Abstract
Cyclo(Phe-Pro) (cFP), produced by the Vibrio species, plays the dual roles of being a signaling molecule and a virulence factor. Acting modes of this compound have recently been characterized at the molecular level. Nevertheless, the method by which this compound passes across biological membranes remains obscure. Using radiolabeled cFP, we examined the kinetics of transport for this compound across membranes using V. vulnificus, Escherichia coli, and sheep red blood cells. We observed that cFP was taken up by these cells in a concentration-dependent manner and was not affected by the addition of the proton ionophore carbonyl cyanide m-chlorophenyl hydrazone (CCCP), suggesting that cFP is taken up by passive transport. The kinetics of uptake of cFP by the above three types of cells revealed no significant differences, indicating that no specific protein is involved in this process. When the intracellular accumulation of cFP in the tested cells was measured, the concentrations did not exhibit significant differences between the 1-min and 10-min time points after cFP was added to the culture. In contrast, the intracellular concentration of fumarate, which is well known to be taken up by cells via active transport, was significantly higher at the 10-min than at the 1-min time point after addition. Taken together, this study shows that cFP is a diffusible molecule that does not require energy for transportation across biological membranes, and that cFP does not need membrane machinery in order to cross membranes and consequently act as a virulence factor or signal.
Key Points
• Kinetics of cFP uptake into cells of V. vulnificus, E. coli, or RBS was studied.
• The uptake was not saturated and required no energy, indicating passive transport.
• The lack of cell specificity in cFP uptake means no specific protein is needed.
• Therefore, the cFP moves across the biological membrane by simple diffusion.
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Funding
This work was supported by grants from the National Research Foundation (NRF) of Korea funded by the Ministry of Science and ICT (2017R1A2B2006966 and 2019R1A2C2084282), Republic of Korea.
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KK and OK designed the research, and NP conducted the experiments. NP and KK wrote the manuscript. YC conducted the experiments for revision. All authors have read and approved the manuscript.
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Park, NY., Cho, Y.B., Kim, O.B. et al. Cyclo(Phe-Pro) produced by Vibrio species passes through biological membranes by simple diffusion. Appl Microbiol Biotechnol 104, 6791–6798 (2020). https://doi.org/10.1007/s00253-020-10646-4
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DOI: https://doi.org/10.1007/s00253-020-10646-4