Exploiting gasdermin-mediated pyroptosis for enhanced antimicrobial activity of phage endolysin against Pseudomonas aeruginosa

Abstract

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_Nterm, facilitating the release of cytokines. Additionally, GSDMD_Nterm 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 P. aeruginosa LPS could enhance the effectiveness of recombinant phage endopeptidase KP27 (peptidoglycan-degrading enzyme) against P. aeruginosa, enabling penetration through OM and bacterial killing synergistically. Our findings demonstrated that recombinant GSDMD alone exhibited antibacterial effects against wild-type P. aeruginosa with smooth LPS, while recombinant GSDMD_Nterm efficiently permeabilized both smooth LPS-bearing and O-chain-deficient P. aeruginosa 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.

Importance: Recombinant GSDMD_Nterm protein was able to efficiently permeabilize P. aeruginosa 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.

Keywords: Pseudomonas aeruginosa LPS; endolysin; gasdermin D; outer membrane permeabilization; pyroptosis.

Fig 1

The experimental pipeline of the study (1). Recombinant gasdermin D (GSDMD) or its N-terminal region for membrane permeability of P. aeruginosa PAO1 and its LPS O-chain-deficient mutant (ΔwbpL) was monitored using NPN uptake assay. Membrane permeability experiments were performed for GSDMDs alone and in the presence of KP27 endolysin (2). Mimicking bacterial infection—gasdermin D activation by P. aeruginosa O10 LPS in A549, HeLa, or THP1 cells was measured by microarray (gene expression) and ELISA (IL-1β) assays (3). The inflammatory properties of LPS isolated from E.coli (control) and tested P. aeruginosa strains were evaluated using immune-serological tests. The patterns of isolated LPS samples (the O-chain presence or absence) were characterized using the SDS-PAGE method (4). LPS-induced pyroptosis alone or in combination with nigericin was tested on the THP1 cell line to obtain active gasdermin D for enhanced KP27 endolysin activity against P. aeruginosa PAO1 strain. LPS was isolated from tested P. aeruginosa strains and E. coli (control). Gasdermin (GSDMD) production was monitored using Western blot, and the antibacterial activity of endolysin was evaluated using culture optical density (OD₆₀₀).

Fig 2

OM permeabilization of P. aeruginosa PAO1 wild type with LPS (S) and its ΔwbpL mutant lacking LPS O-chain (R) by 1.5 µM GSDMD or GSDMD_Nterm recombinant proteins alone or in combination with endolysin (36 µg/mL) calculated as the relative fluorescence units (RFU). The control consisted of untreated P. aeruginosa cells.

Fig 3

Two-color microarray-based gene expression analysis of cell lines treated with P. aeruginosa O10 LPS. The number of up- (A) and downregulated (B) genes in tested cell lines. Heatmaps of KEGG pathway enrichment analysis (PahthfindeR, R package), based on comparison of expression between LPS-exposed and non-exposed cell lines, are presented for A549 (C), HeLa (D), and THP (E).

Fig 4

The induction of pyroptosis in monocytic cells by P. aeruginosa O10 LPS. The GSDMD expression level analysis by RT-qPCR in THP1-Xblue cells after treatment with serial concentrations of O10 LPS. The ACTB gene was used as an internal reference gene, and the ΔΔCT method was applied for relative quantification (*P < 0.05) (A). The IL-1β production by THP1-Xblue cells in the presence of P. aeruginosa O10 LPS measured using ELISA; *P < 0.05 (B). One-way ANOVA with Dunnett’s post-hoc test was used to test the statistically significant differences.

Fig 5

Induction of inflammatory response of THP-1 monocytes by P. aeruginosa LPS. Supernates from 24-h THP-1 cultures stimulated with various concentrations of LPS were evaluated using ELISA for the presence of proinflammatory cytokines (A) IL-8 and (B) TNF. Data are expressed as mean ± SEM from at least two independent experiments performed in two plates at four technical repeats each (two technical repeats per plate). Supernates from duplicates from each plate were pooled and assessed using ELISA. Dashed lines refer to non-stimulated control. One-way ANOVA with post-hoc Tukey test was used to test the differences. *P < 0.05, **P < 0.01, ***P < 0.0005. As a positive control, LPS from O10 strains was used.

Fig 6

The OD₆₀₀ of P. aeruginosa PAO1 culture growth was measured in TSB supplemented with 2× diluted THP1-Null2 cell line supernatant, either alone or with 36 µg/mL of KP27 endolysin (E) after 18 h of incubation at 37°C. The THP1-Null2 line was first pre-treated with 1 µg/mL of LPS (S) and/or 10 µM nigericin (N). C, bacterial culture treated with THP1-Null2 supernatant; C_E, bacterial culture treated with THP1-Null2 supernatant and endolysin; C_N, bacterial culture treated with THP1-Null2 supernatant after nigericin stimulation; C_EN, bacterial culture treated with THP1-Null2 supernatant after nigericin stimulation and supplemented with endolysin; S, bacterial culture treated with THP1-Null2 supernatant after LPS stimulation; S_E, bacterial culture treated with THP1-Null2 supernatant after LPS stimulation and supplemented with endolysin; S_N, bacterial culture treated with THP1-Null2 supernatant after LPS and nigericin stimulation; S_EN, bacterial culture treated with THP1-Null2 supernatant after LPS and nigericin stimulation and supplemented with endolysin. Color columns show particular LPS samples used for pyroptosis induction. The experiments were done in three biological repeats, and statistical data were established by one-way ANOVA with post-hoc Dunnett’s test. The P-values are presented in the bottom table.