Song Xiumei, Zhou Han, Wang Jingwen
State Key Laboratory of Genetic Engineering, School of Life Sciences, Department of Infectious Diseases, Zhongshan Hospital, Fudan University, Shanghai, China.
Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University, Shanghai, China.
PLoS Biol. 2025 Jan 6;23(1):e3002967. doi: 10.1371/journal.pbio.3002967. eCollection 2025 Jan.
The peritrophic matrix (PM) acts as a physical barrier that influences the vector competence of mosquitoes. We have previously shown that gut microbiota promotes PM formation in Anopheles stephensi, although the underlying mechanisms remain unclear. In this study, we identify that the cell wall components of gut commensal bacteria contribute to PM formation. Oral administration of primary cell wall components from both gram-positive and gram-negative bacteria, such as diaminopimelic acid-peptidoglycan (DAP-PGN), lysine-peptidoglycan (Lys-PGN), and lipopolysaccharides (LPS), to mosquitoes, after depleting their gut microbiota with antibiotics, restores the down-regulated expression of the peritrophin1 (Per1) gene, which encodes a structural protein of the PM. Moreover, this administration rescues PM formation upon blood ingestion. PGN and LPS are well-known ligands of innate immune signaling pathways in animals. In mosquitoes, the Toll and IMD (immune deficiency) pathways are the 2 major innate immune signaling pathways. We next knocked down the expression of 2 receptors, Pgrp-s1 and Pgrp-lc, as well as 2 transcription factors, Rel1 and Rel2, which are involved in the Toll and IMD pathways, respectively. Double knockdown of Pgrp-s1 and Pgrp-lc, or Rel1 and Rel2, compromised Per1 expression. Additionally, through dual-luciferase assays and supershift electrophoretic mobility shift assays (EMSAs), we identified a 15-bp binding motif (ATAGACACGAGCACC) for Rel1 and Rel2 in the Per1 promoter region. To further explore the role of individual Toll and IMD pathways in the regulation of Per1 expression, we specifically inhibited the activity of each pathway. While inhibition of the Toll pathway by knocking down Pgrp-s1 or Rel1 did not affect Per1 expression, knockdown of Pgrp-lc or Rel2 in the IMD pathway significantly down-regulated Per1 expression. These findings suggest that the IMD pathway plays a major role in regulating Per1 expression in An. stephensi. In summary, our study uncovers a novel role for bacterial cell wall components in regulating PM formation through activation of mosquito immune signaling pathways.
围食膜（PM）作为一种物理屏障，影响蚊子的媒介能力。我们之前已经表明，肠道微生物群促进斯氏按蚊中PM的形成，但其潜在机制仍不清楚。在本研究中，我们确定肠道共生细菌的细胞壁成分有助于PM的形成。在用抗生素耗尽蚊子的肠道微生物群后，给蚊子口服革兰氏阳性和革兰氏阴性细菌的主要细胞壁成分，如二氨基庚二酸肽聚糖（DAP-PGN）、赖氨酸肽聚糖（Lys-PGN）和脂多糖（LPS），可恢复围食膜蛋白1（Per1）基因的下调表达，该基因编码PM的一种结构蛋白。此外，这种给药方式可挽救吸血后PM的形成。PGN和LPS是动物先天免疫信号通路中众所周知的配体。在蚊子中，Toll和IMD（免疫缺陷）通路是两条主要的先天免疫信号通路。接下来，我们敲低了分别参与Toll和IMD通路的2种受体Pgrp-s1和Pgrp-lc，以及2种转录因子Rel1和Rel2的表达。Pgrp-s1和Pgrp-lc或Rel1和Rel2的双重敲低损害了Per1的表达。此外，通过双荧光素酶测定和超迁移电泳迁移率变动分析（EMSA），我们在Per1启动子区域鉴定出Rel1和Rel2的一个15碱基对结合基序（ATAGACACGAGCACC）。为了进一步探究单个Toll和IMD通路在调节Per1表达中的作用，我们特异性抑制了每条通路的活性。虽然通过敲低Pgrp-s1或Rel1抑制Toll通路不影响Per1表达，但在IMD通路中敲低Pgrp-lc或Rel2会显著下调Per1表达。这些发现表明，IMD通路在调节斯氏按蚊中Per1表达中起主要作用。总之，我们的研究揭示了细菌细胞壁成分通过激活蚊子免疫信号通路在调节PM形成中的新作用。
