Tydén Eva, Skarin Moa, Andersson-Franko Mikael, Sjöblom Matilda, Höglund Johan
Department of Biomedical Sciences and Veterinary Public Health, Division of Parasitology, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden.
Department of Biomedical Sciences and Veterinary Public Health, Division of Parasitology, Swedish University of Agricultural Sciences, S-750 07 Uppsala, Sweden.
Mol Biochem Parasitol. 2016 Jan-Feb;205(1-2):22-8. doi: 10.1016/j.molbiopara.2016.02.004. Epub 2016 Feb 12.
Anthelmintic resistance (AR) to macrocyclic lactones (ML) has been described in Parascaris of horses world-wide. In contrast, benzimidazoles (BZ) are still effective, although reduced efficacy to this drug class was recently reported. The mode of action of BZ is binding to β-tubulin, which prevents polymerisation of microtubules. In this study, β-tubulin gene expression of isotypes 1 and 2 was investigated at seven time points (0, 6, 24, 72, 96 and 120 h) during embryogenesis and in adult worms. In addition, an in ovo larval developmental test was developed to study β-tubulin gene expression of both isotypes in parasacaris eggs after exposure to different concentrations of thiabendazole (TBZ) for five days at 25 °C. A strong pattern of differential expression of β-tubulin and isotype 1 was observed in all stages, while isotype 2 expression was mainly found at an early phase of the embryogenesis. For isotype 1, a 5-fold increase was observed during the first 48 h, but gene expression gradually decreased after 72, 96 and 120 h. Isotype 2 was only expressed during the first 24h, followed by a 130-fold decrease at (time points) 72, 96 and 120 h. The in ovo larval developmental test, in which we exposed initially unembryonated eggs to increased concentrations of TBZ, did affect isotype 1 gene expression but not isotype 2. This assumes that each isotype has specific functions in different life stages. This is in agreement with the 'multi-tubulin' hypothesis, which states that different tubulin isotypes are required for specialised microtubule functions. Isotype 1 is the most likely drug target for BZs, as isotype 2 was only expressed at very low levels later in development. Increasing concentrations of TBZ altered β-tubulin isotype 1 gene expression after exposure of the eggs for five days, but this was not seen for isotype 2.
大环内酯类药物（ML）的抗蠕虫药耐药性（AR）在全球范围内的马副蛔虫中均有报道。相比之下，苯并咪唑类药物（BZ）仍然有效，尽管最近有报道称该类药物的疗效有所降低。BZ的作用方式是与β-微管蛋白结合，从而阻止微管聚合。在本研究中，在胚胎发育的七个时间点（0、6、24、72、96和120小时）以及成虫阶段，研究了同型1和同型2的β-微管蛋白基因表达。此外，开发了一种卵内幼虫发育试验，以研究在25℃下将不同浓度的噻苯达唑（TBZ）暴露五天后，副蛔虫卵中两种同型β-微管蛋白基因的表达情况。在所有阶段均观察到β-微管蛋白和同型1的强烈差异表达模式，而同型2的表达主要出现在胚胎发育的早期阶段。对于同型1，在最初的48小时内观察到5倍的增加，但在72、96和120小时后基因表达逐渐下降。同型2仅在最初的24小时内表达，随后在72、96和120小时（时间点）下降了130倍。在卵内幼虫发育试验中，我们将最初未受精的卵暴露于浓度不断增加的TBZ中，这确实影响了同型1基因的表达，但未影响同型2。这表明每种同型在不同的生命阶段具有特定的功能。这与“多微管蛋白”假说一致，该假说认为不同的微管蛋白同型对于特定的微管功能是必需的。同型1最有可能是BZ的药物靶点，因为同型2在发育后期仅以非常低的水平表达。在将卵暴露五天后，浓度不断增加的TBZ改变了β-微管蛋白同型1的基因表达，但同型2未出现这种情况。
