Crosslinking of a Peritrophic Matrix Protein Protects Gut Epithelia from Bacterial Exotoxins

Abstract

Transglutaminase (TG) catalyzes protein-protein crosslinking, which has important and diverse roles in vertebrates and invertebrates. Here we demonstrate that Drosophila TG crosslinks drosocrystallin, a peritrophic matrix protein, to form a stable fiber structure on the gut peritrophic matrix. RNA interference (RNAi) of the TG gene was highly lethal in flies and induced apoptosis of gut epithelial cells after oral infection with Pseudomonas entomophila. Moreover, AprA, a metalloprotease secreted by P. entomophila, digested non-crosslinked drosocrystallin fibers, but not drosocrystallin fibers crosslinked by TG. In vitro experiments using recombinant drosocrystallin and monalysin proteins demonstrated that monalysin, a pore-forming exotoxin of P. entomophila, was adsorbed on the crosslinked drosocrystallin fibers in the presence of P. entomophila culture supernatant. In addition, gut-specific TG-RNAi flies had a shorter lifespan than control flies after ingesting P. entomophila, whereas the lifespan after ingesting AprA-knockout P. entomophila was at control levels. We conclude that drosocrystallin fibers crosslinked by TG, but not non-crosslinked drosocrystallin fibers, form an important physical barrier against exotoxins of invading pathogenic microbes.

Molecular and Functional Analysis of Pore-Forming Toxin Monalysin From Entomopathogenic Bacterium Pseudomonas entomophila

“…Drosophila adults combat these effectors using a cross-linked drosocrystallin (dcy) protein, which works as physical barrier blocking the permeation of macromolecules (>500 kDa) in the peritrophic matrix (24,25). Cleaved Monalysin shows cytotoxic activity, probably by forming pores in the plasma membrane of host cells, leading to disrupted membrane permeability and cell death (23).…”

Transglutaminase-catalyzed incorporation of polyamines masks the DNA-binding region of the transcription factor Relish

“…Genome analysis of Drosophila identified a single TG gene encoding an 87-kDa protein that functions in both intracellular and extracellular spaces, although there is no authentic secretion signal sequence in the N terminus. Drosophila TG forms clots and traps invading pathogenic microbes (10,11) and cross-links drosocrystallin on the peritrophic matrix, a semipermeable barrier structure in insects, to form a stabilized fiber structure against toxic proteases released by orally infected pathogenic bacteria (12). Moreover, RNAi directed against the TG gene in Drosophila leads to a pupal semilethal phenotype, abnormal morphology, and shorter lifespan compared with non-RNAi flies (8,13).…”

Temperature Dramatically Shapes Mosquito Gene Expression with Consequences for Mosquito-Zika Virus Interactions

“…Although the PM is semi-permeable, it is thought to form a barrier and protect the midgut from pathogens (e.g. viruses (Wang and Granados, 2000), bacteria (Kuraishi et al, 2011; Jin et al, 2019), malaria (Rodgers et al, 2017) and protozoa (Weiss et al, 2014)) and other harmful substances present in the insect gut after a blood meal (Wang et al, 2004; Shibata et al, 2015).…”