Crosslinking of a Peritrophic Matrix Protein Protects Gut Epithelia from Bacterial Exotoxins
Fig 4
Covalently crosslinked drosocrystallin fibers are resistant to proteolytic digestion and trap a pore-forming exotoxin, monalysin.
(A) A schematic of the assay for the fiber formation of drosocrystallin. Briefly, wild-type drosocrystallin (Dcy) was incubated on cover glass in the presence or absence of TG and Ca2+ or EDTA for 1 h. The culture supernatant from P. entomophila (Pe sup) containing protease AprA was added on the cover glass with or without monalysin (Mnl). After incubation, proteins on the cover glass were fixed with paraformaldehyde and detected by immunofluorescence. (B) Wild-type drosocrystallin was incubated on coverslip glass and observed by immunofluorescence microscopy. Wild-type drosocrystallin was detected using an anti-His tag antibody and CF488-conjugated secondary antibody (green). Representative data from at least five experiments are shown. (C) Wild-type drosocrystallin was incubated with or without TG on coverslip glass in the presence of Ca2+, and then 2-fold serial dilutions of the culture supernatant from P. entomophila (Pe sup) were added. The concentration of the culture supernatant is indicated. Bars indicate the mean and standard deviations of the mean gray values from four independent experiments (upper panel). Lower panels show representative data of drosocrystallin fibers formed in the presence or absence of TG and Pe sup. Wild-type drosocrystallin was detected by anti-His tag antibody and CF568-conjugated secondary antibody (red). (D) Wild-type drosocrystallin was incubated with or without TG on coverslip glass in the presence of Ca2+, and then wild-type monalysin was added in the presence (Pe sup +) or absence (Pe sup −) of the culture supernatant from P. entomophila. Wild-type drosocrystallin was detected by anti-His tag antibody and CF568-conjugated secondary antibody (red), and wild-type monalysin was detected by anti-histidine affinity tag (HAT) antibody and CF488-conjugated secondary antibody (green). One representative experiment from at least five independent experiments is shown.