Zinc is an essential nutrient for all cells due to its key role as a catalytic and structural cofactor. Competition for zinc and other nutrient metal ions is critical for determining the outcome of host- pathogen interactions. Metalloregulatory proteins sense both zinc deficiency and excess and are required for bacterial acclimation to changes in zinc availability. In Bacillus subtilis, a Gram positive model organism, zinc sufficiency is sensed by Zur (zinc uptake repressor) and zinc excess by CzrA. When zinc levels are limiting for growth, Zur repression is relieved resulting it increased zinc uptake (mediated by the ZnuABC transporter), Zn mobilization by displacement of small, ribosome-associated zinc proteins (L31 and L33), and replacement of a key zinc dependent enzyme (FolE) by a non-zinc requiring alternate enzyme. In the next project period, we will investigate the detailed molecular mechanisms of zinc sensing and define the major components of the labile zinc pool in the cell. Preliminary results have led to five core hypotheses that form the foundation of the Specific Aims.
Aim 1 a will address the hypothesis that regulation by Zur involves a graded response as the cell transitions from zinc sufficiency to deficiency, and will test the role of negative cooperativity in zinc-binding to Zur in expanding the range of zinc responsiveness.
In Aim 1 b, we will adapt genetically encoded fluorescent reporters to monitor free zinc levels in vivo and seek to reconcile the exceptionally high (sub-picomolar) zinc affinity of the Zur and CzrA sensors with emerging evidence consistent with free zinc levels in the mid-picomolar range.
Aim 2 will address the role of the low molecular weight thiol bacillithiol (BSH) as the major buffer of the labile zinc pool (Aim 2a), the role of small, dispensable ribosome-associated proteins as the major storage form of zinc (Aim 2b), and the roles of these two zinc pools in metallation of FolE, a zinc metalloenzyme sensitive to zinc availability, and other key zinc enzymes. Limitation for zinc by calprotectin, and likely other factors, is a key part of the human innate immune response. Since all of the key zinc homeostasis factors described here are conserved in many important human pathogens, these studies will provide key background information to facilitate the ultimate development of antimicrobial agents that affect metal ion homeostasis.

Public Health Relevance

Mammalian hosts synthesize molecules (e.g. siderocalin, calprotectin) that sequester metals and thereby restrict the growth of invading pathogens. These studies will define the responses of Bacillus subtilis to zinc limitation and explore mechanisms of zinc storage and buffering. These results have broad implications for key bacterial pathogens with similar genetics and physiology, including Staphylococcus aureus and pathogenic streptococcal and enterococcal strains, and will thereby contribute directly to improved strategies for preventing and managing bacterial infectious disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
2R01GM059323-13A1
Application #
8630431
Study Section
Macromolecular Structure and Function A Study Section (MSFA)
Program Officer
Chin, Jean
Project Start
2000-02-01
Project End
2018-02-28
Budget Start
2014-03-01
Budget End
2015-02-28
Support Year
13
Fiscal Year
2014
Total Cost
$318,865
Indirect Cost
$103,601
Name
Cornell University
Department
Microbiology/Immun/Virology
Type
Schools of Earth Sciences/Natur
DUNS #
872612445
City
Ithaca
State
NY
Country
United States
Zip Code
14850
Helmann, John D (2014) Specificity of metal sensing: iron and manganese homeostasis in Bacillus subtilis. J Biol Chem 289:28112-20
Chandrangsu, Pete; Dusi, Renata; Hamilton, Chris J et al. (2014) Methylglyoxal resistance in Bacillus subtilis: contributions of bacillithiol-dependent and independent pathways. Mol Microbiol 91:706-15
Ma, Zhen; Chandrangsu, Pete; Helmann, Tyler C et al. (2014) Bacillithiol is a major buffer of the labile zinc pool in Bacillus subtilis. Mol Microbiol 94:756-70
McGuire, Amanda M; Cuthbert, Bonnie J; Ma, Zhen et al. (2013) Roles of the A and C sites in the manganese-specific activation of MntR. Biochemistry 52:701-13
Smaldone, Gregory T; Antelmann, Haike; Gaballa, Ahmed et al. (2012) The FsrA sRNA and FbpB protein mediate the iron-dependent induction of the Bacillus subtilis lutABC iron-sulfur-containing oxidases. J Bacteriol 194:2586-93
Smaldone, Gregory T; Revelles, Olga; Gaballa, Ahmed et al. (2012) A global investigation of the Bacillus subtilis iron-sparing response identifies major changes in metabolism. J Bacteriol 194:2594-605
Faulkner, Melinda J; Ma, Zhen; Fuangthong, Mayuree et al. (2012) Derepression of the Bacillus subtilis PerR peroxide stress response leads to iron deficiency. J Bacteriol 194:1226-35
Gaballa, Ahmed; MacLellan, Shawn; Helmann, John D (2012) Transcription activation by the siderophore sensor Btr is mediated by ligand-dependent stimulation of promoter clearance. Nucleic Acids Res 40:3585-95
Ma, Zhen; Lee, Jin-Won; Helmann, John D (2011) Identification of altered function alleles that affect Bacillus subtilis PerR metal ion selectivity. Nucleic Acids Res 39:5036-44
Faulkner, Melinda J; Helmann, John D (2011) Peroxide stress elicits adaptive changes in bacterial metal ion homeostasis. Antioxid Redox Signal 15:175-89

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