PGRP-LA regulates peritrophic matrix synthesis and influences trypanosome infection outcomes in tsetse flies

Abstract

Peptidoglycan Recognition Proteins (PGRPs) are conserved pattern-recognition receptors that detect microbe-associated molecular patterns (MAMPs) and activate host immune responses. Compared to other dipterans, the tsetse fly (Glossina morsitans morsitans) genome encodes only five PGRPs-PGRP-LA, -LB, -LC, -SA, and -SB - far fewer than most dipterans, likely reflecting its sterile blood diet and streamlined microbiota. Here, we identify PGRP-LA as a critical regulator of peritrophic matrix (PM) integrity in the cardia (proventriculus), the tissue responsible for PM production. The PM is a chitinous sleeve-like barrier that separates the midgut epithelium from the ingested bloodmeal, supporting digestive homeostasis and infection resistance. We show that pgrp-la is prominently expressed in the cardia, transiently induced after a bloodmeal in newly eclosed flies, and reinduced following subsequent feedings, likely in response to blood-constituents or mechanical stretch. This induction is sustained during microbial exposure and prolonged in trypanosome-infected flies. RNAi-mediated reduction of pgrp-la significantly increased the prevalence of midgut trypanosome infections, indicating a protective role during early infection. PGRP-LA did not mediate infection resistance via canonical IMD pathway signaling, as its silencing did not affect antimicrobial peptide expression. Instead, PGRP-LA modulated the expression of PM-associated genes and gut barrier integrity. Silencing pgrp-la reduced PM structure, increased midgut weights and enhanced fly survival following oral challenge with entomopathogen Serratia marcescens, likely due to earlier epithelial immune responses through a compromised PM. Similar phenotypes were observed when flies were fed anti-PGRP-LA antibodies, supporting a structural role for PGRP-LA. In addition, soluble variant surface glycoproteins (sVSGs) from trypanosomes and knockdown of microRNA-275 (miR-275), also decreased pgrp-la expression, suggesting that PGRP-LA is part of a broader regulatory network, including the miR-275/Wingless signaling. Collectively, our results identify PGRP-LA as novel regulator of PM biogenesis and vector competence in tsetse, expanding the functional repertoire of PGRPs in insect gut barrier maintenance beyond canonical immune signaling pathways. Author SummaryInsect vectors such as tsetse flies can be infected with pathogens that cause devastating disease in mammals. To protect themselves insect vectors rely on pattern recognition receptors (PRRs) that detect pathogens and activate the production of antimicrobial peptides (AMPs). Physical barriers in the gut also play an important role in limiting infections. One such barrier is the peritrophic matrix (PM), a sleeve-like structure that lines the insect gut and separates the blood meal and its contents from the underlying cells. For trypanosome parasites, which cause sleeping sickness in humans, the PM is the first barrier they must traverse to colonize the tsetses gut. In this study, we identified a PRR, PGRP-LA, that, unlike related proteins in other insects that activate AMPs, regulates the integrity of the tsetses protective PM barrier. When PGRP-LA was disrupted, the gut barrier weakened, and flies became more susceptible to trypanosome infection. Our work highlights a previously unrecognized role for PGRP-LA in maintaining gut barrier integrity and suggest that targeting this pathway could be a strategy to help reduce parasite transmission.