Animal Parasitic Diseases Laboratory, USDA-ARS, Beltsville, MD, USA.
Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee, USA.
Parasit Vectors. 2021 Nov 19;14(1):580. doi: 10.1186/s13071-021-05010-y.
Haemonchus contortus is a blood-feeding, gastrointestinal nematode (GIN) that causes significant economic losses to the small ruminant industry worldwide. Despite extensive efforts, our understanding of the molecular mechanisms used by GIN to evade host immune responses is limited. Cathepsin B-like proteins (CBPs) are members of the cysteine protease family and are involved in parasite invasion and thus provide viable vaccine candidates.
In silico comparative analysis was used to identify conserved proteins among a subset of clade V parasitic nematodes with emphasis on blood-feeding worms, among which CBPs appeared prominently. We identified and characterized two novel CBPs designated Hc-CBP-1 and Hc-CBP-2. Rabbit anti-recombinant (r) Hc-CBP-1 and rHc-CBP-2 were used to detect the presence of native proteins in the excretory secretory products (ESP) and in worm tissues of adult H. contortus. Peptide arrays of rHc-CBP-1 and rHc-CBP-2 were screened with the homologous and heterologous anti-sera and with sera from dexamethasone-treated (Dex) and non-treated (Dex) H. contortus-infected animals to identify key immunogenic peptides. Gene transcription of Hc-cbp-1 and Hc-cbp-2 was also performed on H. contortus-infected animals treated with Dex. Finally, the mature recombinant proteins were used to assess their abilities to modulate cell functions.
Immunohistochemistry showed that both Hc-CBP-1 and Hc-CBP-2 are present on the brush borders of the intestine; Hc-CBP-2 was also present in the hypodermis of the body wall. Peptide displays screened with rabbit anti-rHc-CBP-1 and anti-rHc-CBP-2 revealed regions within the proteins where dominant and overlapping epitopes prevailed. ELISA results were consistent with only Hc-CBP-1 being present in H. contortus adult ESPs. H. contortus from Dex animals exhibited a threefold increase in Hc-cbp-2 transcript while Hc-cbp-1 expression did not change. In contrast, comparisons of immunoreactivities of rHc-CBP-1 and rHc-CBP-2 peptide arrays to sera from Dex and Dex animals primarily showed changes in Hc-CBP-1 binding. Lastly, rHc-CBP-1 suppressed mRNA expression of bovine peripheral blood mononuclear cell cytokines/activation markers, including TNFα, IL-1, IL-6 and CD86.
These results suggest that as secreted and cryptic proteins, respectively, Hc-CBP-1 and Hc-CBP-2 influence cellular and immunological activities that have interesting dynamics during infection and may provide viable immune-related targets for attenuating H. contortus infectivity.
捻转血矛线虫是一种吸血的胃肠道线虫（GIN），它给全球小反刍动物产业造成了巨大的经济损失。尽管已经进行了广泛的研究，但我们对 GIN 逃避宿主免疫反应所使用的分子机制的理解仍然有限。组织蛋白酶 B 样蛋白（CBPs）是半胱氨酸蛋白酶家族的成员，参与寄生虫的入侵，因此是可行的疫苗候选物。
通过计算机分析比较，从一组亚目寄生虫线虫中鉴定出保守蛋白，重点是吸血线虫，其中 CBPs 尤为突出。我们鉴定并表征了两种新的 CBP，分别命名为 Hc-CBP-1 和 Hc-CBP-2。兔抗重组（r）Hc-CBP-1 和 rHc-CBP-2 用于检测捻转血矛线虫成虫的排泄分泌产物（ESP）和虫体组织中天然蛋白的存在。rHc-CBP-1 和 rHc-CBP-2 的肽阵列用同源和异源抗血清以及地塞米松处理（Dex）和未处理（Dex）捻转血矛线虫感染动物的血清进行筛选，以鉴定关键免疫原性肽。还对 Dex 处理的捻转血矛线虫感染动物进行了 Hc-cbp-1 和 Hc-cbp-2 的基因转录。最后，使用成熟的重组蛋白来评估它们调节细胞功能的能力。
免疫组织化学显示，Hc-CBP-1 和 Hc-CBP-2 均存在于肠道的刷状缘上；Hc-CBP-2 也存在于体壁的皮下组织中。用兔抗 rHc-CBP-1 和 rHc-CBP-2 筛选肽显示，蛋白质内存在优势和重叠表位。ELISA 结果与 H. contortus 成虫 ESP 中仅存在 Hc-CBP-1 一致。地塞米松处理的捻转血矛线虫中 Hc-cbp-2 的转录增加了三倍，而 Hc-cbp-1 的表达没有变化。相比之下，用 Dex 和 Dex 动物的血清对 rHc-CBP-1 和 rHc-CBP-2 肽阵列的免疫反应性进行比较，主要显示 Hc-CBP-1 结合的变化。最后，rHc-CBP-1 抑制了牛外周血单核细胞细胞因子/激活标志物的 mRNA 表达，包括 TNFα、IL-1、IL-6 和 CD86。
这些结果表明，Hc-CBP-1 和 Hc-CBP-2 分别作为分泌蛋白和隐蔽蛋白，影响细胞和免疫活性，在感染过程中具有有趣的动态变化，并且可能为减轻捻转血矛线虫感染提供可行的免疫相关靶点。
