Department of Immunobiology, Institute of Biological Sciences, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19 St., 20-033 Lublin, Poland.
Department of Analytical Biochemistry, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7 St., 30-387 Krakow, Poland.
J Insect Physiol. 2021 May-Jun;131:104239. doi: 10.1016/j.jinsphys.2021.104239. Epub 2021 Apr 23.
The work presents identification of antimicrobial peptides and proteins (AMPs) in the hemolymph of Galleria mellonella larvae infected with two Pseudomonas aeruginosa strains (ATCC 27,853 and PA18), differing in the profile of secreted proteases. The insects were immunized with bacteria cultivated in rich (LB) and minimal (M9) media, which resulted in appearance of a similar broad set of AMPs in the hemolymph. Among them, 13 peptides and proteins were identified, i.e. proline-rich peptides 1 and 2, lebocin-like anionic peptide 1 and anionic peptide 2, defensin/galiomicin, cecropin, cecropin D-like peptide, apolipophoricin, gallerimycin, moricin-like peptide B, lysozyme, apolipophorin III, and superoxide dismutase. Bacterial strain- and/or medium-dependent changes in the level of proline-rich peptide 1, anionic peptide 1 and 2, moricin-like peptide B, cecropin D-like and gallerimycin were observed. The analysis of the expression of genes encoding cecropin, gallerimycin, and galiomicin indicated that they were differently affected by the bacterial strain but mainly by the medium used for bacterial culture. The highest expression was found for the LB medium. In addition to the antibacterial and antifungal activity, proteolytic activity was detected in the hemolymph of the P. aeruginosa-infected insects. Based on these results and those presented in our previous reports, it can be postulated that the appearance of AMPs in G. mellonella hemolymph can be triggered not only by P. aeruginosa pathogen associated molecular patterns (PAMPs) but also by bacterial extracellular proteases secreted during infection. However, although there were no qualitative differences in the set of AMPs depending on the P. aeruginosa strain and medium, differences in the level of particular AMPs synthesized in response to the bacteria used were observed.
该工作鉴定了在感染两种不同分泌蛋白酶谱的铜绿假单胞菌（ATCC 27853 和 PA18）的家蚕幼虫血淋巴中的抗菌肽和蛋白质（AMPs）。昆虫用在丰富（LB）和最小（M9）培养基中培养的细菌进行免疫，导致血淋巴中出现类似的广泛的 AMP 集。其中，鉴定出 13 种肽和蛋白质，即富含脯氨酸的肽 1 和 2、lebocin 样阴离子肽 1 和阴离子肽 2、防御素/加利米辛、cecropin、cecropin D 样肽、载脂蛋白、gallerimycin、moricin 样肽 B、溶菌酶、载脂蛋白 III 和超氧化物歧化酶。观察到依赖于细菌菌株和/或培养基的富含脯氨酸肽 1、阴离子肽 1 和 2、moricin 样肽 B、cecropin D 样和 gallerimycin 的水平变化。编码 cecropin、gallerimycin 和 galiomicin 的基因的表达分析表明，它们受细菌菌株的不同影响，但主要受用于细菌培养的培养基影响。LB 培养基的表达最高。除了具有抗菌和抗真菌活性外，在感染铜绿假单胞菌的昆虫血淋巴中还检测到蛋白水解活性。基于这些结果和我们之前报告的结果，可以假设 AMPs 在 G. mellonella 血淋巴中的出现不仅可以由铜绿假单胞菌病原体相关分子模式（PAMPs）触发，还可以由感染期间分泌的细菌细胞外蛋白酶触发。然而，尽管根据铜绿假单胞菌菌株和培养基， AMPs 的种类没有定性差异，但观察到针对使用的细菌合成的特定 AMPs 的水平存在差异。
