Resistance formation to nitro drugs in Giardia lamblia: No common markers identified by comparative proteomics

Abstract

In order to elucidate the question whether resistance to nitro drugs in G. lamblia is due to common resistance markers, trophozoites of three resistant G. lamblia strains, namely C4, 1062ID10, and 713M3 were grown in the presence of the two nitro drugs metronidazole and nitazoxanide and compared to their corresponding wild-types WBC6, 106, and 713 by mass spectometry shotgun analysis of their proteomes. Depending on the strain and the nitro drug, more than 200 to 500 differentially expressed proteins were identified, but there were no common patterns across strains and drugs. All resistant strains underwent antigenic variation with distinct surface antigens like variant surface proteins or cysteine rich proteins depending on strain and nitro compound. A closer look on enzymes involved in nitroreduction and detoxification of nitro radicals, NO or O₂ suggested the existence of distinct strategies for each drug and each strain. Therefore, we conclude that resistance to nitro drugs in G. lamblia is not correlated with a specific pattern of differentially expressed proteins and therefore seems not to be the result of a directed process.

Keywords: Adaptation; Drug susceptibility; Resistance; Tolerance.

Graphical abstract

Fig. 1

Principal component analysis of proteome data set from nitro resistantG. lambliatrophozoites (C4, 10621D10, 713M3) and their corresponding wild-types (WBC6, 106, 713). Trophozoites of the resistant strains were grown in the presence of 50 μM metronidazole (MET; red symbols) or 50 μM nitazoxanide (NTZ; blue symbols) and compared to their respective wild-types (green symbols) by MS shotgun analysis as described in Materials and methods. For each strain, all technical and biological (square, circle, diamond) replicates are shown. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Venn diagram detailing the number of differentially expressed proteins in trophozoites of wild-type (WT) and nitro drug-resistant (RES) strains. Trophozoites of the resistant strains were grown in the presence of metronidazole (MET) or nitazoxanide (NTZ; both 50 μM) and subjected to MS shotgun analysis as described in Materials and methods.

Fig. 3

Quantitative assessments of the major surface antigens. Trophozoites of the resistant strains were grown in the presence of metronidazole (MET) or nitazoxanide (NTZ; both 50 μM) and subjected to MS shotgun analysis as described in Materials and methods. For all proteins, mean values ± standard errors for LFQ intensities in three biological replicates are shown.