Strategies for Acinetobacter baumannii to Maintain Zinc Homeostasis

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Project Summary All organisms must maintain a balance of nutrient metals to survive, including zinc (Zn). These metals are required as catalytic and structural cofactors for a variety of proteins, but in excess can lead to the generation of reactive oxygen species or inactivation of non-cognate enzymes through mismetallation. Therefore, tight control of metal levels through import, efflux, and storage is important for optimal growth and survival. Due to this requirement, bacterial metal homeostasis mechanisms are attractive targets for novel therapeutics. This proposal seeks to inform the development of metal-based therapies by identifying mechanisms used by the opportunistic pathogen Acinetobacter baumannii to prevent metal imbalance. Previous work in our laboratory has identified zigA which encodes a putative Zn metallochaperone with increased expression upon Zn starvation mediated by calprotectin, a metal sequestering protein of the innate immune system. Loss of ZigA results in a severe fitness defect upon calprotectin exposure, indicating the essentiality of ZigA under these conditions. Since ZigA is critical for A. baumannii to grow under Zn limitation, we hypothesized that proteins that receive metal from ZigA (clients) are equally important in mediating Zn stress. To identify these clients, I performed a genome- wide transposon mutagenesis screen in Zn limiting conditions comparing WT and ΔzigA libraries to identify genes whose fitness is influenced by Zn deficiency and that modulate the fitness of a ΔzigA mutant. I discovered several genes through this genetic interaction method and chose A1S_3027 for further characterization. A1S_3027 encodes a lytic transglycosylase that is predicted to tailor the bacterial cell wall. Strains lacking A1S_3027 are sensitized to Zn deficiency, and this can be reversed upon addition of ZnCl2. We hypothesize that to ensure the integrity of the cell envelope in conditions of Zn starvation, ZigA interacts with A1S_3027 to regulate its function. Characterizing A1S_3027 and its coordination with ZigA will be tested in two specific aims. In Specific Aim 1, I will study the biochemical properties and function of A. baumannii A1S_3027 using biochemical, genetic, and functional assays to probe how A1S_3027 helps to maintain Zn homeostasis. Experiments proposed in Specific Aim 2 will determine the functional role of A. baumannii A1S_3027 in maintaining appropriate nutrient metal balance by employing A1S_3027-deficient strains in a series of in vitro and in vivo experiments. Taken together, these aims will determine the impact of metal imbalance on A. baumannii pathogenesis and provide the first description of the contribution of a Zn metallochaperone and its clients to microbial virulence. Therapeutics that modulate bacterial metal levels will synergize with the immune system’s defenses, and A1S_3027 may be an attractive target for such metal-focused therapeutics.