Crustacean Molecular Biology and Genomics Division, Biomaterials and Biotechnology in Animal Health Lab, Department of Animal Health and Management, Alagappa University, Karaikudi, 630 004, Tamil Nadu, India.
Centre for Animal Science Research and Extension Services, Foundation for Innovative Research in Science and Technology, Kelavannanvilai, NGO Colony Road, Nagercoil, Tamil Nadu, India.
Microb Pathog. 2020 Oct;147:104253. doi: 10.1016/j.micpath.2020.104253. Epub 2020 Jun 25.
Haemocyanin (Hc) is a non-specific innate immune protein present in the haemolymph of arthropods and molluscs. In the current study, we characterized the structural and immunological properties of Hc from grooved tiger shrimp, Penaeus semisulcatus. Hc was isolated from the haemolymph of P. semisulcatus by gel filtration column chromatography using Sephadex G-100. High-performance liquid chromatography of the purified Hc emerged as a single peak through a retention time of 3.3 min demonstrating the homogeneity nature of the protein. X-ray diffraction analysis revealed a distinct peak at 31.7° indicating the crystalline character of the purified Hc. Circular dichroism spectra of the purified Hc displayed negative ellipticity bands close to 225 nm and 208 nm representing β-sheet secondary structure. The purified Hc agglutinated sheep RBCs, yeast Saccharomyces cerevisiae and fungal Candida albicans. In addition, the purified Hc displayed antibacterial activity against Gram-positive bacteria (Bacillus thuringiensis and Bacillus pumilis) and Gram-negative bacteria (Vibrio parahaemolyticus and Vibrio alginolyticus) with a minimal inhibitory concentration of 50 μg/ml. Antibiofilm activity revealed the potential of purified Hc to inhibit biofilm formation of both Gram-positive and Gram-negative bacterial pathogens. Furthermore, live/dead staining of biofilms demonstrated the reduced viability of bacterial cells after exposure to the purified Hc. In silico molecular modeling was carried out using the sequence of Hc from SwissProt and molecular docking was performed with the cell surface components found in Gram-positive and Gram-negative bacteria. Overall our study demonstrates the involvement of Hc in the native immune reaction of P. semisulcatus by eliciting pathogen recognition. Thus, Hc could enhance disease resistance against pathogenic infection in shrimp aquaculture.
血蓝蛋白（Hc）是存在于节肢动物和软体动物血淋巴中的一种非特异性先天免疫蛋白。在本研究中，我们对斑节对虾（Penaeus semisulcatus）的 Hc 的结构和免疫学特性进行了表征。Hc 从斑节对虾的血淋巴中通过 Sephadex G-100 凝胶过滤柱层析分离得到。通过保留时间为 3.3 分钟的高效液相色谱，纯化的 Hc 呈现单一峰，表明该蛋白质具有均一性。X 射线衍射分析表明，在 31.7°处出现明显的峰，表明纯化的 Hc 具有结晶性质。纯化的 Hc 的圆二色性光谱显示负椭圆率带接近 225nm 和 208nm，代表β-折叠二级结构。纯化的 Hc 凝集绵羊 RBCs、酵母 Saccharomyces cerevisiae 和真菌 Candida albicans。此外，纯化的 Hc 对革兰氏阳性菌（苏云金芽孢杆菌和短小芽孢杆菌）和革兰氏阴性菌（副溶血弧菌和溶藻弧菌）显示出抗菌活性，最小抑菌浓度为 50μg/ml。抗生物膜活性表明，纯化的 Hc 具有抑制革兰氏阳性和革兰氏阴性细菌病原体生物膜形成的潜力。此外，生物膜的死活染色表明，暴露于纯化的 Hc 后，细菌细胞的活力降低。使用 SwissProt 中的 Hc 序列进行了计算机分子建模，并与革兰氏阳性和革兰氏阴性细菌中的细胞表面成分进行了分子对接。总体而言，我们的研究表明，Hc 通过引发病原体识别参与了斑节对虾的天然免疫反应。因此，Hc 可以增强虾类养殖中对致病性感染的抗病能力。
