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Immunology and Infection

칸디다 알비칸스 위장관에서 최면 모포 발생을 연구하는 Ex vivo 분석

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

이 연구에서 기장 균질 추출물과 면역형광 스테인링을 사용하여 기술된 전 생체 분석법은 기관에서 칸디다 알비칸스의 최면 형태 발생을 검사하는 새로운 방법을 나타낸다. 이 방법은 창자에 있는 형태유전학 전이를 통제하는 환경 신호를 조사하기 위하여 이용될 수 있습니다.

Abstract

위장(GI) 관내 칸디다 알비칸스 최면 형태발생은 다양한 환경 신호에 의해 엄격하게 제어되며, 이 기회성 곰팡이 병원균의 보급 및 병원성 발생에 중요한 역할을 한다. 그러나, 생체 내 기관에서 곰팡이 최해를 시각화하는 방법은 이러한 형태 발생 과정을 제어하는 환경 신호의 이해를 제한하는 도전적이다. 여기에 설명된 프로토콜은 장 동형 추출물에서 최면 형태 발생의 시각화를 위한 새로운 ex vivo 방법을 보여줍니다. 전 생체 분석기를 사용하여, 이 연구는 항생제 처리된 마우스에게서 cecal 내용이, 그러나 처리되지 않은 대조마우스에서, 창자 내용에서 C. albicans 최면 형태발생을 승진시키는 것을 보여줍니다. 또한, 항생제 처리된 마우스로부터 세칼 내용물의 특정 군을 다시 첨가하면 자궁 내형성 전 생체내를 분화한다. 종합하면, 이 프로토콜은 기관에서 C. albicans 최면 형태발생을 제어하는 환경 신호를 식별하고 조사하는 새로운 방법을 나타냅니다.

Introduction

칸디다 알비칸스는 일반적으로 기만적이지만 면역 절인 개인1,2,3,4,5,6,7,8,9,10,11,12, 12, 12에서생명을 위협하는 감염을 유발할 수 있는 악성 형태로 형태학적 변화를 겪을 수 있습니다. C. 알비칸은 항진균 치료2,14,15에서도40\u201260% 사망률을 가진 전신 외장 감염의 주요 원인이다. C. albicans는 여성 생식시스템(16,17)및 건강한 개인의구강(18)과 위장관(GI)19,20,전신 감염의 대부분을 기관에서 유래하는 등 상이한 숙주 틈새에 거주하고 있지만, 또한, 전신 감염의 근원은 종종 GI요로 확인된다21,22,23,24,25,26,27,28,29,30,31,32, 33,34. C. 기관의 알비칸 병원성은 광범위한 요인에 의해 영향을 받습니다. 그러나, 독성에 필요한 주요 특징은 효모 세포 형태에서 악성 최음세포형태(35,36,37,38,39,40,41,42, 43,44)로의 전환이다. C. 알비칸스 감염 시 기관으로부터의 부착 및 보급은 기막 효모에서 악성 최면으로 전환하는 능력과 매우 관련이 있어 곰팡이가 침습적 질환44,45,47,48,49,50,51,52,53을유발할 수 있다.

n-아세틸글루코사민을 포함한 창자에 있는 다양한 요인은 C. albicans에의하여 최면 형성을 조절합니다. 따라서, 기관54,55,56에서이 곰팡이 병원균의 최면 형태발생에 관한 지식의 격차를 좁히는 것이 중요하다. 최근 증거에 따르면 다양한 장 대사산물이 체외57,58,59,60에서 C. 알비칸의 최면 형태 발생을 차별화적으로 제어한다는 것을 나타낸다. 그러나, 기술적 제약은 생체 내 의 자궁 내의 C. 알비칸최면 형성, 특히 염색 효모 및 최면 세포 및 최면 발달의 정량적 분석을 연구하려고 할 때 문제를 제시한다. 기관에서 C. 알비칸스 최면 형태발생을 이해하기 위해, 전 생체 내 방법은 균질 최면 형태발생에 대한 대사산물의 효과를 연구하기 위해 마우스로부터 균질화 된 장 함량의 용용 추출물을 사용하여 개발되었다. C. albicans GI 감염에 저항하고 영향을 받기 쉬운 마우스에서 창자 견본을 이용하는, 이 방법은 GI 관에 있는 곰팡이 최면 형태발생에 대사 산물, 항생제 및 xenobiotics의 효력을 확인하고 공부하는 것을 도울 것입니다.

Protocol

모든 동물 프로토콜은57이전에 설명된 바와 같이 중서부 대학 기관 동물 관리 및 사용 위원회 (IACUC)에 의해 승인되었다. 미드웨스턴 대학의 기관 동물 관리 및 사용 위원회는 MWU IACUC 프로토콜 #2894 따라이 연구를 승인했습니다. MWU 동물 관리 정책은 실험실 동물의 인도적 관리 및 사용에 대한 공중 보건 서비스 (PHS) 정책과 동물 복지법 (AWA)에 명시된 정책을 따릅니다. <p class="jov…

Representative Results

이러한 결과는 탄가마니실험실(60)의 이전 연구 결과와 함께 C. 알비칸이 위장에서 가져온 장용 균질 추출물에서 전 생체을 재배할 때, 치료되지 않은 대조군 및 항생제 처리 마우스의 소장 및 대형 장, C. 알비칸스는 일반적으로 효모 형태학(도1B)으로개발된다는 것을 나타낸다. 그러나, 항생제 처리된 마우스에서 세칼 추출물에서 재배될 때, <em…

Discussion

여기에서 기술된 방법은 기관에 있는 C. albicans 최면 형태발생에 대한 항생제, 규정식, xenobiotic 및 치료 충격의 효력을 조사하는 새로운 방법을 제시합니다. 전신 감염의 대부분은 기관21,22,23,24,25,26,27,</su…

Disclosures

The authors have nothing to disclose.

Acknowledgements

저자는 미드웨스턴 대학 세포 및 분자 코어 연구 시설에서 자원과 지원을 인정합니다.

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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