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Biologia

定量肽对白色念珠菌的抗真菌活性

Published: January 13, 2023
doi:
10.3791/64416
, ,
1Department of Chemical and Biomolecular Engineering,University of Maryland

Summary

该协议描述了一种获取肽和其他化合物（例如小分子抗真菌剂）抗真菌活性的定量数据的方法，用于对抗 白色念珠菌。它使用光密度而不是计数集落形成单位来量化生长抑制，从而节省时间和资源。

Abstract

对 白色念珠菌 进行抗真菌药敏试验的传统方法既耗时又缺乏定量结果。例如，一种常见的方法依赖于在琼脂平板上用不同浓度的抗真菌分子处理的细胞，然后计数菌落以确定分子浓度与生长抑制之间的关系。这种方法需要许多板和大量时间来计数菌落。另一种常见的方法通过目视检查用抗真菌剂处理的培养物来确定抑制生长所需的最低浓度，从而消除平板和菌落计数;然而，目视检查只能产生定性结果，并且丢失了亚抑制浓度下生长的信息。该协议描述了一种测量 白色念珠菌 对抗真菌肽的敏感性的方法。通过依靠培养物的光密度测量，该方法减少了获得不同肽浓度下培养物生长定量结果所需的时间和材料。使用适当的缓冲液在96孔板中将真菌与肽孵育，对照代表无生长抑制和完全生长抑制。与肽孵育后，将所得细胞悬液稀释以降低肽活性，然后生长过夜。过夜生长后，测量每个孔的光密度并与阳性和阴性对照进行比较，以计算每个肽浓度下产生的生长抑制。使用该测定的结果与使用在琼脂平板上接种培养物的传统方法的结果相当，但该协议减少了塑料浪费和计数菌落所花费的时间。尽管该协议的应用集中在抗真菌肽上，但该方法也适用于测试具有已知或疑似抗真菌活性的其他分子。

Introduction

白色念珠 菌是人类微生物群的成员，该微生物群定植于许多位置，包括口腔、皮肤、胃肠道和阴道1.对于因人类免疫缺陷病毒（HIV）和免疫抑制治疗等疾病而免疫功能低下的患者，白色念珠菌的定植可能导致局部或全身 念珠菌 病2，3。使用目前可用的小分子抗真菌疗法，如两性霉素 B、唑类或棘白菌素类药物，可能会因溶解度和毒性问题以及感染对治疗药物的耐药性而变得复杂4，5。由于当前抗真菌剂的局限性，研究人员不断寻找对 白色念珠菌具有活性的新抗真菌分子。

抗菌肽（AMP）是当前小分子抗真菌剂6，7，8的潜在替代品，并且与小分子药物相比，被认为不易产生耐药性9。AMP是一组不同的肽，但它们通常是阳离子的，具有广泛的活性10，11，12。具有抗白色念珠菌活性的AMP包括来自组抑素和cecropin家族13，14，15的众所周知的肽，以及最近描述的肽，如ToAP2，NDBP-5.7和组抑素5变体K11R-K17R 16，17。由于它们具有治疗念珠菌感染的潜力，因此识别和设计针对白色念珠菌的新AMP是许多研究小组的重要目标。

作为开发靶向白色念珠菌的有效AMP（和其他抗真菌剂）的过程的一部分，体外测试通常用于鉴定有前途的肽。测试针对白色念珠菌的抗真菌活性的方法通常涉及在96孔板中用连续稀释的AMP（缓冲液或培养基）孵育细胞。有几种方法可用于评估孵育后的抗真菌活性。临床实验室标准协会描述的一种技术使用对孔浊度的纯目视评估来确定完全抑制生长的最小浓度（MIC）（对于选定的抗真菌剂，如唑类和棘白菌素类药物至少抑制50%），并且不提供亚MIC浓度下生长的定量18.另一种常用的方法包括通过将孔的内容物铺在琼脂平板上，孵育平板，然后计数平板上菌落形成单位（CFU）的数量来量化AMP孵育后的活力。该方法已用于评估许多肽，包括基于组抑素5的肽LL-37和人乳铁蛋白19，20，21。该技术需要相对大量的琼脂和大量的平板，并且涉及平板上CFU的繁琐计数。为了获得更多定量数据，同时产生更少的塑料废物并避免计数CFU，可以使用孔的内容物在另一个96孔板中接种新鲜培养基。孵育新接种的板后，可以通过在吸光度酶标仪上测量600nm处的光密度（OD600）来量化生长。该方法已用于测定组抑素5及其降解片段和细胞穿透肽17，22，23，24，25的抗真菌活性。

