Kuc-Ciepluch Dorota, Ciepluch Karol, Augustyniak Daria, Guła Grzegorz, Maciejewska Barbara, Kowalik Artur, Jop Ewelina, Drulis-Kawa Zuzanna, Arabski Michał
Division of Medical Biology, Jan Kochanowski University in Kielce, Kielce, Poland.
Department of Pathogen Biology and Immunology, University of Wroclaw, Wroclaw, Poland.
mSystems. 2025 Jan 21;10(1):e0110624. doi: 10.1128/msystems.01106-24. Epub 2024 Dec 23.
Pyroptosis is an inflammatory immune response of eukaryotic cells to bacterial lipopolysaccharide (LPS) and other pathological stimuli, leading to the activation of the gasdermin D (GSDMD) and secretion of pore-forming domain GSDMD, facilitating the release of cytokines. Additionally, GSDMD exhibits antibacterial properties through interactions with bacterial outer membranes (OM). We explored alternative antimicrobial strategy to determine whether inducing natural pyroptosis via GSDMD activation by LPS could enhance the effectiveness of recombinant phage endopeptidase KP27 (peptidoglycan-degrading enzyme) against , enabling penetration through OM and bacterial killing synergistically. Our findings demonstrated that recombinant GSDMD alone exhibited antibacterial effects against wild-type with smooth LPS, while recombinant GSDMD efficiently permeabilized both smooth LPS-bearing and O-chain-deficient potentially synergizing with endolysin KP27. Transcriptomic analyses revealed the activation of the immune system pathways in response to LPS, mainly in monocytic cells, in contrast to epithelial A549 or HeLa cell lines. LPS-induced pyroptosis in monocytes led to GSDMD cleavage and the release of interleukins, regardless of the nature/origin of the LPS used. However, the pyroptosis stimulation by LPS in the antibacterial assay was not effective enough for bacterial OM permeabilization and enhancement of endolysin activity. We assume that leveraging pyroptosis induction in monocytic cells to augment the bactericidal activity of endolysins may be limited.
Recombinant GSDMD protein was able to efficiently permeabilize outer membranes and increase endolysin activity against bacteria, producing either long LPS O-chains or lack them entirely. The obtained results suggest the limited possibility of using the natural process of pyroptosis occurring in monocytic cells to enhance the bactericidal effect of recombinant phage endolysins against Gram-negative bacteria infection.
细胞焦亡是真核细胞对细菌脂多糖（LPS）和其他病理刺激的一种炎症免疫反应，导致gasdermin D（GSDMD）活化和成孔结构域GSDMD的分泌，促进细胞因子的释放。此外，GSDMD通过与细菌外膜（OM）相互作用表现出抗菌特性。我们探索了一种替代抗菌策略，以确定通过LPS激活GSDMD诱导天然细胞焦亡是否能增强重组噬菌体内切酶KP27（肽聚糖降解酶）对[细菌名称未给出]的有效性，使其能够协同穿透外膜并杀死细菌。我们的研究结果表明，单独的重组GSDMD对具有光滑LPS的野生型[细菌名称未给出]表现出抗菌作用，而重组GSDMD能有效使携带光滑LPS和缺乏O链的[细菌名称未给出]的外膜通透化，可能与内溶素KP27协同作用。转录组分析显示，与上皮A549或HeLa细胞系相比，LPS主要在单核细胞中激活免疫系统途径。无论所使用的LPS的性质/来源如何，LPS诱导的单核细胞焦亡都会导致GSDMD裂解和白细胞介素的释放。然而，在抗菌试验中，LPS对焦亡的刺激对于细菌外膜通透化和增强内溶素活性来说不够有效。我们认为，利用单核细胞中的细胞焦亡诱导来增强内溶素的杀菌活性可能是有限的。
重组GSDMD蛋白能够有效使[细菌名称未给出]的外膜通透化，并增加对内溶素对细菌的活性，这些细菌要么产生长LPS O链，要么完全缺乏它们。所获得的结果表明，利用单核细胞中发生的天然细胞焦亡过程来增强重组噬菌体内溶素对革兰氏阴性菌感染的杀菌作用的可能性有限。
