Name: quantifying the antifungal activity of peptides against candida albicans
Uploaded: 2023-01-13T14:00:00
Description: Watch this Scientific Journal Video about Quantifying the Antifungal Activity of Peptides Against Candida albicans at JoVE.com

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。

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
<ol><li>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...

<ol>
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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...

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。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>96-well plates (round bottom)</td>
			<td>VWR</td>
			<td>10062-902</td>
			<td></td>
		</tr>
		<tr>
			<td>Absorbance microplate reader</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any available microplate reader is sufficient</td>
		</tr>
		<tr>
			<td>C. albicans strain SC5314</td>
			<td>ATCC&#160;</td>
			<td>MYA-2876</td>
			<td>Other C. albicans may also be used</td>
		</tr>
		<tr>
			<td>Hemocytometer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Can be used to make a standard curve relating cell number to OD600</td>
		</tr>
		<tr>
			<td>Microplate shaker</td>
			<td>VWR</td>
			<td>2620-926</td>
			<td></td>
		</tr>
		<tr>
			<td>Peptide(s)</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Peptides can be commercially synthesized by any reliable vendor; a purity of &#8805;95% and trifluoroacetic acid salt removal to hydrochloride salt are recommended</td>
		</tr>
		<tr>
			<td>Reagent reservoirs for multichannel pipettors</td>
			<td>VWR</td>
			<td>18900-320</td>
			<td>Simplifies pipetting into multiwell plates with multichannel pipettor</td>
		</tr>
		<tr>
			<td>Sodium phosphate, dibasic</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td>For making NaPB</td>
		</tr>
		<tr>
			<td>Sodium phosphate, monobasic</td>
			<td>Fisher Scientific</td>
			<td>BP329-500</td>
			<td>For making NaPB</td>
		</tr>
		<tr>
			<td>UV spectrophotometer</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Any available UV spectrophotometer is sufficient</td>
		</tr>
		<tr>
			<td>YPD medium powder</td>
			<td>BD Life Sciences</td>
			<td>242820</td>
			<td>May also be made from yeast extract, peptone, and dextrose</td>
		</tr>
	</tbody>
</table>

quantifying the antifungal activity of peptides against candida albicans

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。

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...

Watch this Scientific Journal Video about Quantifying the Antifungal Activity of Peptides Against Candida albicans at JoVE.com

Quantifying the Antifungal Activity of Peptides Against Candida albicans

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。

定量肽对白色念珠菌的抗真菌活性

Traditional methods for performing antifungal susceptibility testing for Candida albicans are time-consuming and lack quantitative results. For example, a ...

肽正在作为新型抗真菌分子进行研究。该协议可用于研究对人类真菌病原体白色念珠菌的抗真菌功效。该技术提供了抗真菌活性的定量数据，同时与需要在琼脂上创建的其他方法相比，减少了实验时间和塑料浪费。除了测试针对白色念珠菌的活性外，该协议还可用于测试针对其他酵母形成细胞的活性并测试小分子抗真菌剂的活性首先将所需的白色念珠菌菌株接种到培养管中的10毫升液体酵母提取物 - 蛋白胨 - 葡萄糖或YPD培养基中。在旋转摇床上以 30 摄氏度和 230 rpm 的速度培养培养过夜。传代培养白色念珠菌的过夜培养物，并在600纳米波长下生长至约1.0至1.2的光密度。将每种肽溶解在无菌水中并将其稀释至所需最高浓度的两倍，然后将40微升所需的肽储备溶液添加到圆底96孔板中每行的第一孔中。接下来，向含有肽的行的第2至12列中加入20微升无菌超纯水。要将肽储备溶液连续稀释到板中至第10列，请将20微升从第1列转移到第2列，并通过上下移液进行混合。重复稀释过程直至第10列，该色谱柱将含有40微升肽溶液，稀释度为1至512。完成后，从第 10 列中取出 20 微升肽溶液并丢弃。将白色念珠菌传代培养物转移到15毫升离心管中，并在室温下以3， 900g离心3分钟。通过移液或倾析除去上清液。用1毫升2毫摩尔磷酸钠缓冲液重悬沉淀，并将悬浮液转移到1.7毫升离心管中。通过离心沉淀细胞并丢弃上清液。再次，将沉淀重悬于1毫升2毫摩尔磷酸钠缓冲液中，再洗涤2次，将洗涤的细胞留在1毫升磷酸钠缓冲液中。确定洗涤细胞悬液的细胞密度，并在2毫摩尔磷酸钠缓冲液中将悬浮液稀释至每毫升5乘以10至第5个细胞。取稀释的白色念珠菌细胞悬液，每行的第1至10列和第12列中加入20微升，然后在第11列中加入20微摩尔的2毫摩尔磷酸钠缓冲液。盖上板1并将其放入30摄氏度的培养箱中30分钟。准备一个新的 96 孔培养板以量化活力，方法是向 2 号板的所有孔中加入 100 微升 YPD，然后将 100 微升 2 毫摩尔磷酸钠缓冲液添加到板 2 的所有孔中。要稀释板1中的样品，请从培养箱中取出板1，并在每个孔中加入280微升1毫摩尔磷酸钠缓冲液。用移液管充分混合内容物后，将8微升从板1转移到板2的相应孔中。完成后，将覆盖的板 2 放在微量滴定板振荡器上，在 350 rpm 下在 30 摄氏度下放置 17 小时。通过上下移液混合板2中的所有孔，确保所有细胞都重新悬浮。使用吸光度读板器获得板2中每个孔的600纳米波长的光密度读数。将活力降低绘制为肽浓度的函数，以计算生长抑制百分比并确定肽对白色念珠菌生长的影响。将肽与白色念珠菌细胞孵育后，使用集落形成单位或CFU计数方法和结合光密度测量的所述方案定量抗真菌活性。使用光密度读数的数据具有高度可重复性，如重复的小标准偏差所示。在执行此协议时，通过在30摄氏度下孵育细胞来保持酵母形式的白色念珠菌细胞，因为光密度测量在菌丝细胞中不可靠。这种方法的效率使研究人员能够通过测试更多的肽和肽浓度来对肽活性进行高三重研究。

