Graduate Program in Veterinary Science, Department of Animal Parasitology, Federal Rural University of Rio de Janeiro, Seropédica, RJ 23790-000, Brazil.
Graduate Program in Cellular and Molecular Biology, Federal University of Rio Grande do Sul, Porto Alegre, RS 90610-000, Brazil.
Microbiol Res. 2021 Jul;248:126753. doi: 10.1016/j.micres.2021.126753. Epub 2021 Apr 7.
Menadione (MND) is known to induce oxidative stress in fungal cells. Here, we explore how exposure to this molecule alters conidial enzyme activities, fungal efficacy against Rhipicephalus microplus, and mycelial secretion (secretome) of an isolate of Metarhizium anisopliae sensu lato. First, the fungus was exposed to different MND concentrations in potato-dextrose-agar (PDA) to determine the LC by evaluating conidia germination (38μM). To ensure high cell integrity, a sublethal dose of MND (half of LC) was added to solid (PDA MND) and liquid media (MS MND). Changes in colony growth, a slight reduction in conidia production, decreases in conidial surface Pr1 and Pr2 activities as well as improvements in proteolytic and antioxidant (catalase, superoxide dismutase, and peroxidase) conidial intracellular activities were observed for PDA MND conidia. Additionally, PDA MND conidia had the best results for killing tick larvae, with the highest mortality rates until 15 days after treatment, which reduces both LC and LT, particularly at 10 conidia mL. The diversity of secreted proteins after growth in liquid medium + R. microplus cuticle (supplemented or not with half of MND LC), was evaluated by mass spectrometry-based proteomics. A total of 654 proteins were identified, 31 of which were differentially regulated (up or down) and mainly related to antioxidant activity (catalase), pathogenicity (Pr1B, Pr1D, and Pr1K), cell repair, and morphogenesis. In the exclusively MS MND profile, 48 proteins, mostly associated with cellular signaling, nutrition, and antioxidant functions, were distinguished. Finally, enzymatic assays were performed to validate some of these proteins. Overall, supplementation with MND in the solid medium made conidia more efficient at controlling R. microplus larvae, especially by increasing, inside the conidia, the activity of some infection-related enzymes. In the liquid medium (a consolidated study model that mimics some infection conditions), proteins were up- and/or exclusively-regulated in the presence of MND, which opens a spectrum of new targets for further study to improve biological control of ticks using Metarhizium species.
甲萘醌（MND）已知可诱导真菌细胞中的氧化应激。在这里，我们探讨了暴露于该分子如何改变分生孢子酶活性、真菌对 Rhipicephalus microplus 的功效以及粘帚霉属（Metarhizium anisopliae sensu lato）分离株的菌丝体分泌（分泌组）。首先，将真菌暴露于不同浓度的甲萘醌在土豆-葡萄糖-琼脂（PDA）中，通过评估分生孢子萌发（38μM）来确定 LC。为确保细胞完整性高，添加亚致死剂量的 MND（LC 的一半）到固体（PDA MND）和液体培养基（MS MND）中。观察到 PDA MND 分生孢子的菌落生长变化、产孢量略有减少、Pr1 和 Pr2 表面活性降低以及细胞内蛋白酶和抗氧化剂（过氧化氢酶、超氧化物歧化酶和过氧化物酶）活性提高。此外，PDA MND 分生孢子在杀死蜱幼虫方面效果最好，处理后 15 天内死亡率最高，LC 和 LT 均降低，尤其是在 10 个分生孢子 mL 时。在液体培养基+ R. microplus 角质层（添加或不添加 MND LC 的一半）中生长后，通过基于质谱的蛋白质组学评估分泌蛋白的多样性。共鉴定出 654 种蛋白质，其中 31 种被差异调节（上调或下调），主要与抗氧化活性（过氧化氢酶）、致病性（Pr1B、Pr1D 和 Pr1K）、细胞修复和形态发生有关。在单独的 MS MND 图谱中，区分出 48 种主要与细胞信号转导、营养和抗氧化功能有关的蛋白质。最后，进行了酶活性测定以验证其中一些蛋白质。总的来说，在固体培养基中添加 MND 可使分生孢子更有效地控制 R. microplus 幼虫，特别是通过增加分生孢子内一些与感染相关的酶的活性。在液体培养基中（模拟一些感染条件的综合研究模型），在存在 MND 的情况下，蛋白质被上调和/或专门调节，这为进一步研究利用粘帚霉属物种进行 ticks 的生物防治开辟了一系列新的靶标。
