Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2024

Involvement of prophage genes in modifying the sugar composition of the K58 capsule produced by Acinetobacter baumannii (#99)

Nowshin Sharar 1 , Cristina De Castro 2 , Ruth Hall 3 , Johanna Kenyon 4
  1. School of Biomedical Sciences, Faculty of Health, Queensland University of Technology, Brisbane, QLD, Australia
  2. Department of Agricultural Sciences, University of Naples Federico II, Naples, Italy
  3. School of Life and Environmental Sciences, The University of Sydney, Camperdown, NSW, Australia
  4. School of Pharmacy and Medical Sciences, Griffith University, Gold Coast, QLD, Australia

Capsular polysaccharide (CPS) is an important therapeutic target for the critical priority pathogen, Acinetobacter baumannii. As genes at the chromosomal ‘K locus’ (KL) generally direct CPS synthesis, genetic analysis can assist with predicting CPS composition to develop tailored therapies and assist with identifying genes outside the KL that may influence the CPS type produced, knowledge of which can enhance CPS typing strategies. This study aims to identify and characterise CPS genes outside the KL in the genome of the clinical isolate, BAL062. This isolate includes the KL58 locus with psaABCDEF genes for synthesis of 5,7-di-N-acetylpseudaminic acid (Pse). However, the CPS was shown to include the novel non-2-ulosonic acid form, 5,7-di-N-acetyl-8-epipseudaminic acid (8ePse). Comparative whole genome analysis was used to identify candidate 8ePse genes in the BAL062 genome. A range of bioinformatics tools were used to assess genetic context and distribution. An A. baumannii-E. coli shuttle vector was constructed using Gibson assembly to clone the genes. Carbohydrate analysis using NMR spectroscopy was conducted to assess the non-2-ulosonic acid in the CPS of MRSN31468. Two candidate genes, designated epaA/epaB, for converting Pse to 8ePse were identified in a prophage region in BAL062 genome. Further analysis showed that these genes were also carried by diverse strains that have KL with Pse genes. An A. baumannii-E. coli shuttle vector including an A. baumannii-specific ori, epaA/epaB and the putative promoter, was constructed and cloned into MRSN31468. Carbohydrate analysis of the non-2-ulosonic acid in the CPS demonstrated that the strain produces 8ePse. Genes for converting Pse to 8ePse were identified in a prophage and their role in CPS synthesis was confirmed. This finding increases our knowledge of CPS biosynthesis and assists with improving the predictive capability of CPS typing tools.