Presentation Information
[P03-349]Design strategy for novel antifungal drug based on the phase partitioning property of giant fibrous proteins
○Ayasa Nagatani1, Shogo Yoshimoto2, Toki Taira3, Katsutoshi Hori2, Noriho Kamiya1,4 (1. Department of Applied Chemistry, Graduate School of Engineering, Kyushu Univ. (Japan), 2. Department of Biomolecular Engineering, Graduate School of Engineering, Nagoya Univ. (Japan), 3. Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus (Japan), 4. Division of Biotechnology, Center for Future Chemistry, Kyushu Univ. (Japan))
Keywords:
Antifungal activit,bioconjugation,chitinase,fibrous protein,aqueous two-phase system
[Purpose]
Cells contain highly crowded molecular environments in which spatial organization is required to regulate multiple biochemical reactions efficiently. Polyethylene glycol (PEG)/dextran (Dex) aqueous two-phase systems (ATPS) have been employed as simplified models of crowded intracellular environments. In these systems, concentrating proteins of interest (POIs) in the Dex-rich phase often enhances enzymatic activity and molecular recognition. However, current strategies to induce partitioning into the Dex-rich phase are often limited by low transport efficiency and poor controllability. We found that Cstalk, the trimeric protein AtaA from Acinetobacter sp. Tol 5, partitions into the Dex-rich phase. Here, we propose the giant fibrous protein as a novel recruiter to facilitate selective partitioning of POIs into polysaccharide-rich environments. Using the SpyTag (ST)–SpyCatcher (SC) system, we enabled precise control of conjugation stoichiometry and evaluated the effect of the recruiter-to-POI ratio on phase selectivity. We further investigated whether this strategy could be applied to antifungal applications by targeting polysaccharide-rich fungal cell walls.
[Methods]
Recombinant SC-Cstalk-SC and POI-ST were expressed in E. coli BL21(DE3). Conjugates were prepared by reacting in 20 mM sodium phosphate buffer (pH 7.4) via the ST-SC reaction. The effect of the reaction molar ratio (SC:ST) on antifungal activity was examined. When 1 µM SC-Cstalk-SC was reacted with 1 µM GlxChiB-ST, the condition was defined as SC:ST = 6:1.
[Results and consideration]
We first used muGFP as a model POI to evaluate the recruiter function of Cstalk. While muGFP alone showed no clear phase preference, Cstalk–muGFP conjugates exhibited partitioning into the Dex-rich phase. We demonstrated that Cstalk can transport proteins into polysaccharide-rich environments while preserving its intrinsic phase preference. We next extended this concept to natural targets by considering fungal cell walls as polysaccharide-rich environments. Given its ability to recruit proteins into such phases, Cstalk was expected to enable targeted delivery of antifungal enzymes (GlxChiB) to the fungal cell wall. Accordingly, Cstalk–GlxChiB conjugates were constructed and evaluated using Trichoderma viride. Among the tested conditions, the 6:6 conjugate exhibited enhanced antifungal activity compared with GlxChiB alone. Localization analysis revealed that GlxChiB alone primarily targeted filamentous hyphae, whereas both Cstalk and Cstalk–GlxChiB conjugates accumulated on spores. These findings indicate that enhanced antifungal activity arises from selective delivery of GlxChiB to spores via Cstalk-mediated recruitment.
[Conclusion]
We demonstrated that the giant fibrous protein Cstalk functions as a unique recruiter that selectively drives POIs into the Dex-rich phase in PEG/Dex ATPS. By applying this recruitment system to antifungal enzymes, we successfully enhanced the antifungal performance of GlxChiB.
Cells contain highly crowded molecular environments in which spatial organization is required to regulate multiple biochemical reactions efficiently. Polyethylene glycol (PEG)/dextran (Dex) aqueous two-phase systems (ATPS) have been employed as simplified models of crowded intracellular environments. In these systems, concentrating proteins of interest (POIs) in the Dex-rich phase often enhances enzymatic activity and molecular recognition. However, current strategies to induce partitioning into the Dex-rich phase are often limited by low transport efficiency and poor controllability. We found that Cstalk, the trimeric protein AtaA from Acinetobacter sp. Tol 5, partitions into the Dex-rich phase. Here, we propose the giant fibrous protein as a novel recruiter to facilitate selective partitioning of POIs into polysaccharide-rich environments. Using the SpyTag (ST)–SpyCatcher (SC) system, we enabled precise control of conjugation stoichiometry and evaluated the effect of the recruiter-to-POI ratio on phase selectivity. We further investigated whether this strategy could be applied to antifungal applications by targeting polysaccharide-rich fungal cell walls.
[Methods]
Recombinant SC-Cstalk-SC and POI-ST were expressed in E. coli BL21(DE3). Conjugates were prepared by reacting in 20 mM sodium phosphate buffer (pH 7.4) via the ST-SC reaction. The effect of the reaction molar ratio (SC:ST) on antifungal activity was examined. When 1 µM SC-Cstalk-SC was reacted with 1 µM GlxChiB-ST, the condition was defined as SC:ST = 6:1.
[Results and consideration]
We first used muGFP as a model POI to evaluate the recruiter function of Cstalk. While muGFP alone showed no clear phase preference, Cstalk–muGFP conjugates exhibited partitioning into the Dex-rich phase. We demonstrated that Cstalk can transport proteins into polysaccharide-rich environments while preserving its intrinsic phase preference. We next extended this concept to natural targets by considering fungal cell walls as polysaccharide-rich environments. Given its ability to recruit proteins into such phases, Cstalk was expected to enable targeted delivery of antifungal enzymes (GlxChiB) to the fungal cell wall. Accordingly, Cstalk–GlxChiB conjugates were constructed and evaluated using Trichoderma viride. Among the tested conditions, the 6:6 conjugate exhibited enhanced antifungal activity compared with GlxChiB alone. Localization analysis revealed that GlxChiB alone primarily targeted filamentous hyphae, whereas both Cstalk and Cstalk–GlxChiB conjugates accumulated on spores. These findings indicate that enhanced antifungal activity arises from selective delivery of GlxChiB to spores via Cstalk-mediated recruitment.
[Conclusion]
We demonstrated that the giant fibrous protein Cstalk functions as a unique recruiter that selectively drives POIs into the Dex-rich phase in PEG/Dex ATPS. By applying this recruitment system to antifungal enzymes, we successfully enhanced the antifungal performance of GlxChiB.
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