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The so-far putative serine protease inhibitor dipetalogastin-like (GmSPID) was found in one fraction obtained after separation of hemolymph by RP-HPLC. Its amount depended on the immune status of the insect: it significantly increased after oral (10^3 CFU) and intrahemocelic (10 and 50 CFU) infection with entomopathogenic bacteria <jats:italic>Pseudomonas entomophila<\/jats:italic>. This was accompanied by up-regulation of the respective gene in the fat body of infected larvae. GmSPID was purified to homogeneity and characterised as a protein with immune properties. Among the three proteases tested, i.e. trypsin, elastase, and thermolysin, the strongest inhibition was observed toward trypsin. No inhibition toward the metalloproteinase thermolysin was detected, confirming that GmSPID is an inhibitor of serine proteases. Additionally, GmSPID was shown to have antimicrobial properties. At the concentration of 7 \u00b5M and 15 \u00b5M, it acted against <jats:italic>Pseudomonas entomophila<\/jats:italic>, <jats:italic>Pseudomonas aeruginosa<\/jats:italic>, <jats:italic>Bacillus thuringiensis<\/jats:italic>, <jats:italic>Escherichia coli<\/jats:italic>, and <jats:italic>Candida albicans<\/jats:italic> but not against <jats:italic>Staphylococcus aureus<\/jats:italic>. Moreover, with the use of atomic force, scanning, and transmission electron microscopy techniques, we present the effect of the GmSPID protein on the surface properties, shape, and ultrastructure of <jats:italic>P. entomophila<\/jats:italic> cells. The protein caused modest perforation of the cellular membrane, contributing to loss of its integrity. The mode of the GmSPID protein action as an antimicrobial compound and its role in <jats:italic>G. mellonella<\/jats:italic> immunity are discussed.<\/jats:p>","DOI":"10.1038\/s41598-025-08159-z","type":"journal-article","created":{"date-parts":[[2025,7,6]],"date-time":"2025-07-06T05:10:16Z","timestamp":1751778616000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":3,"title":["Serine protease inhibitor dipetalogastin-like from Galleria mellonella is involved in insect immunity"],"prefix":"10.1038","volume":"15","author":[{"given":"Jakub","family":"Kordaczuk","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Micha\u0142","family":"Su\u0142ek","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Pawe\u0142","family":"Mak","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Bo\u017cena","family":"Pawlikowska-Pawl\u0119ga","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Iwona","family":"Wojda","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"297","published-online":{"date-parts":[[2025,7,6]]},"reference":[{"key":"8159_CR1","doi-asserted-by":"publisher","first-page":"81","DOI":"10.1007\/978-3-030-41769-7_4","volume":"94","author":"I Wojda","year":"2020","unstructured":"Wojda, I., Cytry\u0144ska, M., Zdybicka-Barabas, A. & Kordaczuk, J. 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