A comparative characterisation of commercially available lipid-polymer nanoparticles formed from model membranes

Journal article

Antibiotic resistance is a major health issue of this century with resistant bacterial infections causing ten million deaths yearly by 2050. One of the approaches to tackle this problem is to block the antibiotic resistance genes from spreading by targeting conjugation, one of the main horizontal gene transfer mechanisms bacteria use to acquire these genes. This process encompasses the transfer of a DNA molecule from a donor to a recipient organism, and is operated by a Type 4 Secretion System (T4SS) which have been found in more than 20 bacteria species. This work investigates components involved in the Gram-positive T4SS encoded by the tcp locus from the pathogenic bacteria Clostridium perfringens. The tcp T4SS is composed of TcpK and TcpM in charge of binding the DNA molecule, a complex forming a channel used by the DNA formed by TcpA, TcpC, TcpG and TcpH. Outside the cell, the transfer is facilitated by the cell adhesin CnaC and the sortase SrtD. Other proteins such as the putative ATPase TcpF, the membrane proteins TcpD and TcpE and the soluble protein TcpN are known to be involved although their functions remain unknown. The subcomplex formed by the TcpH and TcpA components was successfully expressed recombinantly in E. coli and purified using chromatography methods. However, this subcomplex was found to be unstable, therefore, the proteins TcpC and TcpG were then successfully co-expressed with TcpA and TcpH to stabilise the complex. The proteins of interest were shown to co-elute after a purification of the membrane fraction. The sample characterisation by mass photometry suggest that the complex TcpACGH is 950kDa big and thus could correspond to a stoichiometry of six TcpA, two TcpH, three TcpC and six TcpG. In addition, a construct containing the ATPase domain of TcpF, another essential tcp component was purified from an E. coli expression system. The characterisation allowed to conclude for the first time that the ATPase domain is active without the N-terminal half of the protein. The construct was also found to be able to oligomerise including the expected hexameric form. It was found that the addition of an ATP analogue did not stabilise the hexameric form. This work explored the components of the T4SS in a Gram-positive bacteria to expand our knowledge around the conjugation mechanism in such bacteria. Continuation of this work could lead to a better understanding of the dissemination of antibiotic resistance in the bacterial populations

Authors: Sawczyc, H; Heit, S; Watts, A

• Publication status: Published
• Peer review status: Peer reviewed
• Publisher: Springer
• Journal: European Biophysics Journal with Biophysics Letters
• Volume: 52
• Issue: 1-2
• Pages: 39-51
• Publication date: 2023-02-14
• DOI: 10.1007/s00249-023-01632-5
• EISSN: 1432-1017
• ISSN: 0175-7571
• Language: English
• Keywords: Materials science; Bilayer; Nanoparticle; POPC; Lipid bilayer; Copolymer; Nanotechnology; Polymer; Organic chemistry; Maleic acid; Chemical engineering; Chemistry; Chromatography; Membrane