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.
Highlights
Gut epithelia are the first line of defense against invading microorganisms
In the absence of the culture supernatant, wild-type monalysin colocalized with both the TG-dependent crosslinked fibers and the Ca2+-induced non-covalent associated fibers of wildtype drosocrystallin (Fig 4D, left and right panels of Pe sup −). These findings indicate that the non-covalent fiber formation of drosocrystallin leads to co-localization of monalysin and that the protease-resistant drosocrystallin fibers crosslinked by TG, but not non-crosslinked drosocrystallin fibers, trap monalysin released from P. entomophila in the presence of proteases such as metalloprotease AprA
There is no genetic evidence for involvement of these peritrophic matrix proteins with multiple chitin-binding domains in peritrophic matrix formation in Drosophila, but the TG-catalyzed crosslinked fibers could promote the formation of a rigid peritrophic matrix structure to protect against exotoxins
Summary
Intestinal homeostasis is ensured by a subtle balance between bacteria and host immunity. Gut epithelial barriers, such as the mucus layer in mammals and the peritrophic matrix in invertebrates, have a protective function for the host, as they are impermeable to invading intestinal microbes. In the fly Drosophila melanogaster, transglutaminase (TG), a molecular glue involved in protein-protein covalent bond formation, is essential for peritrophic matrix formation by converting the peritrophic protein drosocrystallin into a stable fiber-like structure and inhibition of pathogenic bacteria. Knockdown of the TG gene led to increased permeability of the peritrophic matrix and greatly increased the PLOS Pathogens | DOI:10.1371/journal.ppat.1005244. TG contributes to form a stable fiber-like barrier on the peritrophic matrix and increase tolerance to pathogenic microorganisms
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