Incorporation of macrophage immune stresses into an assay for drug tolerance in intracellular Mycobacterium tuberculosis

Abstract

Development of new and improved tuberculosis (TB) chemotherapies is hampered by antibiotic resistance and drug tolerance by Mycobacterium tuberculosis (Mtb). Phenotypic drug tolerance, a phenomenon where Mtb populations 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 tolerant Mtb are urgently required to improve TB treatment regimens. Here, we report the development of a 96-well plate assay capable of identifying anti-Mtb drugs with activity against drug tolerant Mtb in 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 an Mtb strain constitutively expressing luciferase. Following drug exposure, differences in bacterial survival were measured by bacterial outgrowth after lysis of the host macrophages. By monitoring Mtb luciferase 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 the Mtb sub-populations in the infected host. Author summaryTB treatment is a lengthy process which at minimum takes 6-9 months in cases of drug sensitive Mtb. 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 tolerant Mtb inside 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 bacteria in vivo. By infecting the modified macrophages with an Mtb strain expressing luciferase followed by drug addition, our assay was able to confirm that immune related stress induce drug tolerance to Mtb, 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.