Title: 1345 - Mechanisms of Drug Tolerance During Starvation in Mycobacterium tuberculosis
Alisha Aagesen (Presenter)
University of Minnesota
Anna Tischler, University of Minnesota
Objectives: Tuberculosis (TB), caused by Mycobacterium tuberculosis, is a serious pulmonary infection that can spread to other areas of the body, resulting in extrapulmonary TB. Oral manifestations of TB can involve the salivary glands, tonsils, uvula, tongue, floor of the mouth, soft palate, gingiva, lips, and hard palate. Mtb infections can be challenging to treat due to physiologically heterogeneous populations of cells, which include both antibiotic-susceptible and “persister” cells. Persisters tolerate cidal concentrations of antibiotics and return to an antibiotic-sensitive state once the antibiotic pressure is relieved. Starvation is known to contribute to persister formation. However, the mechanism(s) responsible for promoting Mtb persister formation during starvation are unknown.
Methods: We demonstrate that starved Mtb cultures contain at least 10-fold more persisters than exponentially growing cultures, suggesting the starvation response may mediate Mtb persister formation. To identify mechanisms involved in promoting Mtb persister formation during starvation, we took an unbiased high-throughput transposon (Tn) insertion sequencing approach, Tn-seq, to identify Tn mutants with decreased persister frequency during starvation. Using an arrayed library of approximately 4000 Mtb Tn mutants, we screened for persister phenotypes during starvation using Illumina sequencing. Pools containing ~500 Tn mutants were starved by growing to stationary phase or in Pi-limiting broth (input pool) and exposed to different anti-tubercular drug combinations for nine days prior to recovery of surviving cells on agar (output pool).
Results: Reads from input and output pools were mapped to the Mtb genome to determine individual Tn mutant fitness. Tn mutants with decreased fitness will be tested individually for their contribution to Mtb persister formation followed by complementation analyses.
Conclusions: Understanding Mtb persister formation could reveal new targets for drug development that would eliminate persisters, thus potentially shortening TB treatment. Knowledge gained from this work could also be applied to other difficult-to-treat pathogens infecting the head, neck, and oral cavity.
This abstract is based on research that was funded entirely or partially by an outside source:
NIH grants 1DP2AI112245-01 (NIAID) and T90 DE022732 (NIDCR)
The submitter must disclose the names of the organizations with which any author have a relationship, the nature of the relationship, and the clinical or research area involved. The following is submitted: NONE