Lighting up the bad bugs: fluorescent labelling of the bacterial resistance factor ArnT with commercial fluorophores Prachi Bendale 1 and Abigail Hopkins 1 Sheiliza Carmali 1 , Mohammad Arefian 2 , Ben Collins 2 ,Mark Sutton 3 , Miguel A. Valvano 4 & Gerd K. Wagner 1 1 School of Pharmacy, Queen’s University Belfast, 2 School of Biological Sciences, Queen’s University Belfast, 3 UK Health Security Agency, 4 Wellcome-Wolfson Institute for Experimental Medicine, Queen’s University Belfast Antimicrobial resistance (AMR) is a major threat to global health predicted to lead to 10 million deaths per year by 2050 if no action is taken [1]. Operationally simple technologies for the diagnostic labelling of bacterial resistance factors are therefore of great interest to study molecular, cellular, and environmental factors driving AMR development. One such resistance factor is the enzyme 4-amino-4-deoxy-l-arabinose transferase (ArnT). ArnT is involved in membrane lipopolysaccharide remodelling of Gram-negative pathogens such as Klebsiella pneumoniae , Pseudomonas aeruginosa , and Burkholderia cenocepacia , resulting in resistance to polymyxins, last-resort antibiotics employed to treat multidrug antimicrobial resistant infections [2]. In this project, we have investigated the use of commercially available fluorophores as chemical tools for the diagnostic labelling of ArnT. Analysis of sequence and structural data for ArnT from K. pneumoniae , P. aeruginosa , B. cenocepacia and Cupriavidus metallidurans identified several lysine residues as potential target sites for the covalent attachment of fluorescent probes. To exploit this opportunity, we assembled a small library of commercially available fluorophores with different lysine-reactive electrophiles and established a workflow for protein labelling. Relevant experimental parameters such as incubation time and fluorophore concentration were optimised using bovine serum albumin as a model protein. Application of our protocol to the B. cenocepacia ArnT [3] allowed the desired in-gel fluorescent detection of ArnT after electrophoretic separation. Protein mass spectrometry confirmed the covalent attachment of selected fluorophores and identified their attachment sites. Finally, we rationalised the observed reactivities using a recently developed algorithm for the prediction of site and sequence of protein modifications with lysine-reactive reagents [4]. Our results demonstrate that ArnT can be readily labelled with commercially available fluorophores, providing a basis for the rational development of bespoke labelling reagents for this enzyme as novel diagnostic tools in the fight against antimicrobial resistance. References 1. O’Neill (2016) Tackling Drug-Resistant Infections Globally: final report and recommendations 2. Petrou et al. (2018) Structures of aminoarabinose transferase ArnT suggest a molecular basis for lipid A glycosylation. Science , 351 (6273), 608-612. 3. Tavares-Carreon et al. (2015) Burkholderia cenocepacia and Salmonella enterica ArnT proteins that transfer 4-amino-4- deoxy-l-arabinose to lipopolysaccharide share membrane topology and functional amino acids. Sci. Rep. , 5, doi.org/10.1038/ srep10773 4. Patel, A. et al. (2022) Automated prediction of site and sequence of protein modification with ATRP initiators. PLoS One , doi. org/10.1371/journal.pone.0274606
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