Departamento de Parasitología, Facultad de Farmacia, Universidad de Granada, 18071 Granada, Spain.
Vet Parasitol. 2011 Dec 29;183(1-2):95-102. doi: 10.1016/j.vetpar.2011.07.002. Epub 2011 Jul 12.
Proteases have a significant role in the life cycle of parasites and the pathogen-host relationship, being regarded as important virulence factors. In the parasitic nematode Hysterothylacium aduncum proteolytic activity was measured during in vitro development from third larval stage (L3) to mature adult, using DQ red casein as a fluorogenic substrate. Proteolytic activity was detected in all the developmental stages studied and at all pH values within the range employed (2.0-7.5). The assay with specific inhibitors permitted the determination of metalloprotease activity, and, to a lesser extent, that of aspartate- and cysteine-protease. Serine-protease activity was the lowest of those studied. In L3 recently collected from the host fish (L3-0 h), the greatest activity was found at an optimum pH of 4.0 and was mainly inhibited by 1,10-phenathroline (metalloprotease inhibitor). This metalloprotease activity in L3-0 h (infective stage) may be related to the invasion of the host tissues by this larva. In the other developmental stages, the greatest protease activity was found at pH 5.5, although at pH 4.0 a lower activity peak was detected. On the other hand, our data show that the proteolytic activity of the nematode varies according to the presence of pepsin (an aspartic-protease) in the culture medium. Thus, at pH 4.0, activity was greater in the absence of pepsin, with increasing aspartic-protease activity. Together with the detection of aspartic-, cysteine- and metallo-protease (enzymes involved in digestion in invertebrates) in all the developmental stages of the parasite taking place in the digestive tract of the host fish, this allows us to suggest that the pepsin in the culture medium mimics the predigestion conditions in the habitat of the worm within the host and that the activity detected may have, amongst others, a digestive function.
蛋白酶在寄生虫的生命周期和病原体-宿主关系中起着重要作用，被认为是重要的毒力因子。在寄生线虫海鞘弯曲杆菌中，使用 DQ 红色酪蛋白作为荧光底物，测量了从第三幼虫期（L3）到成熟成虫的体外发育过程中的蛋白水解活性。在研究的所有发育阶段和所采用的 pH 值范围内（2.0-7.5）都检测到了蛋白水解活性。使用特异性抑制剂的测定允许确定金属蛋白酶活性，并且在较小程度上确定天冬氨酸和半胱氨酸蛋白酶活性。研究中发现丝氨酸蛋白酶活性最低。在最近从宿主鱼中收集的幼虫（L3-0 h）中，在最适 pH 值为 4.0 时发现最大活性，并且主要被 1,10-邻菲咯啉（金属蛋白酶抑制剂）抑制。这种 L3-0 h（感染阶段）中的金属蛋白酶活性可能与该幼虫对宿主组织的侵袭有关。在其他发育阶段，最大蛋白酶活性在 pH 5.5 时发现，尽管在 pH 4.0 时检测到较低的活性峰。另一方面，我们的数据表明，根据培养基中胃蛋白酶（一种天冬氨酸蛋白酶）的存在，线虫的蛋白水解活性会发生变化。因此，在 pH 4.0 时，在没有胃蛋白酶的情况下活性更大，天冬氨酸蛋白酶活性增加。与在宿主鱼的消化道中发生的寄生虫所有发育阶段中检测到的天冬氨酸、半胱氨酸和金属蛋白酶（参与无脊椎动物消化的酶）一起，这使我们可以假设培养基中的胃蛋白酶模拟了蠕虫在宿主体内栖息地的预消化条件，并且检测到的活性可能具有消化功能等。
