Harris Mark, Mora-Montes Héctor M, Gow Neil A R, Coote Peter J
Centre for Biomolecular Sciences, School of Biology, University of St Andrews, The North Haugh, St Andrews KY16 9ST, UK.
School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK.
Microbiology (Reading). 2009 Apr;155(Pt 4):1058-1070. doi: 10.1099/mic.0.026120-0.
The outermost layer of the Candida albicans cell wall is enriched with mannosylated glycoproteins. We have used a range of isogenic glycosylation mutants of C. albicans, which are defective to varying degrees in cell wall protein mannosylation, to investigate the role of the outermost layer of the yeast cell wall in mediating the fungicidal action of the cationic, alpha-helical antimicrobial peptide dermaseptin S3(1-16) [DsS3(1-16)]. The degree of phosphomannan loss, and concomitant reduction in surface negative charge, from the series of glycosylation mutants correlated with reduced levels of peptide binding to the cells. In turn, the reduced peptide binding correlated with enhanced resistance to DsS3(1-16). To ascertain whether DsS3(1-16) binds to negatively charged phosphate, we studied the effect of exogenous glucosamine 6-phosphate, and glucosamine hydrochloride as a negative control, on the antifungal efficacy of DsS3(1-16). Glucosamine 6-phosphate retarded the efficacy of DsS3(1-16), and this was attributed to the presence of phosphate, because addition of identical concentrations of glucosamine hydrochloride had little detrimental effect on peptide efficacy. Fluorescence microscopy with DsS3(1-16) tagged with fluorescein revealed that the peptide binds to the outer surface of the yeast cell, supporting our previous conclusion that the presence of exterior phosphomannan is a major determinant of the antifungal potency of DsS3(1-16). The binding of the peptide to the cell surface was a transient event that was followed by apparent localization of DsS3(1-16) in the vacuole or dissemination throughout the entire cytosol. The presence of glucosamine 6-phosphate clearly reduced the proportion of cells in the population that showed complete cytosolic staining, implying that the binding and entry of the peptide into the cytosol is significantly reduced due to the exogenous phosphate sequestering the peptide and reducing the amount of peptide able to bind to the surface phosphomannan. In conclusion, we present evidence that an antimicrobial peptide, similar to those employed by cells of the human immune system, has evolved to recognize molecular patterns on the surface of pathogens in order to maximize efficacy.
白色念珠菌细胞壁的最外层富含甘露糖基化糖蛋白。我们使用了一系列白色念珠菌的同基因糖基化突变体，这些突变体在细胞壁蛋白甘露糖基化方面存在不同程度的缺陷，以研究酵母细胞壁最外层在介导阳离子α-螺旋抗菌肽皮肤防御素S3(1-16)[DsS3(1-16)]的杀真菌作用中的作用。一系列糖基化突变体中磷酸甘露聚糖的损失程度以及随之而来的表面负电荷减少与肽与细胞结合水平的降低相关。反过来，肽结合减少与对DsS3(1-16)的抗性增强相关。为了确定DsS3(1-16)是否与带负电荷的磷酸盐结合，我们研究了外源性6-磷酸葡萄糖胺以及作为阴性对照的盐酸葡萄糖胺对DsS3(1-16)抗真菌功效的影响。6-磷酸葡萄糖胺延缓了DsS3(1-16)的功效，这归因于磷酸盐的存在，因为添加相同浓度的盐酸葡萄糖胺对肽的功效几乎没有不利影响。用荧光素标记的DsS3(1-16)进行荧光显微镜观察发现，该肽结合到酵母细胞的外表面，支持了我们之前的结论，即外部磷酸甘露聚糖的存在是DsS3(1-16)抗真菌效力的主要决定因素。肽与细胞表面的结合是一个短暂的事件，随后DsS3(1-16)明显定位在液泡中或扩散到整个细胞质中。6-磷酸葡萄糖胺的存在明显降低了群体中显示完全细胞质染色的细胞比例，这意味着由于外源性磷酸盐螯合肽并减少了能够结合到表面磷酸甘露聚糖上的肽量，肽与细胞质的结合和进入显著减少。总之，我们提供的证据表明，一种抗菌肽，类似于人类免疫系统细胞所使用的抗菌肽，已经进化到能够识别病原体表面的分子模式，以最大限度地提高功效。
