Kachaev Zaur M, Ghassah Mona, Musabirov Anton A, Shaposhnikov Alexander V, Toropygin Ilya Y, Ulianova Yulia A, Stepanov Nikita G, Chmykhalo Victor K, Shidlovskii Yulii V
Laboratory of Gene Expression Regulation in Development, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia.
Laboratory of Gene Expression Regulation in Development, Institute of Gene Biology, Russian Academy of Sciences, 119334 Moscow, Russia; School of Biological and Medical Physics, Moscow Institute of Physics and Technology (National Research University), 141701 Dolgoprudny, Russia.
J Invertebr Pathol. 2025 Jul;211:108315. doi: 10.1016/j.jip.2025.108315. Epub 2025 Mar 13.
The canonical model of immune response activation in Drosophila suggests that the IMD pathway is activated by Gram-negative (Gram (-)) bacteria, while the Toll pathway is activated by both Gram-positive bacteria (Gram (+)) and fungi. However, the mechanisms by which these pathogens promote cross-activation of these pathways remain controversial. In addition, the mechanisms of cross-activation in S2 cell culture remain unstudied. In this study, we investigated the role of two Gram (+) bacteria (Micrococcus luteus and Bacillus subtilis) and fungal spores (Metarhizium anisopliae) in activating the IMD pathway in S2 cell cultures. Cells were treated with Escherichia coli as a control to ensure the specificity of IMD pathway activation. Our results demonstrated a significant involvement of M. luteus in the activation of the IMD pathway in S2 cell cultures. This is evidenced by the marked activation of IMD pathway-dependent genes, as well as the proteolytic cleavage of the Relish protein, which serves as a key transcription factor for this pathway. We also observed a strong recruitment of Relish to the promoters of antimicrobial peptide (AMP) genes, along with a partial recruitment to the genes encoding peptidoglycan recognition proteins (PGRPs). Furthermore, RNA interference targeting Relish resulted in a significant reduction in the transcription levels of all AMP genes and most PGRPs. Similarly, we analyzed the contributions of B. subtilis and M. anisopliae to the cross-activation of the IMD pathway. Our data indicate that both B. subtilis and M. anisopliae also activate the IMD pathway, albeit to a lesser extent compared to M. luteus. At the same time, fungal spores exhibited minimal influence on the activation of the IMD pathway when compared to Gram (+) bacteria. Thus, we have investigated in detail the mechanisms of cross-activation of the immune response in S2 cell culture, suggesting that Relish may play a critical role in inducing a humoral immune response in Drosophila S2 cells, primarily against M. luteus and to a lesser extent against B. subtilis and M. anisopliae.
果蝇免疫反应激活的经典模型表明，IMD途径由革兰氏阴性（Gram (-)）细菌激活，而Toll途径由革兰氏阳性细菌（Gram (+)）和真菌激活。然而，这些病原体促进这些途径交叉激活的机制仍存在争议。此外，S2细胞培养中交叉激活机制尚未得到研究。在本研究中，我们调查了两种革兰氏阳性细菌（藤黄微球菌和枯草芽孢杆菌）和真菌孢子（绿僵菌）在S2细胞培养中激活IMD途径的作用。用大肠杆菌作为对照处理细胞，以确保IMD途径激活的特异性。我们的结果表明，藤黄微球菌在S2细胞培养中对IMD途径的激活有显著作用。这通过IMD途径依赖性基因的显著激活以及Relish蛋白的蛋白水解切割得到证明，Relish蛋白是该途径的关键转录因子。我们还观察到Relish强烈募集到抗菌肽（AMP）基因的启动子，同时部分募集到编码肽聚糖识别蛋白（PGRP）的基因。此外，靶向Relish的RNA干扰导致所有AMP基因和大多数PGRP转录水平显著降低。同样，我们分析了枯草芽孢杆菌和绿僵菌对IMD途径交叉激活的作用。我们的数据表明，枯草芽孢杆菌和绿僵菌也激活IMD途径，尽管与藤黄微球菌相比程度较小。同时，与革兰氏阳性细菌相比，真菌孢子对IMD途径激活的影响最小。因此，我们详细研究了S2细胞培养中免疫反应交叉激活的机制，表明Relish可能在诱导果蝇S2细胞的体液免疫反应中起关键作用，主要针对藤黄微球菌，对枯草芽孢杆菌和绿僵菌的作用较小。
