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Abstract
Introduction
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Immunologie et infection

胃腸管における カンジダ・アルビカンス ・ヒphphal形態形成を研究するエクスビボアッセイ

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

腸ホモジネート抽出物および免疫蛍光染色を用いた本研究で説明したex vivoアッセイは、GI管における カンジダ・アルビカン スの催眠形態形成を調べる新規の方法を表す。この方法は、腸内の形態遺伝学的遷移を調節する環境シグナルを調べるのに利用することができる。

Abstract

胃腸(GI)管におけるカンジダ・アルビカンスの催眠形態形成は、様々な環境シグナルによって厳しく制御され、この日和見真菌病原体の普及および病因において重要な役割を果たしている。しかし、生体内のGI管で真菌性ヒphphaeを可視化する方法は、この形態形成プロセスを制御する環境信号の理解を制限する困難です。ここで説明するプロトコルは、腸管ホモジネート抽出物における催眠形態形成の可視化のための新規のex vivo方法を示す。この研究は、ex vivoアッセイを用いて、抗生物質治療マウスのcecal内容物は、未治療の対照マウスからではなく、腸内のC.アルビカンス催眠形態形成を促進することを示している。さらに、抗生物質処理マウスからcecal内容物に腸代謝物の特定のグループを付加すると、ヒファアル形態形成ex vivoを差し出して調節する。このプロトコルを組み合わせると、このプロトコルは、GI管におけるC.アルビカンス催眠形態形成を制御する環境信号を同定し、調査するための新しい方法を表す。

Introduction

カンディダ・アルビカンスは、通常はコメンサルである日和見的な多形性真菌病原体であるが、免疫不全個体1、2、3、4、5、6、7、8、9、10、11、12、13で生命を脅かす感染症を引き起こす可能性のある悪質な形態に形態変化を受けることができる。C. アルビカンスは全身性結室感染症の主要な原因であり、抗真菌治療2、14、15でも40\u201260%の死亡率を有する。C.アルビカンは、女性の生殖器系16、17、健康な個体18および胃腸(GI)管19、20の口腔を含む異なる宿主ニッチに存在するが、全身感染の大部分は、消化管に由来し、さらに、 全身感染の原因は、しばしば、GI管21、22、23、24、25、26、27、28、29、30、31、32、33、34であることが確認される。C. アルビカンスの消化管の病原性は、幅広い因子の影響を受ける。しかし、病原性に必要な主な特徴は、酵母細胞形態から、病変性のヒュファル細胞形態35、36、37、38、39、40、41、42、43、44への移行である。感染時のGI管からのアルビカンスの付着および播種は、病原酵母から毒性のヒファエに移行する能力と強く関連しており、真菌が侵襲性疾患44、49、50、51、52、53を引き起こすことを可能にする。

腸内の様々な要因, n-アセチルグルコサミンを含む, C. アルビカンによる催眠形成を調節します.したがって、GI管54、55、56におけるこの真菌病原体の催眠形態形成に関する知識のギャップを狭めることが重要である。最近の証拠は、様々な腸代謝産物が、Vitro57、58、59、60のC.アルビカンの催眠形態形成を差し出して制御することを示している。しかし、技術的な制約は、生体内の腸内サンプルでのC.アルビカンス催眠の形成、特に酵母およびヒファエ細胞の染色および催眠の発達の定量的分析を試みる際に問題を提示する。GI管におけるC.アルビカンスの催眠形態形成を理解するために、マウスからの均質化された腸内含量の可溶性抽出物を用いて、真菌性催眠形態形成に対する代謝産物の効果を研究するために、ex vivo法が開発された。この方法は、C.アルビカンスGI感染に耐性があり、感受性のマウスからの腸サンプルを利用して、消化管における真菌性催眠形態形成に対する代謝産物、抗生物質および異種生物系の効果を同定し、研究するのに役立つ。

Protocol

すべての動物プロトコルは、57の前に記載されているように中西部大学の制度的動物の世話と使用委員会(IACUC)によって承認されました。中西部大学の制度的動物ケアおよび使用委員会は、MWU IACUCプロトコル#2894の下でこの研究を承認しました.MWU動物ケア政策は、ヒューマンケアと実験動物の使用に関する公衆衛生サービス(PHS)ポリシーと動物福祉法(AWA)に定められた方針に従…

Representative Results

タンガマニ研究所60 からの以前の知見と共に 、C.アルビカンス が胃から採取された腸ホモゲネート抽出物でex vivoを成長させると、未治療のコントロールおよび抗生物質処理マウスの小腸および大腸が 、C.アルビカンは 一般的に酵母形態で発症することを示している(図1B)。しかし、抗生物質処理マウスからcecal抽出物で増殖すると 、C….

Discussion

ここで説明する方法は、GI管におけるC.アルビカンス催眠形態形成に対する抗生物質、食事、異種および治療的影響の影響を調査する新しい方法を提示する。全身感染の大部分は、GI管21、22、23、24、25、26、27、28、29、30、31、32、33、34およびヒファエ形成に由来するので、Cの播種を促進する重要な毒性因子である。 GI管からのアルビカンは、この形態形?…

Divulgations

The authors have nothing to disclose.

Acknowledgements

著者らは、中西部大学セルラーアンド分子コア研究施設からのリソースと支援を認めている。

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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