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Immunologia e infezioni

前体内分析研究胃 肠道的 念珠菌血脂磷

Published: July 01, 2020
doi:
10.3791/61488
, ,
1College of Veterinary Medicine,Midwestern University, 2Masters in Biomedical Science Program, College of Graduate Studies,Midwestern University, 3Department of Pathology and Population Medicine, College of Veterinary Medicine,Midwestern University

Summary

本研究中描述的利用肠道同质化提取物和免疫荧光染色的外体检测是一种研究胃肠道中念珠菌的催眠形态的新方法。 该方法可用于研究调节肠道形态遗传过渡的环境信号。

Abstract

胃肠道（GI） 的白种病膜形成受到各种环境信号的严格控制，在这种机会性真菌病原体的传播和发病机制中起着重要作用。然而，在体内的GI道道中可视化真菌催眠的方法具有挑战性，这限制了对控制这种形态过程的环境信号的理解。此处描述的协议演示了一种新颖的外生体方法，用于在肠道同质性提取物中可视化催眠形态。使用体外测定，这项研究证明，来自抗生素治疗小鼠的切克萨尔含量，但不是来自未经治疗的对照小鼠，促进肠道内 C.albicans 催眠膜形成。此外，从抗生素治疗的小鼠中加入特定组肠道代谢物，可不同地调节生外膜形成。综合起来，该协议代表了一种识别和研究控制胃肠道中 C.albicans 催眠形态形成的环境信号的新方法。

Introduction

念珠菌是一种机会主义的多态性真菌病原体，通常是共生性的，但可以经历形态变化，形成一种致命的形式，能够引起免疫功能低下的个体1、2、3、4、5、6、7、8、9、10、11、12、13等危及生命的感染。C. albicans是全身性耳部感染的主要原因，即使抗真菌治疗 2、14、15，死亡率也是40~u201260%。虽然C.albicans居住在不同的宿主利基，包括女性生殖系统16，17，口腔健康个体18和胃肠道（GI）19，20，大多数全身感染来自胃肠道，此外， 全身感染的来源通常被证实为胃肠道21、22、23、24、25、26、27、28、29、30、31、32、33、34。GI道区中的二比二丹致病性受多种因素的影响;然而，毒性所需的一个主要特征是，从酵母细胞形态过渡到毒性催眠细胞形态35，36，37，38，39，40，41，42，43，44。感染期间从胃肠道附着和传播的C.albicans与它从共生酵母转化为毒催眠剂的能力高度相关，使真菌能够引起侵入性疾病44，45，46，47，48，49，50，51，52，53。

肠道中的各种因素，包括n-乙酰葡萄糖胺，调节由C.albicans形成催眠。因此，缩小有关这种真菌病原体在胃肠道54、55、56中催眠形态的知识差距至关重要。最近的证据表明，各种肠道代谢物在体外57、58、59、60中对C.albicans的催眠形态进行差分控制。然而，技术约束在尝试研究体内肠道样本中的C.albicans催眠形成时存在问题，特别是染色酵母和催眠细胞以及催眠细胞的定量分析。为了了解胃肠道中C.albicans催眠病的形态，利用小鼠同质化肠道含量的可溶性提取物，开发出一种外生殖方法，以研究代谢物对真菌催眠形态生成的影响。利用来自具有抗药性且易感染C.albicans GI感染的小鼠的肠道样本，此方法将有助于识别和研究代谢物、抗生素和异体生物对胃肠道真菌性脂肪形态生成的影响。

Protocol

所有动物协议都经过中西部大学机构动物护理和使用委员会（IACUC）的批准，如57之前所述。中西部大学机构动物护理和使用委员会根据MWU IACUC协议批准了这项研究#2894。MWU 动物护理政策遵循公共卫生服务 （PHS） 关于实验室动物的人道护理和使用政策以及《动物福利法》中制定的政策。 1. 小鼠学习标准协议 使用雄性和雌性C57BL/6J小鼠至少六周大。补?…

Representative Results

这些结果以及丹加马尼实验室60 的先前发现表明， 当C.albicans 在未经治疗的对照组和抗生素治疗小鼠的胃、小肠和大肠的肠道同质提取物中生长时 ，C.albicans 通常与酵母形态一起发展（图1B）。然而，当从抗生素处理的小鼠的cesal提取物中生长时 ，C.albicans 很容易经历形态生成，导致含有酵母和催眠素形式的样本（图1B）;…

Discussion

本文描述的方法提出了一种新方法，用于研究抗生素、饮食、异种生物和治疗对胃肠道中C.albicans催眠膜形成的影响。由于大部分全身感染来自胃肠道21、22、23、24、25、26、27、28、29、30、31、32、33、34，<sup c…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

作者承认中西部大学细胞和分子核心研究机构的资源和支持。

Materials

1 – 10 µL Pipet Tips Fisher Scientific 02-707-454 Misc
100 – 1000 µL Pipet Tips Fisher Scientific 02-707-400 Misc
20 – 200 µL Pipet Tips Fisher Scientific 02-707-451 Misc
2-methylbutyric acid Sigma 193070-25G hyphal-inhibitory compound
488 goat anti-rabbit IgG Invitrogen (Fisher) A11008 IF Staining secondary ab
Agar Fisher BP1423-500 YPD agar component
Automated Imaging Microscope Keyence BZX700
Candida Albicans Antibody Invitrogen (Fisher) PA1-27158 IF Staining primary ab
cefoperazone Cayman 16113 antibiotic
deoxycholic acid Sigma 30960 hyphal-inhibitory compound
D-Glucose Fisher D16-500 hyphal-promoting compound
forceps Fisher 08-885
lactic acid Alfa Aesar AAAL13242-06 hyphal-inhibitory compound
lithocholic acid Sigma L6250-10G hyphal-inhibitory compound
palmitic acid Sigma P5585-10G hyphal-inhibitory compound
Paraformaldehyde Alfa Aesar A11313 IF Staining fixative
Phosphate-buffered saline (PBS), 10x Alfa Aesar J62692 PBS component
p-tolylacetic acid SCBT sc-257959 hyphal-inhibitory compound
sebacic acid Sigma 283258-250G hyphal-inhibitory compound
sharp ended scissors Fisher 28301
sterile Milli-Q water N/A N/A Misc
YPD Broth BD Biosciences 242810 YPD agar component
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Tags

Ex Vivo AssayCandida AlbicansHyphal MorphogenesisGastrointestinal TractGut MetabolitesEnteric PathogensDissectionGI Tract ExtractionGut Content CollectionC. Albicans CulturePBS ResuspensionGut Content HomogenizationMicroscopy Analysis

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Monasky, R., Villa, S., Thangamani, S. An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract. J. Vis. Exp. (161), e61488, doi:10.3791/61488 (2020).
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