关键词: 黑腹果蝇, 先天免疫, 吞噬作用, pap2g, 细胞骨架, cdc42

Abstract: 【Aim】The innate immune system is the core defense mechanism of Drosophila melanogaster against pathogen invasion, among which cellular immunity is mainly mediated by D. melanogaster blood cells. This study aims to clarify the molecular mechanism of phosphatidic acid phosphatase type 2G (Pap2g) in the phagocytosis of D. melanogaster macrophages, so as to provide a theoretical basis for exploring its function in the innate immunity of D. melanogaster. 【Methods】 Based on transcriptomic sequencing data, the upregulated genes in the macrophage-like Schneider 2 cell line of D. melanogaster during the phagocytosis of apoptotic cells (ACs) were identified, and pap2g was selected as the target gene. RT-qPCR was employed to detect the changes in the pap2g mRNA levels following exposure of Schneider 2 cells to 1×10⁷ ACs, 1×10⁵ CFU of Staphylococcus aureus and 1×10⁵ CFU of Pseudomonas aeruginosa. RNA interference (RNAi) was utilized to silence pap2g, and its effects on the longevity of D. melanogaster infected with S. aureus and P. aeruginosa were determined. RT-qPCR was also used to measure the changes in mRNA levels of immune signaling pathway-related genes, including NF-κB, CecA1, Def and Drs in adult D. melanogaster, following inhibition of pap2g expression. Laser confocal microscopy was applied to observe the changes in the phagocytic rates of Schneider 2 cells towards ACs and S. aureus after pap2g RNAi. Additionally, laser confocal microscopy was used to observe the alterations in the filopodial morphology of macrophages in the 3rd instar larvae of D. melanogaster after pap2g RNAi. The interaction between Pap2g and cell division cycle 42 (Cdc42) was confirmed using yeast two-hybrid assay and Western blotting. The impact of pap2g RNAi on the expression level of Cdc42 and the change in the expression level of Pap2g following cdc42 RNAi were verified by Western blotting. 【Results】 The expression of pap2g in D. melanogaster Schneider 2 cells was significantly upregulated during phagocytosis. Silencing pap2g by RNAi resulted in shortened longevity of D. melanogaster following bacterial infection, and downregulation of key immune signaling pathway-related genes, including NF-κB, CecA1, Def and Drs in D. melanogaster adults. Furthermore, RNAi-mediated silencing of pap2g reduced the phagocytic rates of Schneider 2 cells towards ACs and S. aureus to 41.20%±2.31% and 38.90%±3.65%, respectively. RNAi-mediated silencing of pap2g reduced the number of filopodia in the macrophages of the 3rd instar larvae of D. melanogaster from 15±1 to 8±1 and shortened the filopodial length from (2.900±0.458) μm to (0.667±0.153) μm. Pap2g interacted with Cdc42, and the expression level of Cdc42 was reduced following silencing of pap2g in Schneider 2 cells by RNAi. A similar decrease in the expression level of Pap2g in Schneider 2 cells was also observed upon RNAi of cdc42.【Conclusion】 Pap2g plays an important role in the innate immunity of D. melanogaster, particularly during the phagocytic process, when its expression is significantly upregulated. Pap2g interacts with Cdc42 to regulate the dynamic changes of filamentous actin (F-actin), thereby influencing the number and length of cellular filopodia. Downregulation of pap2g leads to impaired phagocytic capacity in D. melanogaster macrophages, highlighting its importance in host defense mechanisms.

Key words: Drosophila melanogaster, innate immunity, phagocytosis, pap2g, cytoskeleton, cdc42