The role of outer membrane proteins in bacterial conjugation

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.