The role of outer membrane proteins in bacterial conjugation

背景耐碳青霉烯类肺炎克雷伯菌(CRKP)的出现使得治疗手段寥寥无几，导致了抗生素耐药危机。碳青霉烯类是最后一种B-内酰胺类药物，用于疑似CRKP感染1。CRKP通过含有碳青霉烯酶；B-内酰胺水解酶1获得抗性。CRKP携带碳青霉烯酶编码的结合质粒，如pKpQIL家族的2，3。这些通常与肺炎克雷伯菌序列类型258(ST258)有关，该序列以引起全球医院暴发而闻名2，3。结合质粒通过细菌结合通过IV型分泌系统(T4SS)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。T4SS是跨越细胞膜的大型蛋白质复合体，通过称为毛丝8，9的细胞外细丝介导DNA或蛋白质的转移，如图110所示。转位(TrA)操纵子编码转位蛋白(TrA)，在大肠杆菌8，9中的接合F-质粒中共同形成T4SS。T4SS外膜(OM)蛋白Tran和Trat在DNA转位11、12、13之前与受体细胞形成细胞表面相互作用。Tran与Trag和潜在的Trau一起参与配对稳定，以稳定转位通道14、15、16。脂蛋白Trat提供对血清杀菌作用的免疫力，并防止供体细胞同时作为受体细胞；表面排除17，18。有证据表明，F-质粒Tran和Trat变体在质粒转移11、13、19、20之前与受体细胞上的OM porin OmpA协同作用。最近对pKpQIL质粒T4SS的研究表明，pKpQIL Tran在质粒转移之前与肺炎克雷伯菌孔蛋白OmpK36在受体细胞上协同作用，尽管直接相互作用尚未得到证实。因此，结合的OM蛋白表现出质粒特异性的细胞表面相互作用，使它们成为新的药物靶标候选。图1-类似F的IV型分泌物系统。基于低温电子显微镜和低温电子断层扫描模型。TrA蛋白(Traa-Trax)用大写字母标记。(图1摘自Bragagnolo等人，2020)10目的拟议的研究项目旨在识别和表征pKpQIL结合过程中结合的OM蛋白Tran和Trat的细胞表面相互作用。生物化学特性首先，我们将优化Tran和Trat在大肠杆菌中的表达和纯化条件，然后利用下拉试验(免疫共沉淀)鉴定它们各自的细胞表面结合伙伴。我们将确认Tran是否参与了DNA转移，预计它类似于B桶蛋白13。我们计划用Tran和一个荧光敏感的探针-DNA来重建脂质体。我们将进行功能分析，以测量DNA荧光或当DNA通过建议的反式结合伙伴OmpK36进入其他脂质体时的猝灭。生物物理表征pKpQIL Tran和Trat结构将通过X射线结晶学进行解析，其中数据收集将在钻石光源进行。或者，我们将利用低温电子显微镜，其中两个数据集之间的分辨率重叠将使准确的结构确定成为可能。还将尝试将Tran和Trat与其各自的结合伙伴结晶。对结合至关重要的残基随后将通过多肽诱变来解开，随后将通过核磁共振进行配体结合研究。这项研究项目将通过与伦敦帝国理工学院弗兰克尔实验室的合作，采取多学科的方法，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.