Ultrastructural and glycoproteomic characterization of Prevotella intermedia: Insights into O-glycosylation and outer membrane vesicles

IF 3.9 3区 生物学 Q2 MICROBIOLOGY MicrobiologyOpen Pub Date : 2024-02-26 DOI:10.1002/mbo3.1401
Xi Ye, Bindusmita Paul, Joyce Mo, Eric C. Reynolds, Debnath Ghosal, Paul D. Veith
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Abstract

Prevotella intermedia, a Gram-negative bacterium from the Bacteroidota phylum, is associated with periodontitis. Other species within this phylum are known to possess the general O-glycosylation system. The O-glycoproteome has been characterized in several species, including Tannerella forsythia, Porphyromonas gingivalis, and Flavobacterium johnsoniae. In our study, we used electron cryotomography (cryoET) and glycoproteomics to reveal the ultrastructure of P. intermedia and characterize its O-glycoproteome. Our cryoET analysis unveiled the ultrastructural details of the cell envelope and outer membrane vesicles (OMVs) of P. intermedia. We observed an electron-dense surface layer surrounding both cells and OMVs. The OMVs were often large (>200 nm) and presented two types, with lumens being either electron-dense or translucent. LC-MS/MS analyses of P. intermedia fractions led to the identification of 1655 proteins, which included 62 predicted T9SS cargo proteins. Within the glycoproteome, we identified 443 unique O-glycosylation sites within 224 glycoproteins. Interestingly, the O-glycosylation motif exhibited a broader range than reported in other species, with O-glycosylation found at D(S/T)(A/I/L/M/T/V/S/C/G/F/N/E/Q/D/P). We identified a single O-glycan with a delta mass of 1531.48 Da. Its sequence was determined by MS2 and MS3 analyses using both collision-induced dissociation and high-energy collisional dissociation fragmentation modes. After partial deglycosylation with trifluoromethanesulfonic acid, the O-glycan sequence was confirmed to be dHex-dHex-HexNAc (HPO3-C6H12O5)-dHex-Hex-HexA-Hex(dHex). Bioinformatic analyses predicted the localization of O-glycoproteins, with 73 periplasmic proteins, 53 inner membrane proteins, 52 lipoproteins, 26 outer membrane proteins, and 14 proteins secreted by the T9SS.

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中间普雷沃茨菌的超微结构和糖蛋白组学特征:对 O-糖基化和外膜囊泡的深入了解。
中间普雷沃特氏菌(Prevotella intermedia)是类杆菌科的一种革兰氏阴性细菌,与牙周炎有关。已知该菌门内的其他菌种也拥有一般的 O-糖基化系统。O-糖蛋白组已在多个菌种中得到表征,包括连翘坦奈氏菌(Tannerella forsythia)、牙龈卟啉单胞菌(Porphyromonas gingivalis)和约翰逊黄杆菌(Flavobacterium johnsoniae)。在我们的研究中,我们利用电子低温显像(cryoET)和糖蛋白组学揭示了中间卟啉单胞菌的超微结构,并描述了其 O 型糖蛋白组的特征。我们的低温电子显微镜分析揭示了介壳虫细胞膜和外膜囊泡的超微结构细节。我们观察到围绕细胞和外膜囊泡的电子致密表层。外膜囊泡通常很大(大于 200 nm),有两种类型,内腔要么是电子致密的,要么是半透明的。通过对中间体部分进行 LC-MS/MS 分析,鉴定出 1655 种蛋白质,其中包括 62 种预测的 T9SS 货物蛋白。在糖蛋白组中,我们在 224 个糖蛋白中发现了 443 个独特的 O-糖基化位点。有趣的是,与其他物种相比,O-糖基化位点的范围更广,在D(S/T)(A/I/L/M/T/V/S/C/G/F/N/E/Q/D/P)处发现了O-糖基化位点。我们发现了一个δ质量为 1531.48 Da 的单一 O-糖基。利用碰撞诱导解离和高能碰撞解离碎片模式,通过 MS2 和 MS3 分析确定了其序列。经三氟甲磺酸部分脱糖基化后,确认 O-糖序列为 dHex-dHex-HexNAc (HPO3 -C6 H12 O5 )-dHex-Hex-HexA-Hex(dHex) 。生物信息学分析预测了 O-糖蛋白的定位,T9SS 可分泌 73 种周质膜蛋白、53 种内膜蛋白、52 种脂蛋白、26 种外膜蛋白和 14 种蛋白质。
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来源期刊
MicrobiologyOpen
MicrobiologyOpen MICROBIOLOGY-
CiteScore
8.00
自引率
0.00%
发文量
78
审稿时长
20 weeks
期刊介绍: MicrobiologyOpen is a peer reviewed, fully open access, broad-scope, and interdisciplinary journal delivering rapid decisions and fast publication of microbial science, a field which is undergoing a profound and exciting evolution in this post-genomic era. The journal aims to serve the research community by providing a vehicle for authors wishing to publish quality research in both fundamental and applied microbiology. Our goal is to publish articles that stimulate discussion and debate, as well as add to our knowledge base and further the understanding of microbial interactions and microbial processes. MicrobiologyOpen gives prompt and equal consideration to articles reporting theoretical, experimental, applied, and descriptive work in all aspects of bacteriology, virology, mycology and protistology, including, but not limited to: - agriculture - antimicrobial resistance - astrobiology - biochemistry - biotechnology - cell and molecular biology - clinical microbiology - computational, systems, and synthetic microbiology - environmental science - evolutionary biology, ecology, and systematics - food science and technology - genetics and genomics - geobiology and earth science - host-microbe interactions - infectious diseases - natural products discovery - pharmaceutical and medicinal chemistry - physiology - plant pathology - veterinary microbiology We will consider submissions across unicellular and cell-cluster organisms: prokaryotes (bacteria, archaea) and eukaryotes (fungi, protists, microalgae, lichens), as well as viruses and prions infecting or interacting with microorganisms, plants and animals, including genetic, biochemical, biophysical, bioinformatic and structural analyses. The journal features Original Articles (including full Research articles, Method articles, and Short Communications), Commentaries, Reviews, and Editorials. Original papers must report well-conducted research with conclusions supported by the data presented in the article. We also support confirmatory research and aim to work with authors to meet reviewer expectations. MicrobiologyOpen publishes articles submitted directly to the journal and those referred from other Wiley journals.
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