该协议描述了如何测试肽的抗真菌活性，并使用OD600 方法来量化由于肽引起的 白色念珠菌 活力的降低。

Protocol

马里兰大学帕克分校机构生物安全委员会（IBC）批准了该议定书中白色 念珠菌 的工作（PN 274）。本研究使用了 白色念珠菌菌 株SC5314（见 材料表）;但是，也可以使用任何其他菌株。 1. 缓冲液、无菌水和培养基的制备 在pH 7.4下制备无菌0.1M磷酸钠缓冲液（NaPB）26 ，并用无菌水稀释至2mM和1mM。对于该协议的大多数…

Representative Results

与电镀样品和计数CFU相比，使用OD600 测量来量化抗真菌肽引起的生长减少可节省大量时间。本协议中描述的方法需要在三个不同的日子内完成这些步骤。在第一天，大约需要1小时来制备缓冲液和培养基（不包括灭菌时间）并接种 白色念珠菌 的起始培养物以进行过夜孵育。在第二天，步骤需要5-6小时（包括传代时间）以准备用于17小时孵育的96孔板。第三天，这些步骤可以在不到1小…

Discussion

该协议描述了一种有效的方法，用于获取有关AMP对真菌病原体 白色念珠菌的抗真菌活性的定量数据。测试肽和其他抗真菌剂的一种常见替代方法是临床实验室标准协会（CLSI）标准M2718中描述的肉汤微量稀释，但该标准侧重于获得定性视觉结果而不是定量结果。另一种替代方法是使用类似于本协议中描述的方法制备肽并将其与细胞孵育，然后将孔的内容物铺在琼脂上以计数…

Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作得到了美国国立卫生研究院（R03DE029270，T32AI089621B），美国国家科学基金会（CBET 1511718），教育部（GAANN-P200A180093）和马里兰大学跨校区种子资助的支持。

Materials

96-well plates (round bottom) VWR 10062-902
Absorbance microplate reader N/A N/A Any available microplate reader is sufficient
C. albicans strain SC5314 ATCC  MYA-2876 Outro C. albicans may also be used
Hemocytometer N/A N/A Can be used to make a standard curve relating cell number to OD600
Microplate shaker VWR 2620-926
Peptide(s) N/A N/A Peptides can be commercially synthesized by any reliable vendor; a purity of ≥95% and trifluoroacetic acid salt removal to hydrochloride salt are recommended
Reagent reservoirs for multichannel pipettors VWR 18900-320 Simplifies pipetting into multiwell plates with multichannel pipettor
Sodium phosphate, dibasic Fisher Scientific BP332-500 For making NaPB
Sodium phosphate, monobasic Fisher Scientific BP329-500 For making NaPB
UV spectrophotometer N/A N/A Any available UV spectrophotometer is sufficient
YPD medium powder BD Life Sciences 242820 May also be made from yeast extract, peptone, and dextrose
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Tags

Keywords: PeptidesAntifungal ActivityCandida AlbicansQuantificationMicrodilution AssayYeast-forming CellsAntifungal AgentsDilution SeriesCell DensitySodium Phosphate Buffer
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Makambi, W. K., Ikonomova, S. P., Karlsson, A. J. Quantifying the Antifungal Activity of Peptides Against Candida albicans. J. Vis. Exp. (191), e64416, doi:10.3791/64416 (2023).
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