The role of outer membrane proteins in bacterial conjugation

背景耐碳青霉烯类肺炎克雷伯菌（CRKP）的出现使得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K有关。肺炎病毒序列类型258（ST 258），其以引起全球医院爆发而闻名2，3。接合质粒通过IV型分泌系统（T4 SS）通过细菌接合在种群之间转移;供体和受体细菌之间的遗传物质交换4，5，6，7。 T4 SS是跨越细胞包膜的大蛋白复合物，其介导DNA或蛋白质通过称为菌毛的细胞外细丝转移8，9，如图110所示。在E. coli8，9. T4 SS外膜（OM）蛋白质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和潜在的TraU一起参与交配对稳定，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;表面排斥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合作11，13，19，20。最近对pKpQIL质粒T4 SS的研究表明，pKpQIL TraN与K. pneumoniae OM孔蛋白OmpK 36在质粒转移前在受体细胞上的表达，尽管直接相互作用尚未得到证实21。因此，共轭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点候选者。 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摘自Bragagnolo等人，（2020年）10 目的研究pKpQIL偶联过程中OM偶联蛋白TraN和TraT在细胞表面的相互作用。 生化特性首先，我们将优化TraN和TraT在大肠杆菌中的表达和纯化条件。coli，然后继续使用下拉测定（免疫共沉淀）鉴定它们各自的细胞表面结合配偶体。我们将确认是否TraN，预测类似于B桶蛋白13，参与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DNA荧光或淬灭，因为DNA通过拟定的TraN结合伴侣OmpK 36与其他脂质体交叉。 生物物理表征pKpQIL TraN和TraT结构将通过X射线晶体学解析，其中数据收集将在Diamond Light Source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使准确的结构测定。还将尝试TraN和TraT与其各自的结合配偶体的结晶。然后通过肽诱变解开结合的关键残基，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的弗兰克尔实验室合作，采取多学科方法，进行必要的分子生物学实验。

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.