Interaction Networks Of A Crucial Cell Wall Enzyme In The Resistant Bacterium Acinetobacter baumannii

Abstract

Acinetobacter baumannii is a common hospital opportunistic pathogen with multidrug resistance, including resistance to last‐resort antibiotics. Many antibiotics today target the cell wall system which is critical for viability. Thus, we sought to better understand cell wall biogenesis in A. baumannii with the goal of finding novel therapeutic targets. L,D‐transpeptidases have been shown to participate in cell wall synthesis by forming unusual peptidoglycan crosslinks. A. baumannii has an L,D‐transpeptidase (Ldt1) that is essential for maintaining the characteristic rod‐shape of the bacterium. However, this transpeptidase and other aspects of cell wall synthesis remain poorly defined. We hypothesized that Ldt1 is important for cell elongation, which is mediated by a multiprotein complex known as the Rod system. To test this hypothesis and define genetic and direct‐protein interactions of Ldt1, we used transposon sequencing (Tn‐seq) and bacterial two‐hybrid techniques. Additionally, we used fluorescent D‐amino acid probes and fluorescence microscopy to visualize differences in active cell wall synthesis sites in WT A. baumannii versus ldt1 deletion mutant. The strongest negative Tn‐seq interactions were with cell division genes, consistent with the hypothesis that Ldt1 is important for the complementary Rod system. The results of the two‐hybrid assay did not show strong direct protein‐protein interaction of Ldt1 with canonical rod‐system components; however, using this method, we mapped novel interactions within the Rod system of A. baumannii. For the fluorescence microscopy, staining conditions of the D‐amino acid probe have been optimized. This study demonstrates methods of elucidating interaction networks in A. baumannii. Through increased understanding of Ldt1 and the Rod system interaction networks, novel synergistic therapeutics can be developed to combat resistant A. baumannii infections.