Cell wall components of gut commensal bacteria stimulate peritrophic matrix formation in malaria vector mosquitoes through activation of the IMD pathway.

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.