present evidence that pglE, which contains heptanucleotide repeats, is responsible for the phase variation between trisaccharide and disaccharide structures in strain C311 musical sharp 3 and other strains. We also present evidence that pglG, pglH and pglB2 are potentially phase variable.
脑膜炎奈瑟菌的菌毛是一种关键的毒力因子，是这种有荚膜生物体的主要黏附素，有助于其对人类宿主的特异性识别。菌毛在翻译后会通过添加O-连接的三糖（Gal (β1-4) Gal (α1-3) 2,4-二乙酰氨基-2,4,6-三脱氧己糖）或O-连接的二糖（Gal (α1,3) GlcNAc）进行修饰。这些结构在脑膜炎球菌致病机制中的作用尚未明确。在之前的研究中，我们鉴定出了两个独立的基因位点，pglA和pglBCD，它们参与菌毛蛋白的糖基化过程。已为这些基因赋予了假定的功能；然而，用于解释所描述结构完整生物合成的基因数量不足，这表明仍有待鉴定其他基因。此外，尚不清楚为何有些菌株表达三糖结构，而有些菌株表达二糖结构。为了找到参与这些结构生物合成的其他基因，我们利用最近公布的A群菌株Z2491和B群菌株MC58脑膜炎奈瑟菌基因组，以及未完成测序的C群菌株FAM18脑膜炎奈瑟菌和淋病奈瑟菌菌株FA1090基因组，基于与已知寡糖生物合成基因的同源性来鉴定参与菌毛蛋白糖基化的新基因。我们鉴定出了一个参与菌毛蛋白糖基化的新基因，命名为pglE，并研究了另外四个基因pglB/B2、pglF、pglG和pglH。菌株调查显示，pglE和pglF存在于所检测的每个菌株中。在菌株C311 #3中未发现pglG、pglH和pglB2的多态性，但在大量临床分离株中存在。在脑膜炎奈瑟菌菌株C311 #3（该菌株具有明确的脂多糖（LPS）和与菌毛相连的聚糖结构）中构建了pglE和pglF的插入突变体。通过蛋白质免疫印迹分析观察到pglE和pglF突变体的菌毛亚基分子的凝胶迁移增加，表明三糖结构被截短。针对C311 #3三糖的抗血清未能与这些pglE和pglF突变体的菌毛蛋白发生反应。与C311 #3野生型相比，pglE突变体糖组成的气相色谱 - 质谱分析显示半乳糖减少。氨基酸序列同源性分析表明pglE和pglF在三糖结构生物合成中具有特定作用。此外，我们提供证据表明，含有七核苷酸重复序列的pglE负责菌株C311 #3和其他菌株中三糖和二糖结构之间的相变。我们还提供证据表明pglG、pglH和pglB2可能是相变可变的。
