Incorporation of macrophage immune stresses into an assay for drug tolerance in intracellularMycobacterium tuberculosis
Abstract
Development of new and improved tuberculosis (TB) chemotherapies is hampered by antibiotic resistance and drug tolerance byMycobacterium tuberculosis(Mtb). Phenotypic drug tolerance, a phenomenon whereMtbpopulations can temporarily survive therapeutic antibiotic concentrations, represents a significant hurdle to TB treatment and is indeed one of the factors responsible for prolonged TB therapy. Assays that can identify compounds with improved efficacy against drug tolerantMtbare urgently required to improve TB treatment regimens. Here, we report the development of a 96-well plate assay capable of identifying anti-Mtbdrugs with activity against drug tolerantMtbin physiologically relevant intracellular environments within macrophages. Primary murine macrophages modified either by immunological activation or specific CRISPR/Cas9 gene knockouts to generate tolerance-inducing environments were infected with anMtbstrain constitutively expressing luciferase. Following drug exposure, differences in bacterial survival were measured by bacterial outgrowth after lysis of the host macrophages. By monitoringMtbluciferase in infected macrophages before, during and after drug treatment, we confirmed earlier observations that host immune stresses trigger induction of drug tolerance. However, while host stresses induced tolerance against some anti-TB compounds, the same host stresses were synergistic with other anti-TB drugs. Our assay provides the ability to profile the activities of anti-TB drugs on bacteria in intracellular host environments which is critical to the rational design of drug combinations that provide optimal coverage of theMtbsub-populations in the infected host.
Author summary
TB treatment is a lengthy process which at minimum takes 6-9 months in cases of drug sensitiveMtb. Long TB treatments are in part, due to drug tolerance phenotypes in the bacterial population which can be a result of immune related stresses on the bacteria in the infected host cells. We used this knowledge to develop a luciferase-based assay which can be used to screen, optimize and discriminate anti-TB drugs with enhanced activity against drug tolerantMtbinside the infected host cells. Primary murine macrophages were modified either by cytokine activation or genetic knockout of certain host genes to mimic immune related stresses experienced by the bacteriain vivo. By infecting the modified macrophages with anMtbstrain expressing luciferase followed by drug addition, our assay was able to confirm that immune related stress induce drug tolerance toMtb, and that the drug tolerance phenotypes are unique to certain drug classes consistent with their known mode of action. The assay provides an important addition to anti-TB drug discovery by providing a means to readily screen new drugs and drug combinations that display improved activity in tolerance inducing environments in a physiologically relevant context.
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