MRC Centre for Medical Mycology, University of Exeter, Exeter, United Kingdom.
Aberdeen Fungal Group, University of Aberdeen, Institute of Medical Sciences, Foresterhill, Aberdeen, United Kingdom.
Microbiol Spectr. 2023 Jun 15;11(3):e0113523. doi: 10.1128/spectrum.01135-23. Epub 2023 May 9.
Fc-C-type lectin receptor (Fc-CTLRs) probes are soluble chimeric proteins constituted of the extracellular domain of a CTLR fused with the constant fraction (Fc) of the human IgG. These probes are useful tools to study the interaction of CTLRs with their ligands, with applications similar to those of antibodies, often in combination with widely available fluorescent antibodies targeting the Fc fragment (anti-hFc). In particular, Fc-Dectin-1 has been extensively used to study the accessibility of β-glucans at the surface of pathogenic fungi. However, there is no universal negative control for Fc-CTLRs, making the distinction of specific versus nonspecific binding difficult. We describe here 2 negative controls for Fc-CTLRs: a Fc-control constituting of only the Fc portion, and a Fc-Dectin-1 mutant predicted to be unable to bind β-glucans. Using these new probes, we found that while Fc-CTLRs exhibit virtually no nonspecific binding to Candida albicans yeasts, Aspergillus fumigatus resting spores strongly bind Fc-CTLRs in a nonspecific manner. Nevertheless, using the controls we describe here, we were able to demonstrate that A. fumigatus spores expose a low amount of β-glucan. Our data highlight the necessity of appropriate negative controls for experiments involving Fc-CTLRs probes. While Fc-CTLRs probes are useful tools to study the interaction of CTLRs with ligands, their use is limited by the lack of appropriate negative controls in assays involving fungi and potentially other pathogens. We have developed and characterized 2 negative controls for Fc-CTLRs assays: Fc-control and a Fc-Dectin-1 mutant. In this manuscript, we characterize the use of these negative controls with zymosan, a β-glucan containing particle, and 2 human pathogenic fungi, Candida albicans yeasts and Aspergillus fumigatus conidia. We show that A. fumigatus conidia nonspecifically bind Fc-CTLRs probes, demonstrating the need for appropriate negative controls in such assays.
Fc-C 型凝集素受体 (Fc-CTLRs) 探针是由 CTLR 的细胞外结构域与人类 IgG 的恒定区 (Fc) 融合而成的可溶性嵌合蛋白。这些探针是研究 CTLR 与其配体相互作用的有用工具，其应用类似于抗体，通常与广泛可用的靶向 Fc 片段的荧光抗体（抗 Fc）结合使用。特别是，Fc-Dectin-1 已被广泛用于研究致病真菌表面β-葡聚糖的可及性。然而，Fc-CTLRs 没有通用的阴性对照，这使得区分特异性结合与非特异性结合变得困难。我们在这里描述了 2 种 Fc-CTLRs 的阴性对照：仅由 Fc 部分组成的 Fc 对照，以及一种预测无法结合β-葡聚糖的 Fc-Dectin-1 突变体。使用这些新探针，我们发现虽然 Fc-CTLRs 实际上对白色念珠菌酵母没有非特异性结合，但烟曲霉静止孢子以非特异性方式强烈结合 Fc-CTLRs。然而，使用我们在这里描述的对照，我们能够证明烟曲霉孢子暴露了少量的β-葡聚糖。我们的数据强调了在涉及 Fc-CTLRs 探针的实验中使用适当的阴性对照的必要性。虽然 Fc-CTLRs 探针是研究 CTLR 与配体相互作用的有用工具，但由于缺乏针对真菌和潜在其他病原体的适当阴性对照，其应用受到限制。我们已经开发并表征了 2 种用于 Fc-CTLRs 测定的阴性对照：Fc 对照和 Fc-Dectin-1 突变体。在本文中，我们用β-葡聚糖颗粒和 2 种人类致病性真菌，白色念珠菌酵母和烟曲霉分生孢子，来描述这些阴性对照的使用。我们表明，烟曲霉分生孢子非特异性地结合 Fc-CTLRs 探针，这表明在这些测定中需要适当的阴性对照。
