Team:Paris Bettencourt/Biocompatibility Testing

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Biocompatibility Testing

Majority of antimicrobial peptides are cationic, known to interact with and destabilize the negatively charged bacterial membrane via forming pores or transmembrane channels . This leads to cellular stress, loss of membrane potential and ultimately cell death. However these antimicrobial peptides can also interact and form pores in mammalian cells as their membranes are also negatively charged, thus leading to cytotoxicity. Therefore it becomes necessary to evaluate the membrane activity of antimicrobial peptides on mammalian cells.

Vesicle-encapsulated fluorescent dyes and quenchers (in our case: calcein and cobalt respectively) that change fluorescence on release are commonly used for studying the permeabilization of membranes by peptides or proteins. Leakage of these fluorescent dyes can be measured and could lend an insight on the mechanisms of permeabilization allowing us to confirm the efficiency of these antimicrobial peptides.

Figure 2:Star core induced vesicle leakage leads to release of fluorescent dye.

As demonstrated in 1995 by Ladokhin et al, Vesicle-encapsulated Calcein leakage allows us to confirm that Star-cores exhibit a membrane specific action. Moreover, we can test for selectivity and biocompatibility by studying the permeabilization of vesicles representing bacterial and mammalian membranes respectively.

Results

Construct: Ferritin-Ovispirin (BBa_K2738006)

Figure 1:Mammalian liposome leakage induced by ferritin-ovispirin StarCore (A) and the kinetics of liposome leakage. (B) Ovispirin (5 uM) and ferritin-ovispirin (3 uM) are used at their MIC concentration obtained from previous results.

Ovispirin alone lyses the liposome indicating damage to mammalian cell membrane. Ferritin-ovispirin has a reduced effect on the liposome leakage indicating an improvement in biocompatibility towards mammalian cell membrane. The Starcore ferritin-ovispirin has a safer profile in terms of membrane activity as compared to ovispirin alone at their respective MIC concentration.

Construct: Lyase cage-Enterocin (Bba_K2738104)

Figure 2:Mammalian liposome leakage induced by lyase cage-Enterocin A StarCore (A) and the kinetics of liposome leakage.(B) Ovispirin (5 uM) and lyase-ovispirin (3.9 uM) are used at their MIC concentration obtained from previous results.

Ovispirin alone lyses the liposome indicating damage to mammalian cell membrane. Lyase-cage-enterocin has a reduced effect on the liposome leakage indicating an improvement in biocompatibility towards mammalian cell membrane. The Starcore ferritin-ovispirin has a safer profile in terms of membrane activity as compared to ovispirin alone at their respective MIC concentration.

Conclusions

Fusing antimicrobial peptides to scaffold proteins not only improves their antibacterial activity but also confers biocompatibility and makes them less effective on mammalian cell membranes. Our results are supported by previous findings from Lam et al. 2016, where they demonstrated that structurally designed antimicrobials are biocompatible while simultaneously exhibiting good antibacterial activity.

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
paris-bettencourt-2018@cri-paris.org