Research

JoVE Journal

Biology

Quantifying the Antifungal Activity of Peptides Against Candida albicans

Photo-Induced Cross-Linking of Unmodified Proteins (PICUP) Applied to Amyloidogenic Peptides

Photo-Induced Cross-Linking of Unmodified Proteins PICUP Applied to Amyloidogenic Peptides

光致交联未修改蛋白（PICUP）适用于淀粉样肽

The assembly of amyloidogenic proteins into toxic oligomers is a seminal event in the pathogenesis of protein misfolding diseases, including Alzheimer's, ...

科研

生物学

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro (Protozoa Culture) and in vivo (Microinjection into Termite Hindgut)

Testing Protozoacidal Activity of Ligand-lytic Peptides Against Termite Gut Protozoa in vitro Protozoa Culture and in vivo Microinjection into Termite Hindgut

廉政白蚁肠道原生动物催化配体肽的Protozoacidal活性测试在体外（原生动物文化）在体内（显微注射到白蚁后肠）

We are developing a novel approach to subterranean termite control that would lead to reduced reliance on the use of chemical pesticides. Subterranean ...

Quantifying Agonist Activity at G Protein-coupled Receptors

量化在G蛋白偶联受体激动剂活性

When an agonist activates a population of G protein-coupled receptors (GPCRs), it elicits a signaling pathway that culminates in the response of the cell ...

Direct Detection of the Acetate-forming Activity  of the Enzyme Acetate Kinase

醋酸形成醋酸酶激酶活性的直接检测

Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily1-5, is responsible for the reversible phosphorylation of ...

Assaying the Kinase Activity of LRRK2 in vitro

Assaying the Kinase Activity of LRRK2 in vitro

含量测定的LRRK2的激酶活性在体外</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Quantifying Single Microvessel Permeability in Isolated Blood-perfused Rat Lung Preparation

在离体血液灌注大鼠肺备量化单微血管通透性

The isolated blood-perfused lung preparation is widely used to visualize and define signaling in single microvessels. By coupling this preparation with ...

Measurement of Larval Activity in the Drosophila Activity Monitor

Measurement of Larval Activity in the Drosophila Activity Monitor

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

Measuring Lactase Enzymatic Activity in the Teaching Lab

实验教学中乳糖酶活性的测定

Understanding how enzymes work, and relating this to real life examples, is critical to a wide range of undergraduate degrees in the biological and ...

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

肉汤稀释体外筛选: 一种简便快速检测新抗真菌化合物的方法

Fungal infections have become an important medical condition in the last decades, but the number of available antifungal drugs is limited. In this ...

Quantifying Spatiotemporal Parameters of Cellular Exocytosis in Micropatterned Cells

定量微模式细胞细胞外细胞异细胞的时态参数

Live imaging of the pHluorin tagged Soluble N-ethylmaleimide-sensitive-factor Attachment protein REceptor (v-SNARE) Vesicle-associated membrane protein 7 ...