Structure Guided Functional Studies of a Lipoprotein Involved in Salmonella Copper Homeostasis

Abstract

Cells from aerobic organisms require copper ions to generate biochemical energy and for certain enzyme‐catalyzed redox reactions. However, recent studies suggest that when bacteria are engulfed by phagosomes within macrophages, Cu2+ is used in high concentrations, among other molecules, to kill the bacterium. To infect a host, bacteria such as Salmonella enterica must survive this toxic environment containing high Cu2+ levels. A recently discovered protein of unknown function, DcrB, plays a role in copper homeostasis within Salmonella enterica, a major cause of food‐borne illness. DcrB is a small lipoprotein anchored to the periplasmic face of the cytoplasmic membrane. Currently, Salmonella phenotypic assays have shown DcrB is necessary for growth in high levels of Cu2+. The hypothesis of this project is that DcrB binds copper and possibly other divalent cations. To test this hypothesis, we used thermal stability assays paired with structure‐based functional studies. We examined the effect of divalent cations on the stability of purified DcrB protein using a fluorescence‐based thermal shift assay and circular dichroism. Furthermore, we are using our recent crystallographic structure of DcrB to identify possible metal ion binding sites and to test whether each hypothesized metal ion binding site is required for the ability of DcrB to confer resistance to Cu2+. For each site, we used site‐directed mutagenesis to generate an allele of dcrB that encodes a DcrB variant lacking the functional side‐chains at the site. Then, we determined whether a multicopy plasmid that contains the mutant allele of dcrB can complement the copper sensitivity of a dcrB knockout mutant of Salmonella enterica. Our results have established the specificity of DcrB for binding divalent cations and have led to the identification of potential metal binding sites, which we anticipate will shed light on the role of DcrB in metal homeostasis in pathogenic bacteria.Support or Funding InformationThis research was supported by a UW‐La Crosse Faculty Research Grant (JFM), a UW‐La Crosse Undergraduate Research Grant (RWS), and the Dean's Distinguished Fellowship (RWS). This research used resources of the Advanced Photon Source, a U.S. Department of Energy Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DEAC0206CH11357. Use of the LS‐CAT Sector 21 was supported by the Michigan Economic Development Corporation and the Michigan Technology Tri‐Corridor (Grant 085P1000817).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.