Tonk Miray, Cabezas-Cruz Alejandro, Valdés James J, Rego Ryan O M, Chrudimská Tereza, Strnad Martin, Šíma Radek, Bell-Sakyi Lesley, Franta Zdeněk, Vilcinskas Andreas, Grubhoffer Libor, Rahnamaeian Mohammad
Biology Centre of the AS CR, Institute of Parasitology, Branišovská 31, 37005, České Budějovice, Czech Republic.
Faculty of Science, University of South Bohemia, Branišovská 31, 37005, České Budějovice, Czech Republic.
Parasit Vectors. 2014 Dec 3;7:554. doi: 10.1186/s13071-014-0554-y.
Ixodes scapularis is the most common tick species in North America and a vector of important pathogens that cause diseases in humans and animals including Lyme disease, anaplasmosis and babesiosis. Tick defensins have been identified as a new source of antimicrobial agents with putative medical applications due to their wide-ranging antimicrobial activities. Two multigene families of defensins were previously reported in I. scapularis. The objective of the present study was to characterise the potential antimicrobial activity of two defensins from I. scapularis with emphasis on human pathogenic bacterial strains and important phytopathogenic fungi.
Scapularisin-3 and Scapularisin-6 mature peptides were chemically synthesised. In vitro antimicrobial assays were performed to test the activity of these two defensins against species of different bacterial genera including Gram-positive bacteria Staphylococcus aureus, Staphylococcus epidermidis, and Listeria spp. as well as Gram-negative bacteria Escherichia coli, Pseudomonas aeruginosa along with two plant-pathogenic fungi from the genus Fusarium. In addition, the tissue-specific expression patterns of Scapularisin-3 and Scapularisin-6 in I. scapularis midgut, salivary glands and embryo-derived cell lines were determined using PCR. Finally, tertiary structures of the two defensins were predicted and structural analyses were conducted.
Scapularisin-6 efficiently killed L. grayi, and both Scapularisin-3 and Scapularisin-6 caused strong inhibition (IC50 value: ~1 μM) of the germination of plant-pathogenic fungi Fusarium culmorum and Fusarium graminearum. Scapularisin-6 gene expression was observed in I. scapularis salivary glands and midgut. However, Scapularisin-3 gene expression was only detected in the salivary glands. Transcripts from the two defensins were not found in the I. scapularis tick cell lines ISE6 and ISE18.
Our results have two main implications. Firstly, the anti-Listeria and antifungal activities of Scapularisin-3 and Scapularisin-6 suggest that these peptides may be useful for (i) treatment of antibiotic-resistant L. grayi in humans and (ii) plant protection. Secondly, the antimicrobial properties of the two defensins described in this study may pave the way for further studies regarding pathogen invasion and innate immunity in I. scapularis.
肩突硬蜱是北美最常见的蜱种，也是包括莱姆病、无形体病和巴贝斯虫病在内的多种可导致人类和动物疾病的重要病原体的传播媒介。蜱防御素因其广泛的抗菌活性，已被确定为具有潜在医学应用价值的新型抗菌剂来源。此前在肩突硬蜱中报道了两个防御素多基因家族。本研究的目的是表征来自肩突硬蜱的两种防御素的潜在抗菌活性，重点关注人类致病细菌菌株和重要的植物致病真菌。
化学合成了肩突硬蜱防御素-3（Scapularisin-3）和肩突硬蜱防御素-6（Scapularisin-6）的成熟肽。进行了体外抗菌试验，以测试这两种防御素对不同细菌属的活性，包括革兰氏阳性菌金黄色葡萄球菌、表皮葡萄球菌和李斯特菌属，以及革兰氏阴性菌大肠杆菌、铜绿假单胞菌，还有镰刀菌属的两种植物致病真菌。此外，使用PCR确定了Scapularisin-3和Scapularisin-6在肩突硬蜱中肠、唾液腺和胚胎衍生细胞系中的组织特异性表达模式。最后，预测了这两种防御素的三级结构并进行了结构分析。
Scapularisin-6能有效杀死格氏李斯特菌，Scapularisin-3和Scapularisin-6均对植物致病真菌禾谷镰刀菌和黄色镰刀菌的孢子萌发有强烈抑制作用（IC50值：约1 μM）。在肩突硬蜱的唾液腺和中肠中观察到了Scapularisin-6基因的表达。然而，仅在唾液腺中检测到Scapularisin-3基因的表达。在肩突硬蜱蜱细胞系ISE6和ISE18中未发现这两种防御素的转录本。
我们的研究结果有两个主要意义。首先，Scapularisin-3和Scapularisin-6的抗李斯特菌和抗真菌活性表明，这些肽可能有助于（i）治疗人类中对抗生素耐药的格氏李斯特菌，以及（ii）植物保护。其次，本研究中描述的两种防御素的抗菌特性可能为进一步研究肩突硬蜱中的病原体入侵和先天免疫铺平道路。
