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. 2023 Dec 13:13:1295311.
doi: 10.3389/fcimb.2023.1295311. eCollection 2023.

Impact of CRAMP-34 on Pseudomonas aeruginosa biofilms and extracellular metabolites

Shiyuan Wang  1   2   3 , Chengjun Ma  1   2   4 , Jinying Long  1   2   4 , Peng Cheng  1   2 , Yang Zhang  2   5 , Lianci Peng  1   2   4 , Lizhi Fu  2   5 , Yuandi Yu  2   5 , Dengfeng Xu  2   5 , Suhui Zhang  2   5 , Jinjie Qiu  2   5 , Yuzhang He  1   2   4 , Hongzao Yang  1   2   4 , Hongwei Chen  1   2   4
Affiliations

Impact of CRAMP-34 on Pseudomonas aeruginosa biofilms and extracellular metabolites

Shiyuan Wang et al. Front Cell Infect Microbiol. .

Abstract

Biofilm is a structured community of bacteria encased within a self-produced extracellular matrix. When bacteria form biofilms, they undergo a phenotypic shift that enhances their resistance to antimicrobial agents. Consequently, inducing the transition of biofilm bacteria to the planktonic state may offer a viable approach for addressing infections associated with biofilms. Our previous study has shown that the mouse antimicrobial peptide CRAMP-34 can disperse Pseudomonas aeruginosa (P. aeruginosa) biofilm, and the potential mechanism of CRAMP-34 eradicate P. aeruginosa biofilms was also investigated by combined omics. However, changes in bacterial extracellular metabolism have not been identified. To further explore the mechanism by which CRAMP-34 disperses biofilm, this study analyzed its effects on the extracellular metabolites of biofilm cells via metabolomics. The results demonstrated that a total of 258 significantly different metabolites were detected in the untargeted metabolomics, of which 73 were downregulated and 185 were upregulated. Pathway enrichment analysis of differential metabolites revealed that metabolic pathways are mainly related to the biosynthesis and metabolism of amino acids, and it also suggested that CRAMP-34 may alter the sensitivity of biofilm bacteria to antibiotics. Subsequently, it was confirmed that the combination of CRAMP-34 with vancomycin and colistin had a synergistic effect on dispersed cells. These results, along with our previous findings, suggest that CRAMP-34 may promote the transition of PAO1 bacteria from the biofilm state to the planktonic state by upregulating the extracellular glutamate and succinate metabolism and eventually leading to the dispersal of biofilm. In addition, increased extracellular metabolites of myoinositol, palmitic acid and oleic acid may enhance the susceptibility of the dispersed bacteria to the antibiotics colistin and vancomycin. CRAMP-34 also delayed the development of bacterial resistance to colistin and ciprofloxacin. These results suggest the promising development of CRAMP-34 in combination with antibiotics as a potential candidate to provide a novel therapeutic approach for the prevention and treatment of biofilm-associated infections.

Keywords: CRAMP-34; Pseudomonas aeruginosa; anti-biofilm peptides; extracellular metabolites; synergistic effect.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Effect of CRAMP-34 on PAO1 pre-biofilm biomass and metabolic viability. (A) PAO1 biofilms were cultured for 72 hours at 37°C in 96-well plates. these biofilms were exposed to CRAMP-34 for 1 hour. Biomass quantification was conducted using crystal violet (CV). (B) After culturing PAO1 biofilms under similar conditions and post-exposure to CRAMP-34, the metabolic viability of the biofilm-associated bacteria was assessed using the formazan-based MTT assay. The significance of results was analyzed using an unpaired two-tailed t-test. Notably, *P < 0.05 and **P < 0.01 when compared to the control group.
Figure 2
Figure 2
CLSM imaging of CRAMP-34 treated PAO1 prebiofilms. PAO1 biofilms were formed for 72 h at 37°C on chambered coverglass slides. biofilms were treated with CRAMP-34 for 1 h at 37°C as described and subsequently stained with SYTO 9 and PI for 20 min in the dark. (A, B) The 3D and orthogonal views biofilm representation in the objective of 20X in the control group. (C, D) The 3D and orthogonal views biofilm representation of CRAMP-34 in the objective of 20X. (E) The total fluorescence intensity of biofilms. (F) represents the number of biofilms. (G) The bottom area of biofilms. (H) represents the volume of biofilms. (I) The total fluorescence intensity of viable or dead bacteria per unit biofilm area. (J) The total fluorescence intensity of viable or dead bacteria per unit biofilm volume. Unpaired t-test (two-tailed) was used to measure statistical significance. *P < 0.05, **P < 0.01 compared with the control group.
Figure 3
Figure 3
Extracellular metabolites in CRAMP-34 treated PAO1 prebiofilms. Three-day-old preformed biofilms were treated by CRAMP-34 at 62.5 μg/mL for 1 hour. There were 6 samples in both the CRAMP-34 group and the control group. The samples were analyzed by LCMS, and the data were extracted and preprocessed using Mas-terView (SCIEX). (A) Volcano plots showed the fold change of extracellular metabolites in CRAMP-34 group (EP) vs control group (EC). The green dots represent significantly down-regulated metabolites (73), the red dots represent significantly up-regulated metabolites (185), and grey dots represent non-significantly changed differential metabolites. (B) Some representative differential metabolites. (C) The bubble diagram was analyzed using KEGG metabolism pathway enrichment. The p value is the significance of enrichment of this metabolic pathway. The ordinate is the name of the metabolic pathway; the abscissa is the rich factor. The larger the rich factor, the more metabolites enriched in the pathway. The size of the dots represents the number of differential metabolites enriched into the pathway.
Figure 4
Figure 4
Effects of Glutamate and Succinate on PAO1 preformed biofilms. The biomass of the preformed 96-h-old biofilms treated at 37°C with different concentrations of glutamate (A) and succinate (B) were measured after 24 h. *P < 0.05 and **P < 0.01 when compared to the control group.
Figure 5
Figure 5
CRAMP-34 combined with antibiotics on biofilm dispersed bacteria. CRAMP-34 was used to disperse the biofilm that was pre-formed for 3 days, and the dispersed cells were collected for testing. (A) The time killing curve (TKC) of CRAMP-34 (1/4MIC) combined with vancomycin (1/2,1/4,1/8 MIC). (B) TKC of CRAMP-34 (1/4MIC) combined with colistin (1/2,1/4,1/8 MIC).
Figure 6
Figure 6
CRAMP-34 delaying antibiotic resistance in dispersed bacteria. The dispersed cells were collected for testing the ability of CRAMP-34 combined with antibiotics to delay the development of drug resistance by 25 generations of continuous induction at the sub inhibitory concentration, with planktonic cells as control. (A) The change multiple of the MIC value of CRAMP-34 combined with ciprofloxacin and colistin against planktonic cells. (B) The change multiple of the MIC value of CRAMP-34 combined with ciprofloxacin and colistin against dispersed cells.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This research was funded by Chongqing Postgraduate Scientific Research Innovation Project (CYS21134, CYS22247), National Center of Technology Innovation for Pigs (NCTIP-XD/B12, NCTIP-XD/C17), Chongqing Technical Innovation and Application Development Special General Project (CSTB2023TIAD-LDX0006), Fundamental Research Funds for Central Universities (XDJK2019B040, SWU-KQ22045), the Project of Shandong Province on the Transformation of Scientific and Technological Achievements (2022LYXZ030). Performance Guidance Project for Scientific research institutions (19538).

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