| Project/Area Number |
22K15454
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 49050:Bacteriology-related
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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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| Project Status |
Granted (Fiscal Year 2023)
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| Budget Amount *help |
¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000)
Fiscal Year 2024: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2023: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2022: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
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| Keywords | Staphylococcus aureus / Pseudomonas aeruginosa / Extracellular vesicles / Extracellular vesicle / Interaction / Pathogenicity |
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| Outline of Research at the Start |
Chronic wound infections, especially by multidrug-resistant organism, becomes a challenge for treatment. Staphylococcus aureus and Pseudomonas aeruginosa are commonly found in chronic wounds. The synergistic effects of these two bacteria were demonstrated. However, their mediators are not yet fully reported. Here, we elucidate the role of extracellular vesicles (EVs) as mediators in the pathogenicity of S. aureus and P. aeruginosa. Understanding these mediators’ functions will be useful for applying the EVs as a target tool for the treatment of chronic wound infections.
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| Outline of Annual Research Achievements |
In FY2022-23, we investigated the effect of EVs derived from S. aureus (SaEVs) and P. aeruginosa (PaEVs) on the pathogenicity of their bacterial counterparts. Our findings revealed that SaEVs had no impact on the growth and antibiotic pattern of P. aeruginosa. Surprisingly, SaEVs were found to enhance LPS production, biofilm formation, and the expression of polysaccharide polymerization-related genes in P. aeruginosa after SaEV treatment. Additionally, SaEVs increased the epithelial cell invasion of P. aeruginosa. Conversely, the uptake of P. aeruginosa by RAW264.7 macrophages was impaired after pretreatment with SaEVs. Regarding the inhibitory effect of PaEVs on S. aureus growth, we studied the mechanism in more detail. Our result revealed a reduction in lactate dehydrogenase 2 and formate acetyltransferase enzyme expression in the pyruvate fermentation pathway of S. aureus after PaEV treatment at both gene and protein levels. Furthermore, this inhibitory effect of PaEVs was abolished by supplementation with pyruvate or oxygen. It indicates that PaEVs inhibit the growth of S. aureus by suppressing the pyruvate fermentation pathway. In a further study, we plan to analyze the molecules in SaEVs and PaEVs that affect the P. aeruginosa pathogenicity and S. aureus growth. The EVs will be sequentially treated with enzymes including lipase, DNase, RNase, and protease. The activity of enzyme-treated EVs will be used to detect the promotion of P. aeruginosa pathogenicity and the inhibition of S. aureus growth.
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| Current Status of Research Progress |
Current Status of Research Progress
1: Research has progressed more than it was originally planned.
Reason
In FY2022-23, PI planed to isolate and purify the extracellular vesicles derived from S. aureus (SaEVs), and P. aeruginosa (PaEVs) culture supernatant. The effects of SaEVs and PaEVs on their respective bacterial counterparts were investigated. PI successfully isolated EVs and studied their effect on bacterial counterparts. SaEVs were found to enhance P. aeruginosa pathogenicity by increasing lipopolysaccharide biosynthesis, promoting biofilm formation, epithelial cell invasion, and impairing macrophage uptake. The mechanism of PaEVs in inhibiting S. aureus growth involved reducing lactate dehydrogenase 2 and formate acetyltransferase enzymes in the pyruvate fermentation pathway. This effect was abolished by supplementing with pyruvate or oxygen, indicating that PaEVs inhibit S. aureus growth by suppressing the pyruvate fermentation pathway. Finally, the PI and colleagues successfully published these two studies in international journals. Therefore, the PI self-evaluates as (1) Progressing More Smoothly Than Initially Planned.
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| Strategy for Future Research Activity |
In FY2023, we observed that SaEVs enhance P. aeruginosa pathogenicity by increasing lipopolysaccharide biosynthesis, promoting biofilm formation, epithelial cell invasion, and impairing macrophage uptake. The mechanism of PaEVs in inhibiting S. aureus involved reducing lactate dehydrogenase 2 and formate acetyltransferase enzymes in the pyruvate fermentation pathway. To identify the active molecules of SaEVs that promote P. aeruginosa pathogenesis and PaEVs that inhibit S. aureus growth. The PI plans to sequentially treat the SaEVs and PaEVs with lipase, DNase, RNase, and protease. After treatment with each enzyme, the activity of SaEVs and PaEVs will be used to detect the ability to promote pathogenicity and inhibit growth in P. aeruginosa and S. aureus, respectively.
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