The role of outer membrane proteins in bacterial conjugation

外膜蛋白在细菌接合中的作用

• 批准号: 2283913
• 金额: --
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2019
• 资助国家: 英国
• 起止时间: 2019 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2283913
• 关键词: role; outer; membrane; proteins; bacterial

Background The emergence of carbapenem-resistant Klebsiella pneumoniae (CRKP) has left very few therapeutic treatments available, leading to an antibiotic resistance crisis. Carbapenems are a last-line B-lactam treatment utilized for suspected CRKP infections1. CRKP acquire resistance through harbouring carbapenemases; B-lactam hydrolysing enzymes1. CRKP carry carbapenemase-encoding conjugative plasmids, such as the pKpQIL-family plasmids2,3. These are commonly associated with the K. pneumoniae sequence type 258 (ST258) which is renowned for causing hospital outbreaks globally2,3. Conjugative plasmids are transferred between populations through a type IV secretion system (T4SS) via bacterial conjugation; the exchange of genetic material between a donor and a recipient bacteria4,5,6,7. T4SSs are large protein complexes spanning the cell envelope that mediate DNA or protein transfer through an extracellular filament known as the pilus8,9, as illustrated in Figure 110. The transfer (tra) operon encodes the Tra proteins that collectively form the T4SS in the conjugative F-plasmid in E. coli 8,9. The T4SS outer membrane (OM) proteins TraN and TraT form cell surface interactions with the recipient cell prior to DNA translocation11,12,13. TraN is involved in mating pair stabilisation alongside TraG and potentially TraU, to stabilise the translocation channel14,15,16. The lipoprotein TraT provides immunity against the bactericidal action of serum and prevents the donor cell from simultaneously acting as a recipient cell; surface exclusion17,18. There is evidence that F-plasmid TraN and TraT variants cooperate with the OM porin OmpA on the recipient cell prior to plasmid transfer11,13,19,20. Recent research into the pKpQIL plasmid T4SS suggests that pKpQIL TraN cooperates with the K. pneumoniae OM porin OmpK36 on the recipient cell prior to plasmid transfer, although direct interactions have not been confirmed21. Therefore, conjugal OM proteins exhibit plasmid-specific cell surface interactions, making them novel drug target candidates. Figure 1 - F-like Type IV secretion system. Based on cryoelectron microscopy and cryoelectron tomography models. Tra proteins (TraA- TraX) are labelled with capital letters. (Figure 1 is taken from Bragagnolo et al., 2020)10 Aim The proposed research project aims to identify and characterise the cell surface interactions of the conjugal OM proteins TraN and TraT during pKpQIL conjugation. Biochemical characterisation Firstly, we will optimise expression and purification conditions of TraN and TraT in E. coli, and then proceed onto identifying their respective cell surface binding partners using pull-down assays (co-immunoprecipitation). We will confirm whether TraN, predicted to resemble a B-barrel protein13, is involved in DNA transfer. We plan to reconstitute liposomes with TraN and a fluorescent sensitive probe - DNA. We will perform functional assays that will measure DNA fluorescence or quenching as DNA crosses over to other liposomes via the proposed TraN binding partner, OmpK36. Biophysical characterisation pKpQIL TraN and TraT structures will be resolved by X-ray crystallography, where data collection will take place at Diamond Light Source. Alternatively, we will take advantage of cryoelectron microscopy, where resolution overlap between the two data sets will enable accurate structure determination. Crystallisation of TraN and TraT with their respective binding partners will also be attempted. Residues critical for binding will then be unravelled by peptide mutagenesis, followed by ligand binding studies by NMR. This research project will take a multidisciplinary approach through collaboration with the Frankel Lab at Imperial College London, to undertake essential molecular biology experiments.

背景 耐碳青霉烯类肺炎克雷伯菌（CRKP）的出现导致可用的治疗方法非常少，导致抗生素耐药性危机。碳青霉烯类是用于疑似 CRKP 感染的最后一线 B-内酰胺治疗1。 CRKP通过携带碳青霉烯酶获得耐药性； B-内酰胺水解酶1。 CRKP 携带编码碳青霉烯酶的接合质粒，例如 pKpQIL 家族质粒2,3。这些通常与肺炎克雷伯菌序列类型 258 (ST258) 有关，该序列因在全球范围内引起医院爆发而闻名2,3。接合质粒通过 IV 型分泌系统 (T4SS) 通过细菌接合在群体之间转移；供体和受体细菌之间遗传物质的交换4,5,6,7。 T4SS 是跨越细胞被膜的大型蛋白质复合物，通过称为菌毛 8,9 的胞外丝介导 DNA 或蛋白质转移，如图 110 所示。转移 (tra) 操纵子编码 Tra 蛋白，这些蛋白在大肠杆菌 8,9 的接合 F 质粒中共同形成 T4SS。 T4SS 外膜 (OM) 蛋白 TraN 和 TraT 在 DNA 易位之前与受体细胞形成细胞表面相互作用11,12,13。 TraN 与 TraG 和可能的 TraU 一起参与交配稳定，以稳定易位通道14,15,16。脂蛋白TraT提供针对血清杀菌作用的免疫力，并防止供体细胞同时充当受体细胞；表面排除17,18。有证据表明，F-质粒 TraN 和 TraT 变体在质粒转移之前与受体细胞上的 OM 孔蛋白 OmpA 协同作用11,13,19,20。最近对 pKpQIL 质粒 T4SS 的研究表明，pKpQIL TraN 在质粒转移之前与受体细胞上的肺炎克雷伯菌 OM 孔蛋白 OmpK36 合作，尽管直接相互作用尚未得到证实21。因此，接合 OM 蛋白表现出质粒特异性细胞表面相互作用，使其成为新的药物靶标候选物。 图 1 - F 样 IV 型分泌系统。基于冷冻电子显微镜和冷冻电子断层扫描模型。 Tra 蛋白 (TraA-TraX) 用大写字母标记。 （图 1 取自 Bragagnolo 等人，2020）10 目的 拟议的研究项目旨在识别和表征 pKpQIL 缀合过程中缀合 OM 蛋白 TraN 和 TraT 的细胞表面相互作用。 生化表征首先，我们将优化 TraN 和 TraT 在大肠杆菌中的表达和纯化条件，然后使用 Pull-down 测定（免疫共沉淀）继续鉴定其各自的细胞表面结合伴侣。我们将确认 TraN（预计类似于 B 桶蛋白 13）是否参与 DNA 转移。我们计划用 TraN 和荧光敏感探针 - DNA 重建脂质体。我们将进行功能测定，测量当 DNA 通过提议的 TraN 结合伴侣 OmpK36 跨越到其他脂质体时的 DNA 荧光或猝灭。 生物物理表征 pKpQIL TraN 和 TraT 结构将通过 X 射线晶体学来解析，数据收集将在钻石光源处进行。或者，我们将利用冷冻电子显微镜，其中两个数据集之间的分辨率重叠将能够实现准确的结构确定。还将尝试 TraN 和 TraT 与其各自的结合伙伴的结晶。然后通过肽诱变来解开对结合至关重要的残基，然后通过 NMR 进行配体结合研究。 该研究项目将通过与伦敦帝国理工学院弗兰克尔实验室合作，采用多学科方法进行重要的分子生物学实验。