酵母孢子在哺乳动物细胞中能够被有效地内化,因为孢子壁包含即使在非吞噬细胞中也能诱导吞噬作用/微胞吞作用的分子。在该项研究中,建立了使用孢子壁材料将微米级的颗粒递送至非吞噬细胞的方法。采用静电吸附或交联的带有表面氨基基团的胶乳珠(直径2 μm)对孢子的高盐洗脱液进行处理。将乳胶珠与HEK293T细胞孵育,以测试它们是否在细胞中被内化。孢子壁衍生的分子可以通过静电相互作用附着到珠子上,也可以诱导乳胶珠子的吞噬。通过将孢子壁材料交联到珠子上可以进一步提高吞噬效率。结果表明,孢子壁洗脱液可以诱导微米级颗粒的吞噬。因此,孢子壁材料可以应用于细胞递送系统,该方法特别有利于将相对较大的颗粒递送至非吞噬细胞。
Yeast spores are efficiently internalized in mammalian cells because the spore wall contains molecules that can induce phagocytosis/micropinocytosis even in nonprofessional phagocytes. In this study, a delivery method of μm sized particles to nonprofessional phagocytes using spore wall materials was built. High salt eluent of spores was either electrostatically attached or crosslinked to latex beads (Φ=2 μm) with surface amino groups. The latex beads were incubated with HEK293T cells to test whether they were internalized in the cells. Spore wall derived molecules can induce uptake of the latex beads even when they were attached to the beads via electrostatic interaction. The uptake efficiency can be improved by crosslinking the spore wall materials to the beads. The results showed that spore wall eluent could induce uptake of μm sized particles. Thus, the spore wall materials could be applied to cell delivery systems. The method would be beneficial particularly to deliver relatively larger particles to nonprofessional phagocytes.
[1] NEIMAN A M.Ascospore formation in the yeast Saccharomyces cerevisiae[J].Microbiology and Molecular Biology Reviews, 2005, 69(4):565-584.
[2] 王晓文, 殷政, 高晓冬, 等.表达人源 HPD 基因对酿酒酵母孢子壁二酪氨酸层正确组装的干扰[J].食品与发酵工业, 2019, 45(15):17-23.
WANG X W, YIN Z, GAO X D, et al.Expression of human HPD gene interferes correct assembly of the dityrosine layer on the spore wall of Saccharomyces cerevisiae[J].Food and Fermentation Industries, 2019, 45(15):17-23.
[3] SHI L B, LI Z J, TACHIKAWA H, et al.Use of yeast spores for microencapsulation of enzymes[J].Applied and Environmental Microbiology, 2014, 80(15):4 502-4 510.
[4] BRIZA P, BREITENBACH M, ELLINGER A, et al.Isolation of two developmentally regulated genes involved in spore wall maturation in Saccharomyces cerevisiae[J].Genes & Development, 1990, 4(10):1 775-1 789.
[5] LONGTINE M S, RD M A K, DEMARINI D J, et al.Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae[J].Yeast, 1998, 14(10):953-961.
[6] NEIMAN A M. Sporulation in the budding yeast Saccharomyces cerevisiae[J].Genetics, 2011, 189(3):737-765.
[7] BASIONY M, YANG Y, LIU G Y, et al.Studies on the properties of the sporulation specific protein dit1 and its product formyl tyrosine[J].Journal of Fungi, 2020, 6(2):77.
[8] JIN Y F, OKAMOTO M, CHIBANA H, et al.Functional characteristics of Svl3 and Pam1 that are required for proper cell wall formation in yeast cells[J].Yeast, 2020, 37(7-8):359-371.
[9] YANG Y, WANG Q, LIU G Y, et al.Studies on the mechanism of non-phagocytic uptake of budding yeast spores in mammalian cells[C].The Joint Annual Meeting of 71st JSDB and 19th PSSJ.KOBE:Office of JSDB and PSSJ, 2019.
[10] HU J W, HAN J Z, LI H R, et al.Human embryonic kidney 293 cells:A vehicle for biopharmaceutical manufacturing, structural biology, and electrophysiology[J].Cells Tissues Organs, 2018, 205(1):1-8.
[11] SAKAGUCHI M, MURATA H, YAMAMOTO K, et al.TIRAP, an adaptor protein for TLR2/4, transduces a signal from RAGE phosphorylated upon ligand binding[J].PLoS One, 2011, 6(8):e23132.
[12] MEDRAÑO-FERNANDEZ I, REYES R, OLAZABAL I, et al.RIAM (Rap1-interacting adaptor molecule) regulates complement-dependent phagocytosis[J].Cellular and Molecular Life Sciences:CMLS, 2013, 70(13):2 395-2 410.
[13] TANG C, ZHANG Y X, TANG D, et al.An easy NaV1.8 channel expression system based on HEK293T cell line[J].Toxicon, 2019, 158(20):518-524.
[14] ALIPANAHI B, DELONG A, WEIRAUCH M T, et al.Predicting the sequence specificities of DNA- and RNA-binding proteins by deep learning[J].Nature Biotechnology, 2015, 33(8):831-838.
[15] GAO C, HANES M S, BYRD-LEOTIS L A, et al.Unique binding specificities of proteins toward isomeric asparagine-linked glycans[J].Cell Chemical Biology, 2019, 26 (4):535-547.
[16] OKADA H, NEIMAN A M, OHYA Y.Assay for spore wall integrity using a yeast predator[J].Cold Spring Harbor protocols, 2016(8):138-142.
[17] LIU G Y, GAO X D, NAKANISHI H.Unique properties of the S.cerevisiae spore wall and its applications[J].Trends in Glycoscience and Glycotechnology, 2020, 32(190):E189-E193.
[18] RAHA S, MERANTE F, PROTEAU G, et al.Simultaneous isolation of total cellular RNA and DNA from tissue culture cells using phenol and lithium chloride[J].Gene Analysis Techniques, 1990, 7(7):173-177.
[19] SATO N, FUJIMURA T, MASUZAWA M, et al.Recombinant Mycobacterium leprae protein associated with entry into mammalian cells of respiratory and skin components[J].Journal of Dermatological Science, 2007, 46(2):101-110.
