A Genomics Approach to Drug Tolerance

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DESCRIPTION (provided by applicant): The overall goal of the current grant is to identify the genes leading to the formation of dormant, multidrug tolerant persister genes. This is a challenging problem and the main impediment to progress in understanding the nature of persistence. This remains an important goal and will be achieved by the end of the funded period of the project. With the persister genes in hand, we then planned to study the detailed molecular mechanism of persister formation in a subsequent phase of the project. However, we recently discovered that persisters can be formed in response to DNA damage, and have identified a toxin/antitoxin module TisAB that apparently leads to dormancy, and is the subject of the proposed 2-year Recovery Act Funds for Competitive Revision Application Notice Number NOT-OD-09-058. This finding provides us with an attractive opportunity to accelerate the speed of the project and describe the first molecular mechanism of persister cell formation within the next two years, rather than wait till the next phase of the project. We also did not plan to study persister resuscitation, assuming that this will be done some time in the future once we know the mechanism by which they are formed. TisB provides us with a great opportunity to address this question in the next two years as well. In the Supplement, we propose to establish the first detailed mechanism of persister formation considerably ahead of schedule. Proposed studies will include isolating TisB-dependent persisters by cell sorting and obtaining their transcriptome; a detailed study of the molecular mechanism of TisB action; and the mechanism by which TisB-dependent persisters resuscitate. In case of TisB, the mechanism of resuscitation is intriguing. TisB does not have a protein antitoxin that could antagonize its action - the antitoxin is an RNA that only affects expression. TisB is a membrane-acting peptide that disrupts the pmf and causes a decrease in ATP. This will produce an overall systems shutdown in the cell and multidrug tolerance. In the absence of an antitoxin, it is not clear how the cell manages to resuscitate from such a state of deep dormancy. A screen for mutants able to grow in a strain ectopically expressing TisB will lead to identification of the resuscitation genes by whole genome sequencing. Apart from finding a persister formation and resuscitation mechanism, this work will for the first time link together two seeming opposite strategies of cell survival - active protection through stress responses; and dormancy. The new paradigm emerging from our studies is that stress causes dormancy, and this will be detailed in the proposed Supplement studies. This study will point to an intriguing possibility of a link between other stress responses and persister formation. Pathogens are exposed to many stress factors in the host environment apart from DNA damaging agents - oxidants, high temperature, low pH, membrane-acting agents. It is possible that all stress responses induce the formation of a small but resilient subpopulation of surviving persisters. The present study will lay the foundation for examining the role of stress responses in causing multidrug tolerance in vivo. Once we properly understand the nature of multidrug tolerance, this will instruct our efforts on finding effective counter-measures. PUBLIC HEALTH RELEVANCE: This project will also address the goals of stimulating the economy by hiring two new Postdoctoral Fellows; providing a Research Assistantship to a graduate student; purchasing capital equipment; and contracting out some of the work of this project.