弥散性念珠菌感染模型
Summary
凝膜状杆菌是一种播散念珠菌病的无脊椎动物模型。在这里, 我们详细介绍了感染协议, 并为模型的有效性提供了支持数据。
Abstract
念珠菌是人类在皮肤、粘膜表面和胃肠道中的常见真菌共生体。在某些条件下,念珠菌可以过度生长他们的自然定位, 导致衰弱的黏膜感染以及危及生命的全身感染, 这是一个主要的研究焦点, 因为他们相关的高死亡率。为研究疾病进展和剖析念珠菌致病性特征, 存在传播感染的动物模型。其中,麦地那龙菌蜡虫感染模型为高通量系统毒力调查提供了一个具有成本效益的实验工具。许多其他细菌和真核感染因子已在g. mellonella中得到有效研究, 以了解致病性, 使其成为一个被广泛接受的模型系统。然而, 用于感染g. mellonella 的方法的变化可以改变表型结果, 并使结果的解释复杂化。在这里, 我们概述了蜡虫模型的好处和缺点, 以研究系统念珠菌的发病机制, 并详细介绍了提高重现性的方法。我们的研究结果突出了g. mellonella 的死亡率动力学范围, 并描述了可以调节这些动力学的变量。最终, 这种方法是一个合乎道德的, 快速的, 具有成本效益的方法来研究在一个模型的传播念珠菌病的毒力。
Introduction
念珠菌是常见的人类共生体, 能够成为严重免疫功能低下和生物功能障碍患者的机会性病原体。虽然许多念珠菌类可以引起疾病,白色念珠菌是传播念珠菌病 1,2最普遍的原因。系统性疾病的原因是白色念珠菌通过直接渗透以前限制性的宿主屏障或在手术部位引入和其他身体接触 3.念珠菌利用一系列致病过程在宿主内引起全身性疾病, 包括丝状、生物膜形成、免疫细胞逃避和逃逸以及铁清除4。体外方法存在, 以调查个人的致病机制, 但动物模型继续提供了最好的选择, 以调查整个疾病结局 5,6。以前的研究详细介绍了许多有希望的体外研究毒力未能在体内繁殖7,8。因此, 仍然需要动物模型来评估体内的毒力.尽管白色念珠菌无法作为例长9自然地殖民小鼠系统, 但大多数疾病模型都依靠老鼠作为人类感染的代名词.无脊椎动物中的传播念珠菌模型包括线虫、果蝇果蝇和蜡虫的线虫, 尽管担心根本的区别在基本生理上, 宿主体温和暴露途径阻碍了它们的广泛接受 10,11 。
最近, g . mellonella蜡虫感染模型已被采用来模拟致病性的细菌和真菌病原体12,13,14。这种模式的优点包括, 与小鼠模型相比, 它的成本相对较低, 吞吐量增加, 易用性, 以及减少对动物慈善的伦理担忧。对于研究人员来说, 这转化为测试多个变量的能力增强、置信区间更强、实验速度更快、动物协议的旁路。在11、15例临床分离株的生物膜形成、丝状化和基因调控所需的基因受到干扰后, 美洛内拉已经成为快速评估白色念珠菌毒力的平台 ,16。最近的研究纳入了使用g. mellonella 进行抗真菌效果的研究, 以评估体内环境下药物活性和耐药性的药代动力学, 否则这些活性和耐药力具有挑战性和耗时17,18。然而, 据报道, 由于实验中的差异很大, 研究小组之间的重复也不一致, 从而产生不同的毒力表型, 这使得对白色念珠菌中白色念珠菌毒力的研究变得更加复杂老鼠和蜡虫之间 11,13,19, 20,21。在这里, 我们概述了一种g. mellonella 协议,以规范白色念珠菌感染, 提高毒力实验的重现性, 并证明与前面描述的小鼠毒力研究的一致性模型。
先前的研究表明, 5号染色体上的白色念珠菌交配样位点 (mtl) 调节细胞身份和交配能力, 类似于酿酒酵母和其他 ascomycete 真菌 22.白色念珠菌分离株的大多数在mtl位点是杂合, 编码每个mtla 和 mtlα等位基因 (mtla/α) 的一个, 因此是无菌的15,23,24. 由于杂合性 (loh) 丢失或突变而失去其中一个mtl等位基因, 会导致纯合mtla或mtlα菌株, 这些菌株可以经历从无菌 “白色” 状态到无菌 “白色” 状态到无菌 “白色” 状态的表型开关。配合主管 “不透明” 状态25。先前的研究表明, mtl杂合性的丧失也会降低不同应变背景的小鼠全身感染模型的毒力26。在这里, 我们详细介绍 g . mellonella模型的传播念珠菌病使用基因相似的实验集, 以描绘mtl杂合性对毒性的贡献。我们表明, mtl 构型影响白色念珠菌的致病性, 其中mtlα菌株的毒性较低, 无论是mtla/α和mtla 细胞, 类似于研究结果在小鼠感染模型26。
Protocol
Representative Results
Discussion
g. mellonella蜡虫模型是快速、可重复分析白色念珠菌毒力的有效工具。这一详细的协议依赖于在一批幼虫中一致地将确定的传染性剂量传递给同一地点。传染性剂量对 g. mellonella 的死亡率有深远的影响, 而从最初到达到收到后10天使用幼虫产生了类似的结果。失去c. albicansmtl 导致毒力下降, 与以前的实验一致的小鼠, 尽管中断 mtlα等位基因并不能改变幼?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
作者希望感谢帕米拉·华盛顿和利亚·安德森在获得用于本研究的大肠杆菌方面的帮助。
Materials
| Galleria mellonella | Snackworms.com | Buy twice as many worms as expected to use | |
| 10 uL, Model 1701 N SYR Cemented needle, 26G, type 2 syringe | Hamilton | 80000 | |
| Petri dish, 100X15 mm, 500 pack | Fisher | FB0875712 | |
| Microcentrifuge tube, 1.7 mL, 500 pack | VWR | 87003-294 | |
| Phosphate Buffered Saline (Biotechnology grade), 500 mL | VWR | 97062-818 | |
| Ethanol absolute, ≥99.5% pure, 500 mL | Millipore Sigma | EM-EX0276-1S | |
| autoclaved ddH2O |
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