The multiple antibiotic resistance (mar) operon of the enteric bacterium Escherichia coli regulates multiple antibiotic resistance and pathogenicity. The goal of the present study is to improve our understanding of how the Mar system achieves its effects. The marRAB operon encodes MarR, a protein whose sole known function is to repress the expression of its own operon and which is inactivated by small "inducer" molecules (ligands);MarA, a master regulatory protein;and MarB, which negatively affects marA expression.
Aim A of the study will characterize how ligands inactivate MarR, and how the putatively periplasmic MarB protein represses marA expression.
Aim A also involves novel observations on the multidrug efflux pump AcrAB-TolC, which is up-regulated by MarA. Inactivation of the AcrAB-TolC pump gives rise to enhanced expression of the genes for AcrAB and for MarA, suggesting that cellular metabolites, normally exported by the pump, may be inducers of acrAB and marRAB. Genetic inactivation of different metabolic pathways led us to identify several metabolites potentially responsible for such effects;their mechanisms of action will be studied. Additional inducing metabolites will also be identified by a variety of techniques including metabolomics. Substrate antibiotics also induce the expression of the AcrAB-TolC pump;the underlying mechanism will be studied.
Aim B investigates the small RNAs (sRNAs) regulated by MarA and by its two homologs SoxS and Rob, which together with MarA, regulate the transcription of at least forty protein-specifying genes directly. Although expression of two sRNAs (MicF and CsrA) has been shown to be regulated by Mar/Sox/Rob, there has of yet been no systematic search to identify similarly- regulated sRNAs. This search will use innovations in transcriptomic technologies.
Aim C arises from observations that MarA/SoxS/Rob enhance the ability of bacteria to persist in the murine kidney in a model of ascending pyelonephritis.
Aim C will focus on the bacterial targets and pathways involved, including that of the carbon storage regulation system (already an identified target of Mar/Sox/Rob), as well as on the host response to the infection. Strains with and without the genes encoding these three regulators will be compared using in vitro cell culture and in vivo histopathology to assess differential expression patterns of the host immune response. Bacterial pathogenic factors such as those affecting attachment and invasion will also be studied. In summary, this proposal investigates the Mar system and its homologs SoxS/Rob of E. coli, which control many genes involved in antibiotic resistance and pathogenicity. A deeper knowledge of this system will contribute to knowing how this bacterium, and other bacteria with homologous regulators, resist antibiotics and host defenses, and thus will provide leads for improved therapeutic approaches.

Public Health Relevance

Antibiotic resistance was named by the World Health Organization as one of the three major public health menaces in this decade. The marRAB operon in Escherichia coli and six other important genera of enteric bacteria is known to play a role in both multidrug resistance and pathogenicity, but much is not understood about the specifics of the mechanisms. We propose the following studies: 1) find how molecular ligands inactivate MarR, the repressor of the transcriptional regulator MarA (which in turn controls many genes in its regulon);2) determine how marB regulates marA;3) describe how the acrAB efflux pump locus (activated by MarA) controls its own synthesis and that of the marRAB operon by effluxing regulatory metabolites;4) look for additional targets of MarA and its homologs among small RNAs;5) find how MarA and its homologs help E. coli to persist during infection of the mouse kidney.

Agency
National Institute of Health (NIH)
Type
Research Project (R01)
Project #
5R01AI056021-30
Application #
8662680
Study Section
Drug Discovery and Mechanisms of Antimicrobial Resistance Study Section (DDR)
Program Officer
Korpela, Jukka K
Project Start
Project End
Budget Start
Budget End
Support Year
30
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Tufts University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02111
Zhang, Wan-Jiang; Xu, Xing-Ran; Schwarz, Stefan et al. (2014) Characterization of the IncA/C plasmid pSCEC2 from Escherichia coli of swine origin that harbours the multiresistance gene cfr. J Antimicrob Chemother 69:385-9
Ruiz, Cristian; Levy, Stuart B (2014) Regulation of acrAB expression by cellular metabolites in Escherichia coli. J Antimicrob Chemother 69:390-9
Vinue, Laura; McMurry, Laura M; Levy, Stuart B (2013) The 216-bp marB gene of the marRAB operon in Escherichia coli encodes a periplasmic protein which reduces the transcription rate of marA. FEMS Microbiol Lett 345:49-55
McMurry, Laura M; Levy, Stuart B (2013) Amino acid residues involved in inactivation of the Escherichia coli multidrug resistance repressor MarR by salicylate, 2,4-dinitrophenol, and plumbagin. FEMS Microbiol Lett 349:16-24
Duval, Valerie; McMurry, Laura M; Foster, Kimberly et al. (2013) Mutational analysis of the multiple-antibiotic resistance regulator MarR reveals a ligand binding pocket at the interface between the dimerization and DNA binding domains. J Bacteriol 195:3341-51
Warner, Douglas M; Duval, Valerie; Levy, Stuart B (2013) The contribution of PmrAB to the virulence of a clinical isolate of Escherichia coli. Virulence 4:634-7
Warner, Douglas M; Levy, Stuart B (2012) SoxS increases the expression of the zinc uptake system ZnuACB in an Escherichia coli murine pyelonephritis model. J Bacteriol 194:1177-85
Lister, Ida M; Raftery, Connor; Mecsas, Joan et al. (2012) Yersinia pestis AcrAB-TolC in antibiotic resistance and virulence. Antimicrob Agents Chemother 56:1120-3
McMurry, Laura M; Levy, Stuart B (2011) The periplasmic protein MppA is not involved in regulation of marA in Escherichia coli. Antimicrob Agents Chemother 55:4939
Ruiz, Cristian; Levy, Stuart B (2011) Use of functional interactions with MarA to discover chromosomal genes affecting antibiotic susceptibility in Escherichia coli. Int J Antimicrob Agents 37:177-8

Showing the most recent 10 out of 24 publications