Proteogenomic annotation of T6SS components identified in Bacteroides fragilis secretome

Bacteroides fragilis (Bf)'s T6SS locus has been characterized and shown to have functional activity in competition experiments. It has been demonstrated that symbiont nontoxigenic Bf strains have a more effective “weapon” effect on pathogenic Bf, which is realized through the activity of effector-immune (E-I) protein pairs. Intensive study of the T6SS structure has led to an understanding of certain issues related to its functional activity, but the exact regulatory mechanisms of E-I protein pair activity remain unclear. Proteomic annotation of T6SS components and detailed descriptions of all immune-effector pairs are currently available. In this research, we performed detailed proteogenomic analysis and subsequent proteomic annotation of the T6SS components of the toxigenic Bf BOB25. Fractionated cells, cultivated media and vesicles were prepared for proteome analysis by HPLC-MS/MS. Proteogenomic annotation and comparative genomic study of the T6SS loci of the toxigenic Bf BOB25 were carried out by comparison with the reference genomes of the following Bf strains: JIM10, NCTC 9343 and 638R. According to the data obtained, T6SS components were represented in all types of the analysed samples. The following components of the T6SS were identified in culture media and cells: ClpV (TssH), TssK, TssC, TssB, Hcp (TssD), and TetR. The predicted effector protein AKA51715.1 (VU15_08315) was also detected in media. The greatest amount of T6SS proteins, including the Hcp protein, was detected in the vesicle samples, which was also observed by TEM. Potential effectors, including AKA51715.1 (VU15_08315), AKA51716.1 (VU15_08320), AKA51728.1 (VU15_08385) and the immune protein AKA51727.1 (VU15_08380), were detected in vesicles. The presence of the immune and effector proteins in the Bf secretome indicates the high activity of the T6SS without bacterial competition. It is possible that the T6SS is also used by bacteria to regulate population size by altering the activity of different repertoires of E-I pairs.

脆弱拟杆菌（Bacteroides fragilis, 下称Bf）的VI型分泌系统（Type VI Secretion System, T6SS）基因座已得到系统表征，并在竞争实验中被证实具备功能活性。已有研究证实，共生性非产毒Bf菌株对致病性Bf具有更为高效的"武器"效应，该效应通过效应蛋白-免疫蛋白（effector-immune, E-I）对的活性得以实现。对T6SS结构的深入研究已使学界对其功能活性相关的若干问题有所认知，但E-I蛋白对活性的确切调控机制仍未阐明。目前已有关于T6SS组分的蛋白质组注释以及所有免疫-效应蛋白对的详细研究成果公开。本研究对产毒Bf菌株BOB25的T6SS组分开展了详尽的蛋白质基因组学分析，并完成了后续的蛋白质组注释。我们通过高效液相色谱-串联质谱（HPLC-MS/MS）制备了分级分离的菌体、培养上清及囊泡，用于蛋白质组学分析。本研究通过与下述Bf菌株的参考基因组（JIM10、NCTC 9343及638R）进行比对，完成了产毒Bf菌株BOB25的T6SS基因座的蛋白质基因组注释与比较基因组学分析。根据所得实验数据，所有分析样本中均检测到T6SS组分的存在。在培养上清与菌体中鉴定出以下T6SS组分：ClpV（TssH）、TssK、TssC、TssB、Hcp（TssD）以及TetR。在培养上清中还检测到了预测的效应蛋白AKA51715.1（VU15_08315）。在囊泡样本中检测到的T6SS蛋白总量最高，其中包括Hcp蛋白，这一结果也通过透射电子显微镜（Transmission Electron Microscopy, TEM）得到了验证。在囊泡中检测到了多种潜在效应蛋白，包括AKA51715.1（VU15_08315）、AKA51716.1（VU15_08320）、AKA51728.1（VU15_08385），以及免疫蛋白AKA51727.1（VU15_08380）。Bf分泌组中免疫蛋白与效应蛋白的存在表明，即便不存在细菌竞争，T6SS也具备较高的活性。推测细菌或可通过改变不同E-I对的活性谱，利用T6SS调控种群规模。