Date Published: June 7, 2018
Publisher: Public Library of Science
Author(s): Abigail L. Reens, Amy L. Crooks, Chih-Chia Su, Toni A. Nagy, David L. Reens, Jessica D. Podoll, Madeline E. Edwards, Edward W. Yu, Corrella S. Detweiler, Samuel I. Miller.
Bacterial efflux pumps transport small molecules from the cytoplasm or periplasm outside the cell. Efflux pump activity is typically increased in multi-drug resistant (MDR) pathogens; chemicals that inhibit efflux pumps may have potential for antibiotic development. Using an in-cell screen, we identified three efflux pump modulators (EPMs) from a drug diversity library. The screening platform uses macrophages infected with the human Gram-negative pathogen Salmonella enterica (Salmonella) to identify small molecules that prevent bacterial replication or survival within the host environment. A secondary screen for hit compounds that increase the accumulation of an efflux pump substrate, Hoechst 33342, identified three small molecules with activity comparable to the known efflux pump inhibitor PAβN (Phe-Arg β-naphthylamide). The three putative EPMs demonstrated significant antibacterial activity against Salmonella within primary and cell culture macrophages and within a human epithelial cell line. Unlike traditional antibiotics, the three compounds did not inhibit bacterial growth in standard microbiological media. The three compounds prevented energy-dependent efflux pump activity in Salmonella and bound the AcrB subunit of the AcrAB-TolC efflux system with KDs in the micromolar range. Moreover, the EPMs display antibacterial synergy with antimicrobial peptides, a class of host innate immune defense molecules present in body fluids and cells. The EPMs also had synergistic activity with antibiotics exported by AcrAB-TolC in broth and in macrophages and inhibited efflux pump activity in MDR Gram-negative ESKAPE clinical isolates. Thus, an in-cell screening approach identified EPMs that synergize with innate immunity to kill bacteria and have potential for development as adjuvants to antibiotics.
Human pathogens have become increasingly resistant to clinical antibiotics. Gram-negative bacterial pathogens are particularly problematic because their outer membranes are impermeable to many chemicals, and because many compounds that do enter the periplasm or cross the cellular membrane are immediately exported by efflux pumps. Multi-drug resistant (MDR) bacteria typically have increased gene copy number and/or production of efflux pumps, features demonstrated to contribute to the failure of clinical antibiotic treatment . For these reasons, compounds that reduce efflux pump activity (efflux pump modulators, EPMs) are under investigation for their potential use in re-sensitizing MDR pathogens to existing antibiotics .
In-cell screens aimed at identifying chemicals that prevent pathogen intracellular replication have been described. For example screens of FDA-approved drug libraries have uncovered modulators of Listeria monocytogenes or Salmonella enterica infection [57,58]. Similar screens were performed with macrophages infected with Mycobacterium tuberculosis  or the yeast Cryptococcus neoformans . There are also many reports of high-content screens aimed at identifying genetic disruptors of host-pathogen interactions [61–63]. SAFIRE adopts what we thought would be the most useful aspects of these earlier studies, including a GFP-expressing Salmonella to track the microbe, the addition of compounds after infection, automated fluorescence microscopy-based image analysis with MATLAB, and estimation of compound toxicity based on manual visualization of cell morphology and MitoTracker staining. In addition, because the SAFIRE assay includes serum, it avoids compounds that are poor candidates for drug development because of high affinity for serum proteins . These features enabled us to develop SAFIRE as a medium-throughput assay useful for identifying small molecules that interfere with the host-pathogen relationship.