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.
