Poster Presentation The 43rd Lorne Conference on Protein Structure and Function 2018

Functional studies of a lipid A phosphoethanolamine transferase in pathogenic Neisseria (#253)

Katherine Lim 1 , Anandhi Anandan 2 , Alice Vrielink 2 , Charlene Kahler 1
  1. School of Biomedical Sciences, University of Western Australia, Perth, WA, Australia
  2. School of Molecular Sciences, University of Western Australia, Perth, WA, Australia

Neisseria meningitidis (Nm) is usually an asymptomatic coloniser of the upper respiratory tract but can cause serious blood and brain infections that are usually manifested as meningitis and septicaemia in susceptible individuals (1). Neisseria gonorrhoeae (Ng) is a sexually transmitted pathogen that usually infects the urogenital tract (2). If left untreated in females, it can lead to pelvic inflammatory disease (3), which can cause infertility and spontaneous abortions (4). Current treatment for gonorrhoea is with antibiotics but widespread global antibiotic resistance has led to the need for alternative treatment methods.

The role of the enzyme phosphoethanolamine transferase A (EptA) is to transfer phosphoethanolamine (PEA) from phosphatidylethanolamine to the lipid A of the lipooligosaccharide (5). EptA is conserved in both Ng and Nm, and is a pivotal virulence factor. Inactivation of EptA causes complete attenuation in mouse and human infection models due to its role in protecting the bacteria from cationic antimicrobial peptides (CAMPs), which are an important component of the innate immune defense (6). Because of these characteristics, EptA is being developed as a therapeutic drug target that will enable the natural immune system to clear the infection. Comparisons between EptA and other members of the alkaline phosphatase superfamily identified several conserved residues in the enzyme active site involved with coordinating the Zn2+ ion and substrate binding (7). These conserved residues were mutated and confirmed to have caused a reduced resistance to polymyxin B. Mutants were purified in the presence of a non-ionic detergent, dodecyl-β-D-maltoside (DDM), that mimics the bacterial cell membrane. To further understand the impact of these mutations, the active site residue mutants were subjected to enzyme activity and biophysical analysis that allowed for the characterisation of each mutant.

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