Project Summary/Abstract Infectious disease treatment failure is a critical health issue. It is predicted to become the leading cause of mortality by 2050, superseding cancer. Treatment failure can be caused by a combination of factors, including active antimicrobial drug resistance, for example via efflux pumps, and pathogen dormancy, tolerance and persistence mechanisms. However, these factors cannot alone account for all cases of treatment failure. Indeed, bioavailability and pharmacokinetics are the 3rd most common cause of failure during drug development. Pharmacokinetic studies, however, have predominantly focused on biofluid and tissue drug levels. This is insufficient in the case of intracellular pathogens, where intracellular drug accumulation is critical to ensure pathogen clearance. Single-cell mass spectrometry (SCMS) studies in the field of cancer drug development have revealed extensive heterogeneity in intracellular drug levels. However, until now, the technology and techniques to safely perform such analyses in the context of intracellular pathogen infection have been lacking. This is a critical gap in our ability to understand infectious disease treatment failure and to guide infectious disease drug development. To address this gap, proposal MPIs have developed a biosafety- compatible SCMS method to quantify intracellular drug and metabolite levels in the context of Trypanosoma cruzi parasite infection. The overall objective of this proposal is to demonstrate the broad applicability of this method to determine the relationship between lower intracellular drug levels and drug metabolism vs failure to clear intracellular pathogens. The central hypothesis of this proposal is that heterogenous intracellular drug levels and intracellular drug metabolism is a key contributor to antimicrobial treatment failure, and that this mechanism can be revealed using novel SCMS approaches with broad applicability. We will test this central hypothesis using three complementary yet independent aims. Aim 1 will use a Trypanosoma cruzi parasite infection system to relate heterogenous antiparasitic drug levels between cells, to in vitro T. cruzi treatment failure. Aim 2 will use the same T. cruzi infection system to relate heterogenous antiparasitic drug metabolism between cells, to in vitro T. cruzi treatment failure. Aim 3 will broaden the applicability of these SCMS techniques to quantify specific antibacterials and antivirals, and to relate their levels to treatment failure in the context of bacterial and viral infection. The proposed research is innovative because it will lead to the development of a new bioanalytical technology to address the critical biological problem of antimicrobial treatment failure. The proposed research is significant because it will lead to an expanded understanding of the mechanisms of treatment failure in infectious diseases.