2022 Fiscal Year Research-status Report
Role of extracellular vesicles as communication mediators between pathogens to potentiate pathogenicity in wound infections
| Project/Area Number |
22K15454
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| Research Institution | Hirosaki University |
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Principal Investigator |
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| Project Period (FY) |
2022-04-01 – 2025-03-31
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| Keywords | Staphylococcus aureus / Pseudomonas aeruginosa / Extracellular vesicles |
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| Outline of Annual Research Achievements |
The co-infection of S. aureus and P. aeruginosa is common in patients with chronic wounds, but little is known about their synergistic effect mediated by extracellular vesicles (EVs). In this study, we investigate the effect of EVs derived from S. aureus (SaEVs) and P. aeruginosa (PaEVs) on the pathogenicity of their bacterial counterparts. In FY2022, we succeeded in the isolation and purification of the EVs from both pathogens. The effects of SaEVs, and PaEVs on their bacterial counterparts included growth, antibiotic resistance, biofilm formation, and pathogenicity were performed. We found that PaEVs inhibited the growth of S. aureus. In contrast, SaEVs did not have an effect on the growth of P. aeruginosa. Because of PaEVs inhibited S. aureus growth, the following experiments such as biofilm formation, antibiotic resistance, and cell infection were not investigated. We found SaEVs did not change the antibiotic-resistance pattern of P. aeruginosa. Interestingly, P. aeruginosa-treated with SaEVs had higher biofilm formation than non-treated P. aeruginosa with SaEVs. In addition, the SaEVs enhanced the human keratinocyte cell line’s invasion of P. aeruginosa. In a further study, we will investigate the molecular mechanisms of SaEVs to promote P. aeruginosa biofilm formation and host cell invasion. Furthermore, the mechanism by which PaEVs inhibit S. aureus will investigate.
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| Current Status of Research Progress |
Current Status of Research Progress
2: Research has progressed on the whole more than it was originally planned.
Reason
During the first fiscal year, the PI did plan to isolate and purify the extracellular vesicles (EVs) obtained from the culture supernatants of S. aureus (SaEVs) and P. aeruginosa (PaEVs). Consequently, the effect of SaEVs and PaEVs on the bacterial counterpart, including the growth, antibiotic resistance, biofilm formation, and infection ability of human keratinocyte cell line (HaCaT), was evaluated. As a result, the PI successfully isolated the EVs from those bacteria by step-gradient ultracentrifugation, which was confirmed by a transmission electron microscope. In addition, the effect of SaEVs, and PaEVs on their bacterial counterparts was studied. We found that SaEVs significantly affect the biofilm formation of P. aeruginosa and enhance the infection ability to HaCaT cell line. In contrast, the PaEVs inhibited the growth of S. aureus.* Taken all together, the PI could achieve the goals mentioned in the research detail of the application form. Therefore, the PI would self-evaluate as (2)Progressing rather smoothly as mentioned above.
* According to PaEVs, the growth of S. aureus was inhibited. The following experiments as biofilm formation, antibiotic resistance, and infection ability were not performed.
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| Strategy for Future Research Activity |
In FY2022, we found the interaction of two pathogens, S. aureus, and P. aeruginosa, via EVs. The SaEVs increased the biofilm formation of P. aeruginosa and enhanced the infection ability of bacteria in human keratinocyte cell lines. In contrast, the PaEVs inhibited the growth of S. aureus. To investigate the molecular mechanism of SaEVs of the pathogenesis of P. aeruginosa and PaEVs inhibited S. aureus growth, the research plan is described below.
1)Observing the fusion of SaEVs and PaEVs with their bacterial counterparts. The EVs will be labeled with octadecyl rhodamine chloride, and the fusion will be observed by a Confocal Quantitative Image Cytometer.
2)Identifying the targeted molecules of SaEVs and PaEVs that affect the pathogenicity of their bacterial counterparts. The proteomic analysis of P. aeruginosa (pretreated with SaEVs) and S. aureus (pretreated with PaEVs) will be performed. The fold change of the proteins in comparison with non-treated bacterial cells will be compared. The gene expression of the candidate proteins will be confirmed by real-time quantitative reverse transcription PCR.
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