Vaccine Program, Human Health Therapeutics Portfolio, National Research Council of Canada, Ottawa, Ontario, Canada.
Vaccine Program, Human Health Therapeutics Portfolio, National Research Council of Canada, Ottawa, Ontario, Canada
J Bacteriol. 2014 Jul;196(14):2627-37. doi: 10.1128/JB.01469-14. Epub 2014 May 9.
In this study, we identify a major spore surface protein, BclA, and provide evidence that this protein is glycosylated. Following extraction of the spore surface, solubilized proteins were separated by one-dimensional PAGE and stained with glycostain to reveal a reactive high-molecular-mass region of approximately 600 kDa. Tandem mass spectrometry analysis of in-gel digests showed this band to contain peptides corresponding to a putative exosporangial glycoprotein (BclA3) and identified a number of glycopeptides modified with multiple N-acetyl hexosamine moieties and, in some cases, capped with novel glycans. In addition, we demonstrate that the glycosyltransferase gene sgtA (gene CD3350 in strain 630 and CDR3194 in strain R20291), which is located immediately upstream of the bclA3 homolog, is involved in the glycosylation of the spore surface, and is cotranscribed with bclA3. The presence of anti-β-O-GlcNAc-reactive material was demonstrated on the surface of spores by immunofluorescence and in surface extracts by Western blotting, although each strain produced a distinct pattern of reactivity. Reactivity of the spore surface with the anti-β-O-GlcNAc antibody was abolished in the 630 and R20291 glycosyltransferase mutant strains, while complementation with a wild-type copy of the gene restored the β-O-GlcNAc reactivity. Phenotypic testing of R20291 glycosyltransferase mutant spores revealed no significant change in sensitivity to ethanol or lysozyme. However, a change in the resistance to heat of R20291 glycosyltransferase mutant spores compared to R20291 spores was observed, as was the ability to adhere to and be internalized by macrophages.
在这项研究中，我们鉴定了一种主要的孢子表面蛋白 BclA，并提供了证据表明该蛋白发生了糖基化。在提取孢子表面后，通过一维 PAGE 分离溶解的蛋白质，并使用糖染色法染色以显示出大约 600 kDa 的反应性高分子质量区域。胶内消化的串联质谱分析表明，该条带包含与假定的外孢子囊糖蛋白（BclA3）相对应的肽，并鉴定了许多用多个 N-乙酰己糖基修饰的糖肽，在某些情况下，还带有新型聚糖。此外，我们证明位于 bclA3 同源物上游的糖基转移酶基因 sgtA（在菌株 630 中为基因 CD3350，在菌株 R20291 中为 CDR3194）参与了孢子表面的糖基化，并且与 bclA3 共转录。通过免疫荧光和表面提取物的 Western blot，证明了孢子表面存在抗-β-O-GlcNAc 反应性物质，尽管每个菌株都表现出不同的反应模式。在 630 和 R20291 糖苷转移酶突变株中，抗-β-O-GlcNAc 抗体与孢子表面的反应性被消除，而通过野生型基因的互补恢复了β-O-GlcNAc 反应性。对 R20291 糖苷转移酶突变体孢子的表型测试表明，对乙醇或溶菌酶的敏感性没有明显变化。然而，与 R20291 孢子相比，观察到 R20291 糖苷转移酶突变体孢子的耐热性发生了变化，并且观察到对巨噬细胞的粘附和内化能力发生了变化。
