RUI: Unraveling the physiological roles of multidrug efflux pumps in bacteria

Overview.
Bacteria are intrinsically equipped with a variety of multidrug efflux pumps. These pumps first gained interest because of their ability to expel antibiotics out of cells, thus conferring resistance to multiple antibiotics. However, recent findings have shown that these pumps have a broad impact on gene expression, metabolism, and bacterial motility, among other physiological processes. A mechanistic understanding of how these pumps can control and coordinate all these fundamental processes is still lacking. The first goal of this proposal is to determine how AcrAB-TolC, which is the main multidrug efflux pump of the model organism Escherichia coli, controls cell physiology and bacterial motility. The second goal is to integrate this research with the education and mentoring of students from backgrounds traditionally underrepresented in science.
Intellectual Merit.
The mechanisms by which multidrug efflux pumps, located in the cell envelope, affect gene expression, motility and other functions, remain an enigma. The overall objective of this proposal is to discover the molecular mechanisms that allow the AcrAB-TolC pump of E. coli to control gene expression, cell physiology and bacterial motility. Our central hypothesis is that the main function of the AcrAB-TolC pump is to efflux cellular metabolites, whereas the transcriptional repressor AcrR acts as the pump sensor and effector. When the activity of the AcrAB-TolC pump is insufficient to prevent the accumulation of cellular metabolites, these metabolites would bind to and inactivate the AcrR repressor. Such inactivation would cause an up-regulation of flagellum biosynthesis genes and an increase in motility, which would facilitate the escape of E. coli cells to a different environment. This hypothesis is based on recent findings from the PI?s laboratory and other groups that show similar metabolic changes, up-regulation of flagellum biosynthesis genes, and increased motility in both AcrAB-TolC-knockout and AcrR-knockout mutants. To attain the overall objective, we will pursue two specific aims: (I) Identify the flagellum and other genes directly regulated by AcrR and how cellular metabolites impact AcrR function; and (II) Identify the cellular metabolites that bind to the AcrAB-TolC pump and how the small protein AcrZ modulates the function of this pump. The expected outcomes of these aims will significantly advance our mechanistic understanding of central role of multidrug efflux pumps in gene expression and physiology of Gram-negative bacteria; and broadly contribute to other fields such as developing new antibiotics and efflux inhibitors to treat antibiotic resistant infections, or improving tolerance to biofuels and other bioengineering applications.
Broader Impacts.
The proposed project will significantly enhance the mentoring and training of students from diverse backgrounds at California State University Northridge (CSUN). CSUN is a primarily undergraduate and minority-serving institution often attended by first-generation college students. Many of our students are eager to participate in STEM research. However, faculty labs do not have enough space to accommodate all requests. To overcome this limitation, this project aims at integrating the PI?s research into the into the Microbial Physiology course that will be taught by the PI each fall. By studying E. coli, which is an affordable and highly tractable model organism, two underrepresented undergraduate students in the PI?s laboratory produced a publication reporting the global role of the AcrAB-TolC pump in cell metabolism. This proposal aims at expanding this preliminary success to the Microbial Physiology students. These students will experience first-hand the excitement of scientific research, while gaining the training and mentoring necessary to increase their access to graduate programs and biosciences careers. In addition, this award will support a junior investigator whom was a first-generation college student in attaining his goals of 1) advancing the fields of efflux and physiology while impacting biomedicine and bioengineering; and 2) providing research opportunities and inspiring students to pursue science careers.