Antibiotic resistance is an escalating problem for modern chemotherapy of bacterial infectious diseases, and, in combination with the deteriorating pipeline of new antibacterials, is creating a clear and urgent danger to public health and national biodefense. Although the mechanisms that facilitate the de novo development, clonal spread, and horizontal transfer of resistance factors are not fully understood, the rapid rate at which antibiotic-resistant bacteria arise is likely due to a combination of mutations introduced during SOS mutagenesis and gene transfer between organisms. Recently, the Escherichia coli RecA protein's activities in SOS induction and homologous recombination have revealed RecA as a crucial player in these phenomena. A combination of primary literature, patent data, and unpublished results demonstrate that RecA mediates a range of phenomena related to bacterial pathogenecity, particularly the development and transmission of antibiotic resistance genes. Although the high conservation of RecA among bacterial species compellingly suggests the possibility that RecA may play similar roles in species other than E. coli, many questions remain as to how the properties of individual variants are related to their specific biological functions. To delimit possible models for the RecA-mediated activities that occur in pathogenic bacteria, we propose three Specific Aims to exploit our recently developed methods for rapid, parallel purification and rigorous characterization of RecA proteins to elucidate the relationships between RecA structure, in vitro activities, and physiologic functions. Briefly, we will (1) systematically define and evaluate structure-function relationships among RecA proteins from 31 pathogenic bacteria using biochemical and cellular activity assays;(2) provide insight into the species-specific molecular mechanisms of RecA- DNA filament activation using directed mutagenesis and substrate analogs;and (3) demonstrate that RecA effectors can be delivered into living bacteria to produce physiological consequences. The successful realization of the Aims will provide (1) substantial and novel insights into the molecular mechanisms by which different RecA proteins from select bacterial pathogens carry out their biological functions;(2) a novel microbiological toolbox that will be central to teasing apart the various roles of RecA in pathogenicity;and (3) novel methods for the delivery of small-molecule RecA effectors into bacterial pathogens.

Public Health Relevance

The bacterial RecA protein is an emerging target for adjuvants for antibiotic chemotherapy that moderate the development and transmission of antibiotic resistance genes and increase the antibiotic therapeutic index. By combining the use of enzyme kinetics and cellular assays with the synthesis of small-molecule ligands to probe structure-function relationships among RecA proteins from select pathogenic bacteria, a greater understanding of these proteins'roles in various aspects of bacterial pathogenicity - including the de novo development and transmission of antibiotic resistance genes - will be obtained. This new knowledge will increase the efficiency of the discovery of small-molecule effectors that address the urgent unmet need to overcome antibiotic resistance in biothreat and other pathogenic bacteria.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM058114-12
Application #
8289578
Study Section
Synthetic and Biological Chemistry A Study Section (SBCA)
Program Officer
Barski, Oleg
Project Start
1998-10-01
Project End
2014-05-31
Budget Start
2012-06-01
Budget End
2014-05-31
Support Year
12
Fiscal Year
2012
Total Cost
$312,860
Indirect Cost
$97,238
Name
University of North Carolina Chapel Hill
Department
Pharmacology
Type
Schools of Pharmacy
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
Wigle, Tim J; Sexton, Jonathan Z; Gromova, Anna V et al. (2009) Inhibitors of RecA activity discovered by high-throughput screening: cell-permeable small molecules attenuate the SOS response in Escherichia coli. J Biomol Screen 14:1092-101
Singleton, Scott F; Roca, Alberto I; Lee, Andrew M et al. (2007) Probing the structure of RecA-DNA filaments. Advantages of a fluorescent guanine analog. Tetrahedron 63:3553-3566
Wigle, Tim J; Singleton, Scott F (2007) Directed molecular screening for RecA ATPase inhibitors. Bioorg Med Chem Lett 17:3249-53
Lee, Andrew M; Wigle, Tim J; Singleton, Scott F (2007) A complementary pair of rapid molecular screening assays for RecA activities. Anal Biochem 367:247-58