Metals are in widespread use to inhibit the growth of microbial populations. In particular, silver is commonly used in hospital settings as an effective broad spectrum biocide that has low toxicity to humans. However, protein systems are in place which enhance survival of microbes under conditions of high metal concentrations and reduce the efficacy of metals as broad spectrum biocides. One of these systems, the Cus system of E. coli, is responsible for sensing and responding to elevated levels of silver(I) and copper(I) in th environment. Despite its public health importance, the mechanisms of copper and silver handling by the Cus system are not well understood. We propose that mechanistic understandings of copper and silver handling will provide a target for future development of drugs and aid in understanding the limitations of metal-based biocides. In our previous work, we have focused on using the Cus system as a model system for a large class of microbial metal resistance systems. We have biochemically and structurally characterized two of the components, CusF and CusB, of the membrane spanning metal efflux system (CusCFBA) that actively removes metals from the cell. Additionally we have developed a novel and powerful technique to monitor metal transfer between proteins. The natural development of this previous work is to integrate these findings with the other proteins in the system.
In aim 1 we will investigate the mechanism of metal transport by the Cus system and test the hypothesis that the Cus system is primarily important for detoxification of the periplasm, through metal transfer from the Cus periplasmic proteins (CusB and CusF) to the membrane-bound Cus proteins (CusA and CusC).
In aim 2, we will test the hypothesis that CusS is activated by metal binding to the periplasmic domain, leading to conformational changes that ultimately result in activation of Cu(I)/Ag(I) resistance systems.. At the conclusion of these studies, we will have a detailed understanding of the microbial counterattack to metal contaminated environments. We will have determined how a specific metal is discriminated from other metals by a sensory and response system, the mechanisms of the transport process, and the further detoxification strategy after transport has occurred. These studies will give us the tools to understand the microbial response to metal biocides and provide strategies for their effective use in controlling bacterial infections in human populations.

Public Health Relevance

Metals are in widespread use as broad spectrum biocides to control the growth of microbial populations, though the defensive mechanisms used by microorganisms to survive metal challenges are not well understood. The effective use of metals in prevention of infections requires an understanding of the systems that handle metals in microorganisms. This project will determine the bacterial response to copper and silver by the Cus system to understand how metals are discriminated, exported, and detoxified, which will provide strategies for the continued use of metals as biocides.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM079192-07
Application #
8735156
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
2007-04-01
Project End
2017-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
7
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Arizona
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
City
Tucson
State
AZ
Country
United States
Zip Code
85721
Affandi, Trisiani; Issaian, Aaron V; McEvoy, Megan M (2016) The Structure of the Periplasmic Sensor Domain of the Histidine Kinase CusS Shows Unusual Metal Ion Coordination at the Dimeric Interface. Biochemistry 55:5296-306
Gudipaty, Swapna A; McEvoy, Megan M (2014) The histidine kinase CusS senses silver ions through direct binding by its sensor domain. Biochim Biophys Acta 1844:1656-61
Chacón, Kelly N; Mealman, Tiffany D; McEvoy, Megan M et al. (2014) Tracking metal ions through a Cu/Ag efflux pump assigns the functional roles of the periplasmic proteins. Proc Natl Acad Sci U S A 111:15373-8
Padilla-Benavides, Teresita; George Thompson, Alayna M; McEvoy, Megan M et al. (2014) Mechanism of ATPase-mediated Cu+ export and delivery to periplasmic chaperones: the interaction of Escherichia coli CopA and CusF. J Biol Chem 289:20492-501
Gudipaty, Swapna Aravind; Larsen, Andrew S; Rensing, Christopher et al. (2012) Regulation of Cu(I)/Ag(I) efflux genes in Escherichia coli by the sensor kinase CusS. FEMS Microbiol Lett 330:30-7
Mealman, Tiffany D; Zhou, Mowei; Affandi, Trisiani et al. (2012) N-terminal region of CusB is sufficient for metal binding and metal transfer with the metallochaperone CusF. Biochemistry 51:6767-75
Jayakanthan, Samuel; Roberts, Sue A; Weichsel, Andrzej et al. (2012) Conformations of the apo-, substrate-bound and phosphate-bound ATP-binding domain of the Cu(II) ATPase CopB illustrate coupling of domain movement to the catalytic cycle. Biosci Rep 32:443-53
Mealman, Tiffany D; Blackburn, Ninian J; McEvoy, Megan M (2012) Metal export by CusCFBA, the periplasmic Cu(I)/Ag(I) transport system of Escherichia coli. Curr Top Membr 69:163-96
Kim, Eun-Hae; Nies, Dietrich H; McEvoy, Megan M et al. (2011) Switch or funnel: how RND-type transport systems control periplasmic metal homeostasis. J Bacteriol 193:2381-7
Mealman, Tiffany D; Bagai, Ireena; Singh, Pragya et al. (2011) Interactions between CusF and CusB identified by NMR spectroscopy and chemical cross-linking coupled to mass spectrometry. Biochemistry 50:2559-66

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