We propose to study the regulation of expression of chromosomally encoded multidrug resistance (MDR) efflux pumps and their roles in antimicrobial resistance in Staphylococcus aureus, with a focus on quinolone resistance. In prior work we identified quinolone resistance caused for increased expression of the NorA, B, and C pumps, tetracycline resistance due to the Tet38 pump, (-lactam resistance due to the AbcA pump, and others have identified biocide resistance due to the MepA pump. We have also identified direct transcriptional regulators of pump expression, MgrA and NorG, which interact with each other and have broad effects. There are five Specific Aims.
Under Aim 1, we will define the interactions of MgrA and NorG in expression of norA and other genes encoding efflux pumps. We specifically hypothesize that phosphorylation of MgrA alters its interactions with NorG and changes its effects on transcription of pump genes. The work will include measuring expression of norA by Northern hybridization in strains with constructed mutations in HprK kinase, and measurements of promoter binding of phosphorylated and unphosphorylated MgrA.
Under Aim 2, we will identify the direct regulators of norB, tet38, and abcA by isolating proteins from cell extracts that bind to immobilized promoter DNA fragments, determining their identity by mass spectroscopy, and generating strains with mutations in and overexpression of the genes so identified. Such strains will be assessed for changes in expression of genes encoding pumps by Northern hybridization and transcriptional profiling.
Under Aim 3, we will evaluate the global effects of NorG by transcriptional profiling of strains with mutations in and overexpression of norG.
Under Aim 4, we will identify other candidate efflux pumps shown to be over-expressed in microarray analyses of regulatory networks and environmental stresses and define their roles in antibiotic resistance. Specific novel candidate pump genes distinct from those listed have been shown to be overexpressed in sarA, mgrA, and rot global regulatory mutants and under conditions evoking the stringent response or growth in a mouse subcutaneous abscess model. These candidate pump genes will be cloned and the effects of their overexpression on susceptibility to antibiotics and biocides will be assessed in order to add to the complement of known efflux resistance mechanisms.
Under Aim 5, we will identify the environmental triggers of increased expression of norB, tet38, and other efflux pump genes identified under Aim 4. We have shown that norB and tet38 are overexpressed in S. aureus abscesses and that knockouts of these genes reduce fitness in the abscess milieu. Thus, we hypothesize that pumps are important for bacterial survival in an abscess in part due to relative protection from antimicrobial peptides, which are present in high concentrations. We will test specifically the role of low iron conditions and high concentrations of antimicrobial peptides as inducers of pump expression by measurement of pump gene RNA levels by Northern blotting, and we will determine the effects of pump overexpression of resistance to antimicrobial peptides in vitro.

Public Health Relevance

Antibiotic resistance in common bacterial pathogens such as Staphylococcus aureus is increasing in hospitals and the community. Efflux pumps serve to protect bacteria from adverse environments. Multidrug (MDR) and quinolone resistance conferred by multidrug resistance efflux pumps can also compromise drug utility and affect patient responses to antibiotics. Thus, defining the full complement of MDR efflux pumps in S. aureus and how they are regulated will provide both practical and fundamental information that are important for understanding of staphylococcal fitness to cause disease in diverse environments and for optimizing antibiotic action and use.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37AI023988-24
Application #
8240971
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Huntley, Clayton C
Project Start
1986-09-01
Project End
2013-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
24
Fiscal Year
2012
Total Cost
$433,694
Indirect Cost
$188,669
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02199
Truong-Bolduc, Que Chi; Villet, Regis A; Estabrooks, Zoe A et al. (2014) Native efflux pumps contribute resistance to antimicrobials of skin and the ability of Staphylococcus aureus to colonize skin. J Infect Dis 209:1485-93
Villet, Regis A; Truong-Bolduc, Que Chi; Wang, Yin et al. (2014) Regulation of expression of abcA and its response to environmental conditions. J Bacteriol 196:1532-9
Truong-Bolduc, Que Chi; Hsing, Liao Chun; Villet, Regis et al. (2012) Reduced aeration affects the expression of the NorB efflux pump of Staphylococcus aureus by posttranslational modification of MgrA. J Bacteriol 194:1823-34
Truong-Bolduc, Que Chi; Bolduc, Gilles R; Okumura, Ryo et al. (2011) Implication of the NorB efflux pump in the adaptation of Staphylococcus aureus to growth at acid pH and in resistance to moxifloxacin. Antimicrob Agents Chemother 55:3214-9
Didier, Jean-Philippe; Villet, Regis; Huggler, Elzbieta et al. (2011) Impact of ciprofloxacin exposure on Staphylococcus aureus genomic alterations linked with emergence of rifampin resistance. Antimicrob Agents Chemother 55:1946-52
Truong-Bolduc, Q C; Dunman, P M; Eidem, T et al. (2011) Transcriptional profiling analysis of the global regulator NorG, a GntR-like protein of Staphylococcus aureus. J Bacteriol 193:6207-14
Truong-Bolduc, Que Chi; Hooper, David C (2010) Phosphorylation of MgrA and its effect on expression of the NorA and NorB efflux pumps of Staphylococcus aureus. J Bacteriol 192:2525-34
Jacoby, George; Cattoir, Vincent; Hooper, David et al. (2008) qnr Gene nomenclature. Antimicrob Agents Chemother 52:2297-9
Truong-Bolduc, Que Chi; Ding, Yanpeng; Hooper, David C (2008) Posttranslational modification influences the effects of MgrA on norA expression in Staphylococcus aureus. J Bacteriol 190:7375-81