S. aureus COG0523s as bacterial zinc metallochaperones critical to pathogenesis

PROJECT SUMMARY/ABSTRACT Staphylococcus aureus is an opportunistic pathogen that is critical to public health and a top cause of infectious death worldwide. To infect humans and cause disease, S. aureus is dependent on the availability of nutrient metals, including zinc (Zn). In response to infection, host factors including the S100 protein calprotectin (CP) act

as effectors of nutritional immunity and sequester nutrient metals. Vertebrate proteins of the COG0523 family have been identified as bona-fide Zn metallochaperones that equip downstream metalloprotein effectors with Zn ions. COG0523s in bacteria are predicted to perform a similar role in maintaining cellular metal homeostasis and

supporting cellular function, particularly in the setting of Zn starvation. This proposal aims to evaluate S. aureus COG0523 orthologs crzA and zigA, define the impact that these genes have on pathogenesis, and elucidate their role in Zn transfer to predicted interaction partners. Preliminary data suggest that crzA and zigA are

regulated by Zn via the transcriptional repressor Zur, that high confidence interaction partners exist for both CrzA and ZigA, and that loss of either COG0523s or predicted binding partners increases sensitivity to DNA damage. This phenotype is pronounced in crzA and zigA mutants exposed to both Zn stress and specific DNA damage.

The central hypothesis of this proposal is that S. aureus COG0523s transfer Zn to metalloproteins involved in DNA damage repair in a Zn- and GTP-dependent manner and that this process is important to S. aureus pathogenesis. To test this hypothesis, biochemical, genetic, and microbiological techniques will

be used to define a model of CrzA and ZigA function in S. aureus. In Aim 1, I will define the mechanism for interaction between COG0523s and their clients and evaluate how metal, candidate clients, and GTP/GDP affect the dynamics of CrzA and ZigA Zn transfer. To accomplish this, I will express and purify COG0523s and their

clients, and define the function and required co-factors of ZigA and CrzA by co-immunoprecipitation, client- specific biochemical assays, and inductively coupled plasma mass spectrometry (ICP-MS). In Aim 2, I will test the hypothesis that COG0523s regulate client activity and affect S. aureus DNA maintenance and that conserved

COG0523 Walker and CXCC motifs are required for ZigA and CrzA function in S. aureus. I will conduct growth curves and survival assays in the presence of DNA damaging agents and in bone-marrow derived macrophages to elucidate the impact of Zn, DNA damage, COG0523s, and the metal/GTP-binding motifs of these proteins on

S. aureus growth and mutagenesis. In Aim 3, I will test the hypothesis that CrzA and ZigA enable S. aureus to circumvent host-imposed Zn restriction. Manipulation of Zn-handling both in the host and pathogen will demonstrate the impact of COG0523s on infection and increase the rigor of our approach. Taken together, the

proposed work will have broad implications for the study of S. aureus pathogenesis. This work has the potential to establish CrzA and ZigA as the first experimentally confirmed bacterial Zn-metallochaperones and continue to develop an understanding of the role of nutritional immunity in therapeutics and clinical practice.