JoVE Journal > Immunology and Infection
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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Een Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in het maag-darmkanaal

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

De ex vivo test beschreven in deze studie met behulp van gut homogenaat extracten en immunofluorescentie kleuring vertegenwoordigt een nieuwe methode om de hyphal morfogenese van Candida albicans in de GI-darmkanaal te onderzoeken. Deze methode kan worden gebruikt om de milieusignalen te onderzoeken die morfogenetische overgang in de darm regelen.

Abstract

Candida albicans hyphalemorfogenese in het maag-darmkanaal (GI) wordt strak gecontroleerd door verschillende milieusignalen, en speelt een belangrijke rol in de verspreiding en pathogenese van deze opportunistische schimmelpathover. Echter, methoden om schimmelhyphae visualiseren in de GI tract in vivo zijn uitdagend die het begrip van milieusignalen beperkt bij het beheersen van dit morfogenese proces. Het hier beschreven protocol toont een nieuwe ex vivo methode voor visualisatie van hyphal morfogenese in darmhomogenaat extracten. Met behulp van een ex vivo test, deze studie toont aan dat cecal inhoud van antibiotica behandelde muizen, maar niet van onbehandelde controle muizen, bevorderen C. albicans hyphal morfogenese in de darminhoud. Verder, het toevoegen van terug specifieke groepen van darmmetabolieten aan de cecal inhoud van met antibiotica behandelde muizen differentieel reguleert hyphalfogenese ex vivo. Samen vertegenwoordigt dit protocol een nieuwe methode om de milieusignalen te identificeren en te onderzoeken die C. albicans hyphal morfogenese in het GI-kanaal controleren.

Introduction

Candida albicans is een opportunistische, polymorfe schimmelpathiek die normaal commensal is, maar een morfologische verandering kan ondergaan in een virulente vorm die levensbedreigende infecties kan veroorzaken bij immuungecompromitteerde personen1,2,3,4,5,6,7,8,9,10,11,12,13. C. albicans is een belangrijke oorzaak van systemische nosocomiale infecties, met een sterftecijfer van 40\u201260% zelfs met schimmeldodende behandeling2,14,15. Hoewel C. albicans zich in verschillende gastheerniches bevindt, waaronder het vrouwelijke voortplantingssysteem16,17,de mondholte van gezonde individuen18 en het maag-darmkanaal19,20, is het merendeel van de systemische infecties afkomstig van het GI-darmkanaal en bovendien, de bron van systemische infectie wordt vaak bevestigd als het GI-kanaal21,22,23,24,25,26,27,28,29,30,31,32,33,34. C. albicans pathogeniteit in het GI-darmkanaal wordt beïnvloed door een breed scala van factoren; een belangrijk kenmerk dat nodig is voor virulentie is echter de overgang van een gistcelmorfologie naar een virulente hyphaltecelmorfologie35,36,37,38,39,40,41,42,43,44. C. albicans gehechtheid en verspreiding van het GI-darmkanaal tijdens infectie wordt sterk geassocieerd met zijn vermogen om over te stappen van een commensale gist in virulente hyphae, waardoor de schimmels invasieve ziekte44,45,46,47,48,49,50,51,52,53kunnen veroorzaken .

Een verscheidenheid van factoren in de darm, met inbegrip van n-acetylglucosamine, reguleren hyphal vorming door C. albicans. Daarom is het van cruciaal belang om de kloof in kennis over de hyphalemorfose van deze schimmelpathogenese in het GI-kanaal54,55,56teverkleinen . Recent bewijs wijst erop dat verschillende darmmetabolieten de hyphalemorfogenese van C. albicans in vitro57,58,59,60differentieel controleren . Echter, technische beperkingen presenteren problemen bij een poging om C. albicans hyphae vorming studie in in vivo darmmonsters, met name kleuring gist en hyphae cellen en kwantitatieve analyse van hyphal ontwikkeling. Om C. albicans hyphale morfogenese in het GI-kanaal te begrijpen, werd een ex vivo-methode ontwikkeld met behulp van oplosbare extracten van gehomogeniseerd darmgehalte van muizen om het effect van metabolieten op schimmelhyphale morfogenese te bestuderen. Gebruikmakend van darmmonsters van muizen die resistent zijn en gevoelig zijn voor C. albicans GI-infectie, zal deze methode helpen om het effect van metabolieten, antibiotica en xenobiotica op schimmelhyphale morfogenese in het GI-darmkanaal te identificeren en te bestuderen.

Protocol

Alle dierprotocollen werden goedgekeurd door midwestern University Institutional Animal Care and Use Committee (IACUC) zoals beschreven vóór57. De Institutional Animal Care and Use Committee van de Midwestern University keurde deze studie onder MWU IACUC Protocol #2894. Het MWU-beleid voor dierverzorging volgt het Beleid van de Ggd voor humane zorg en gebruik van proefdieren en het beleid dat is vastgelegd in de Wet dierenwelzijn (AWA). 1. Muizen bestuderen standaardpro…

Representative Results

Deze resultaten samen met eerdere bevindingen uit het Thangamani laboratorium60 geven aan dat wanneer C. albicans wordt geteeld ex vivo in gut homogenaat extracten uit de maag, dunne darmen en dikke darmen van onbehandelde controle en antibiotica behandelde muizen, C. albicans ontwikkelt zich over het algemeen met een gist morfologie (Figuur 1B). Echter, wanneer geteeld in het cecal extract van met antibiotica behandelde muizen, C. albicans …

Discussion

De hier beschreven methode presenteert een nieuwe manier om het effect van antibiotica, dieet, xenobiotische en therapeutische effecten op C. albicans hyphalmorphogenese in het GI-kanaal te onderzoeken. Aangezien de meeste systemische infecties afkomstig zijn van het GI-kanaal21,22,23,24,25,26,<sup class="…

Disclosures

The authors have nothing to disclose.

Acknowledgements

De auteurs erkennen middelen en ondersteuning van Midwestern University Cellular and Molecular Core Research faciliteit.

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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