CF Research Translation Center and Research Development Program

University of Washington
UW Health Sciences, K-140
Genome Sciences, Box 357710
Seattle, WA 98195

RDP Pilot: Efflux Pumps in Nontuberculosis Mycobacteria

P.I.: Rafael Hernandez, MD, PhD
Assistant Professor,

Nontuberculosis mycobacteria (NTM) are environmental organisms that can cause pulmonary and other infections in susceptible hosts. Cystic fibrosis (CF) patients are at increased risk for NTM infections and recent epidemiologic studies suggest that the prevalence of NTM disease caused by both Mycobacterium avium complex (MAC) and M. abscessus (MABSC) in patients with CF may be increasing. The rapidly growing mycobacteria of the M. abscessus group are of particular concern as they can be associated with rapid decline in lung function and increased risk for poor outcomes following lung transplant. Like treatment for tuberculosis (TB), NTM infections require treatment with many months of potentially toxic antibiotics. There are a very limited number of antibiotics active against NTM, especially for M. abscessus, cure rates are low, and relapse is common. Therefore, new strategies are desperately needed for treatment of NTM infections; targeting bacterial efflux pumps (EPs) represents one such strategy. EPs are membrane transporters with promiscuous substrate specificity, which have been implicated in both intracellular survival and antibiotic tolerance in M. tuberculosis (Mtb). Furthermore, many of the EPs are conserved between Mtb and related NTM pathogens. Recent data from cell and animal models for TB suggest that the addition of efflux pump inhibitors to standard antibiotic regimens offers the potential for shorter and more efficacious treatment courses. The goal of this pilot project is test the hypotheses that EPs conserved between Mtb and NTM serve as virulence factors in NTM, and that EPs mediate both NTM antibiotic tolerance and NTM resistance to killing by macrophages.

Aim 1. Determine the expression dynamics of mycobacterial efflux pumps in NTM pathogens.
Hypothesis: Intracellular residence of NTM induces expression of mycobacterial efflux pumps.
Approach: We will infect a human macrophage cell line with either MAC or M. abscessus complex and isolate bacterial mRNA for RNAseq evaluation of expression of efflux pumps. We then will determine expression of efflux pumps in bacteria isolated directly from sputum to correlate gene expression in our cell culture model with expression of NTM efflux pumps within patients.

Aim 2. Determine whether efflux pump inhibitors will block intracellular growth or survival of NTM pathogens.
Hypothesis: NTM depend on efflux pumps for intracellular survival and/or growth.
Approach: We will infect a human macrophage cell line with MAC or M. abscessus, using both reference and patient derived strains and then treat infected cells with efflux pump inhibitors. Bacterial growth/survival will be assayed by enumerating colony-forming units.

Aim 3. Determine if efflux pumps contribute to either intrinsic antibiotic resistance or induced tolerance in NTM pathogens.
Hypothesis: Efflux pumps contribute to the broad intrinsic resistance of NTM pathogens and also underlie the poor correlation between in vitro susceptibility to antibiotics and in vivo activity.

Approach: We will test the activity of antibiotics with and without the addition of efflux pump inhibitors both in vitro (broth culture) and in infected macrophages. We will also test whether growth of NTM within biofilms induces antibiotic tolerance and whether efflux pump inhibitors can overcome this tolerance.