Differences in protein expression associated with ivermectin resistance in Caenorhabditis elegans.

Dauana Mesquita Sousa,Claudia Maria Leal Bevilaqua,Rodrigo Rodrigues Cambraia De Miranda,Paulo De Tarso Teles Dourado De Aragão Aragão,Mônica Valéria De Almeida Aguiar,Rodrigo Maranguape Silva Da Cunha,Deisianne Rodrigues Da Silva,Ana Cristina De Oliveira Monteiro Moreira,Nivea Maria Ferreira Da Cunha,Marina Duarte Pinto Lobo

doi:10.1590/s1984-29612019013

Abstract

The indiscriminate administration of synthetic anthelmintics such as ivermectin contributes to the selection of subpopulations capable of resisting the drugs' effects. To understand the mechanisms of ivermectin resistance in Caenorhabditis elegans, this study attempted to identify molecular targets. C. elegans lineages that were sensitive and resistant to ivermectin were used. Collected nematodes were added to an extraction buffer and macerated in liquid nitrogen for protein extraction. The extracted proteins were separated according to molecular weight by SDS-PAGE to verify their integrity. Subsequently, proteins from both lineages were separated using two-dimensional electrophoresis. The gels were analyzed and the relevant spots were excised and identified by mass spectrometry (NanoESI-Q-TOF and MASCOT®) and subsequently assessed by GO enrichment and STRING® analyses. The increased expression of proteins associated with high metabolic activity, such as ATP-2 and ENOL-1, which are responsible for ATP synthesis, was observed. Furthermore, proteins with involvement in mediating muscular function (MLC-1, ACT-1, and PDI-2), signaling (FAR-1 and FAR-2), and embryo development (VHA-2) were identified. Protein interaction analysis indicated that the majority of the identified proteins in the resistant lineages participated in the same reaction triggered by ivermectin.

Highlights

Anthelmintic resistance is a problem that plagues livestock production worldwide, causing a billion dollars in losses annually (CHARLIER et al, 2009)
Ivermectin follows the parameters recommended for good anthelmintics, resistance to this drug was reported in the first years after its launch (VAN WYK & MALAN, 1988).There is no explanation to date for ivermectin resistance, only hypotheses that consider changes in the chlorine receptor channels and p-glycoproteins, which are membrane proteins responsible for the cellular efflux of drugs (LESPINE et al, 2012)
It is possible to identify these proteins in proteomic studies, such as two-dimensional electrophoresis (2DE) associated with mass spectrometry, which emphasizes differences in expressiveness and concentrations (HART et al, 2015)

Summary

Introduction

Anthelmintic resistance is a problem that plagues livestock production worldwide, causing a billion dollars in losses annually (CHARLIER et al, 2009). This problem is caused by the parasites’ ability to survive the drugs used to treat helminthiasis (GOOLSBY et al, 2017). Several protein changes occur in response to stress caused by various factors, such as temperature, food, radiation exposure, and chemicals (CEDERGREEN et al, 2016). These changes can even be caused by genetic selection of the subpopulation to resist a stress environment promoted by drug compounds. It is possible to identify these proteins in proteomic studies, such as two-dimensional electrophoresis (2DE) associated with mass spectrometry, which emphasizes differences in expressiveness and concentrations (HART et al, 2015)

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