Transition metal ions play highly specialized roles in biological catalysis and all cells encode regulatory machinery that controls their intracellular availability so that mis-metallation and metal toxicity is avoided. In bacteria, this process is transcriptionally regulated by a panel of "metal sensor" proteins in response to changes in metal bioavailability. The proposed experiments strongly integrate structural and cell biological approaches to elucidate determinants of metal homeostasis and cross-talk in the respiratory pathogen Streptococcus pneumoniae, focusing on zinc, manganese and copper, three metals whose concentrations change dramatically during the course of invasive disease. In the next project period, our specific aims are to 1) Obtain a structural and physicochemical characterization of the novel zinc uptake regulator S. pneumoniae AdcR (adhesin competence repressor). Here we propose NMR, structural and thermodynamic studies of wild-type and mutant AdcRs in an effort to understand allosteric activation of DNA operator binding;2) Perform a direct test of the thermodynamic "set-point" model for zinc homeostasis in S. pneumoniae, which posits that "free" zinc is buffered in a concentration range dictated by the zinc affinities of the uptake (AdcR) and efflux (SczA, streptococcal czcD activator) regulators. Since intracellular metallation status is dictated by free or chelatable zinc, not total zinc, we propose to measure and manipulate free Zn in the cytoplasm using a FRET-based imaging assay;3) Obtain structural and mechanistic insights into copper resistance and trafficking in S. pneumoniae, a process characterized by features not previously observed;and 4) Carry out structural studies of the CuI sensor CsoR and CsoR-related proteins in Mycobacterium tuberculosis and other bacilli. Small angle X-ray scattering, NMR and crystallographic studies are proposed. Since an emerging aspect of nutritional immunity to infection by bacterial human pathogens is the host control of metal availability, these studies lay the foundation for our long-term goal of disrupting metal availability or inducing metal toxicity by developing new antimicrobial agents that selectively target key components of transition metal homeostasis systems.

Public Health Relevance

Bacterial pathogens require metal ions to perform processes related to both normal physiology and life- threatening invasive disease. As such, host control of metal ion availability is a critical component of the host-pathogen interface. Our studies of this process in the important respiratory pathogen Streptococcus pneumoniae may lead to the identification of new antibiotic targets that play key roles in this process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM042569-24
Application #
8501506
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Anderson, Vernon
Project Start
1991-04-01
Project End
2016-04-30
Budget Start
2013-05-01
Budget End
2014-04-30
Support Year
24
Fiscal Year
2013
Total Cost
$322,242
Indirect Cost
$109,942
Name
Indiana University Bloomington
Department
Chemistry
Type
Schools of Arts and Sciences
DUNS #
006046700
City
Bloomington
State
IN
Country
United States
Zip Code
47401
Higgins, Khadine A; Giedroc, David (2014) Insights into Protein Allostery in the CsoR/RcnR Family of Transcriptional Repressors. Chem Lett 43:20-25
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Fu, Yue; Chang, Feng-Ming James; Giedroc, David P (2014) Copper transport and trafficking at the host-bacterial pathogen interface. Acc Chem Res 47:3605-13
Guerra, Alfredo J; Giedroc, David P (2014) Backbone and sterospecific methyl side chain resonance assignments of the homodimeric zinc sensor AdcR (32 kDa) in the apo- and Zn(II)-bound states. Biomol NMR Assign 8:11-4
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Chakravorty, Dhruva K; Wang, Bing; Lee, Chul Won et al. (2013) Solution NMR refinement of a metal ion bound protein using metal ion inclusive restrained molecular dynamics methods. J Biomol NMR 56:125-37

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