Abstract
BackgroundMetronidazole (Mtz) is the frontline drug treatment for multiple anaerobic pathogens, including the gastrointestinal protist, Giardia duodenalis. However, treatment failure is common and linked to in vivo drug resistance. In Giardia, in vitro drug-resistant lines allow controlled experimental interrogation of resistance mechanisms in isogenic cultures. However, resistance-associated changes are inconsistent between lines, phenotypic data are incomplete, and resistance is rarely genetically fixed, highlighted by reversion to sensitivity after drug selection ceases or via passage through the life cycle. Comprehensive quantitative approaches are required to resolve isolate variability, fully define Mtz resistance phenotypes, and explore the role of post-translational modifications therein.FindingsWe performed quantitative proteomics to describe differentially expressed proteins in 3 seminal Mtz-resistant lines compared to their isogenic, Mtz-susceptible, parental line. We also probed changes in post-translational modifications including protein acetylation, methylation, ubiquitination, and phosphorylation via immunoblotting. We quantified more than 1,000 proteins in each genotype, recording substantial genotypic variation in differentially expressed proteins between isotypes. Our data confirm substantial changes in the antioxidant network, glycolysis, and electron transport and indicate links between protein acetylation and Mtz resistance, including cross-resistance to deacetylase inhibitor trichostatin A in Mtz-resistant lines. Finally, we performed the first controlled, longitudinal study of Mtz resistance stability, monitoring lines after cessation of drug selection, revealing isolate-dependent phenotypic plasticity.ConclusionsOur data demonstrate understanding that Mtz resistance must be broadened to post-transcriptional and post-translational responses and that Mtz resistance is polygenic, driven by isolate-dependent variation, and is correlated with changes in protein acetylation networks.
Highlights
Metronidazole (Mtz) is the frontline drug treatment for multiple anaerobic pathogens, including the gastrointestinal protist, Giardia duodenalis
A nonredundant total of 1,571 proteins was identified across the tandem mass tags (TMT) 10plexes analyzed, with 1,220, 1,126, and 1,060 proteins identified in 10plex 1–3, respectively (Table 2)
Substantial genotypic variation was found in differentially expressed protein (DEP) and post-translational marks
Summary
Metronidazole (Mtz) is the frontline drug treatment for multiple anaerobic pathogens, including the gastrointestinal protist, Giardia duodenalis. Our data confirm substantial changes in the antioxidant network, glycolysis, and electron transport and indicate links between protein acetylation and Mtz resistance, including cross-resistance to deacetylase inhibitor trichostatin A in Mtz-resistant lines. Nitroheterocyclics (e.g., metronidazole [Mtz], nitazoxanide, and furazolidone) include “redox-active” pro-drugs that cross the cell membrane via passive diffusion and are enzymatically reduced to cytotoxic intermediates that oxidize biomolecules. This occurs within highly reducing intracellular environments of microaerophilic protists (Giardia duodenalis, Trichomononas vaginalis, and Entameoba histolytica) as well as anaerobic bacteria (Helicobacter pylori, Clostridium difficile, and Bacteroides fragilis) [1, 2]. The efficacy of frontline Mtz treatment ranges from 73 to 100% in Giardia [7], with clinical resistance confirmed [7, 8] and increasing in incidence [9]
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