Membrane associated proteins of two Trichomonas gallinae clones vary with the virulence

Oropharyngeal avian trichomonosis is mainly caused by Trichomonas gallinae, a protozoan parasite that affects the upper digestive tract of birds. Lesions of the disease are characterized by severe inflammation which may result in fatality by starvation. Two genotypes of T. gallinae were found to be widely distributed in different bird species all over the world. Differences in the host distribution and association with lesions of both genotypes have been reported. However, so far no distinct virulence factors of this parasite have been described and studies might suffer from possible co-infections of different genotypes. Therefore, in this paper, we analyzed the virulence capacity of seven clones of the parasite, established by micromanipulation, representing the two most frequent genotypes. Clones of both genotypes caused the maximum score of virulence at day 3 post-inoculation in LMH cells, although significant higher cytopathogenic score was found in ITS-OBT-Tg-1 genotype clones at days 1 and 2, as compared to clones with ITS-OBT-Tg-2. By using one representative clone of each genotype, a comparative proteomic analysis of the membrane proteins enriched fraction has been carried out by a label free approach (Data available via ProteomeXchange: PXD013115). The analysis resulted in 302 proteins of varying abundance. In the clone with the highest initial virulence, proteins related to cell adhesion, such as an immuno-dominant variable surface antigen, a GP63-like protein, an armadillo/beta-catenin-like repeat protein were found more abundant. Additionally, Ras superfamily proteins and calmodulins were more abundant, which might be related to an increased activity in the cytoskeleton re-organization. On the contrary, in the clone with the lowest initial virulence, larger numbers of the identified proteins were related to the carbohydrate metabolism. The results of the present work deliver substantial differences between both clones that could be related to feeding processes and morphological changes, similarly to the closely related pathogen Trichomonas vaginalis.

禽口咽部毛滴虫病主要由禽毛滴虫（Trichomonas gallinae）引起，该原生动物寄生虫可侵染鸟类的上消化道。该病的病灶以严重炎症为特征，严重时可导致宿主因无法进食而死亡。目前已发现禽毛滴虫的两种基因型在全球范围内不同鸟类种群中广泛分布，已有研究报道两种基因型在宿主分布及其与病灶的关联方面存在差异。然而截至目前，尚未明确该寄生虫的特异性毒力因子，且相关研究可能受到不同基因型共感染的干扰。为此，本研究通过显微操作技术构建了代表两种最常见基因型的7株该寄生虫克隆，并对其毒力能力进行了分析。两种基因型的克隆在接种LMH细胞后第3天达到最大毒力评分；相较于ITS-OBT-Tg-2型克隆，ITS-OBT-Tg-1型克隆在接种后第1、2天的细胞致病评分显著更高。本研究选取每种基因型的1株代表性克隆，采用无标记定量蛋白质组学方法对其膜蛋白富集组分进行了比较蛋白质组学分析（相关数据已上传至ProteomeXchange数据库，编号PXD013115）。本次分析共鉴定得到302个丰度存在差异的蛋白质。在初始毒力最高的克隆中，与细胞黏附相关的蛋白质（如免疫显性可变表面抗原、GP63样蛋白、犰狳/β-连环蛋白样重复蛋白）丰度显著更高；此外，Ras超家族蛋白及钙调蛋白的丰度也显著升高，这可能与细胞骨架重排活性增强有关。与之相反，在初始毒力最低的克隆中，鉴定得到的蛋白质更多与碳水化合物代谢相关。本研究结果表明，两株克隆之间存在显著差异，这些差异可能与营养摄取过程及形态变化相关，这一点与亲缘关系相近的病原体阴道毛滴虫（Trichomonas vaginalis）相似。