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Immunologia e infezioni

En Ex vivo-analys att studera Candida albicans Hyphal Morphogenesis i mag-tarmkanalen

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

Ex vivo assay beskrivs i denna studie med gut homogenate extrakt och immunofluorescens färgning representerar en ny metod för att undersöka hyphal morphogenesis av Candida albicans i mag-tarmkanalen. Denna metod kan utnyttjas för att undersöka de miljösignaler som reglerar morfogogenetiska övergången i tarmen.

Abstract

Candida albicans hyfalan morfogenes i mag (GI) tarmkanalen är hårt styrs av olika miljösignaler, och spelar en viktig roll i spridning och patogenesen vid denna opportunistiska svamp patogen. Men metoder för att visualisera svamp hyfera i mag-tarmkanalen in vivo är utmanande som begränsar förståelsen av miljösignaler i att kontrollera denna morfogensprocess. Protokollet som beskrivs här visar en ny ex vivo metod för visualisering av hyphal morphogenesis i gut homogenate extrakt. Med hjälp av en ex vivo analys, denna studie visar att cecal innehåll från antibiotika behandlade möss, men inte från obehandlade kontroll möss, främja C. albicans hyphal morphogenesis i tarmen innehåll. Vidare, lägga tillbaka specifika grupper av gut metaboliter till cecal innehållet från antibiotika-behandlade möss differentially reglerar hyphal morphogenesis ex vivo. Sammantaget representerar detta protokoll en ny metod för att identifiera och undersöka de miljösignaler som styr C. albicans hyfalans morfogenes i mag-tarmkanalen.

Introduction

Candida albicans är en opportunistisk, polymorfa svamp patogen som normalt commensal, men kan genomgå en morfologisk förändring till en virulent form som kan orsaka livshotande infektioner hos immunkomprometteradeindivider 1,2,3,4,5,6,7,8,9,10,11,12,13. C. albicans är en ledande orsak till systemiska nosokomala infektioner, med en 40\u201260% dödlighet även med svampdödande behandling2,14,15. Även C. albicans är bosatt i olika värd nischer inklusive den kvinnliga reproduktiva systemet16,17, munhålan av friska individer18 och mag -tarmkanalen (GI) tarmkanalen19,20, majoriteten av de systemiska infektioner härstammar från mag-tarmkanalen och vidare, källan till systemisk infektion bekräftas ofta vara mag-tarmkanalen21,22,23,24,25,26,27,28,29,30,31,32,33,34. C. albicans patogenicitet i mag-tarmkanalen påverkas av ett brett spektrum av faktorer; emellertid, en ha som huvudämnekännetecken som är nödvändig för virulens, är övergången från en jästcellmorfologi in i en virulent hyphalcellmorfologi35,36,37,38,39,40,41,42,43,44. C. albicans fastsättning och spridning från mag-tarmkanalen under infektion är starkt förknippad med sin kapacitet att övergången från en commensal jäst i virulent hyfa, vilket gör att svamparna att orsaka invasiv sjukdom44,45,46,47,48,49,50,51,52,53.

En mängd olika faktorer i tarmen, inklusive n-acetylglucosamin, reglera hyphal formation av C. albicans. Därför är det avgörande att minska klyftan i kunskap när det gäller hyfalans morphogenesis av denna svamp patogen imag-tarmkanalen 54,55,56. Nya bevis tyder på att olika gut metaboliter differentially kontrollera hyphal morfogens av C. albicans in vitro57,58,59,60. Dock, tekniska begränsningar presentera frågor när man försöker studera C. albicans hyferbildning i in vivo gut prover, särskilt färgning jäst och hyphae celler och kvantitativ analys av hyphal utveckling. För att förstå C. albicans hyfalans morfogenes i mag-tarmkanalen, en ex vivo metod utvecklades med hjälp av lösliga extrakt av homogeniserade gut innehåll från möss att studera effekten av metaboliter på svamp hyphal morphogenesis. Utnyttja gut prover från möss som är resistenta och mottagliga för C. albicans GI infektion, denna metod kommer att bidra till att identifiera och studera effekten av metaboliter, antibiotika och xenobiotika på svamp hyfalan morfogenes i GI-tarmkanalen.

Protocol

Alla animaliska protokoll godkändes av Midwestern University Institutional Animal Care and Use Committee (IACUC) enligt beskrivningen före57. Den institutionella Animal Care and Use kommittén vid Midwestern University godkänt denna studie enligt MWU IACUC Protocol #2894. MWU:s djurvårdspolitik följer Folkhälsovårds (PHS) policy för human vård och användning av försöksdjur och den politik som fastställs i djurskyddslagen (AWA). 1. Möss studera standardprotok…

Representative Results

Dessa resultat tillsammans med tidigare resultat från Thangamani laboratoriet60 visar att när C. albicans odlas ex vivo i tarmen homogenate extrakt tas från magen, tunntarmen och tjocktarmen av obehandlade kontroll och antibiotika-behandlade möss, C. albicans i allmänhet utvecklas med en jäst morfologi (Figur 1B). Men när odlas i cecalextraktet från antibiotikabehandlade möss, genomgår C. albicans lätt morfogenes, vilket resultera…

Discussion

Den metod som beskrivs här presenterar ett nytt sätt att undersöka effekten av antibiotika, kost, xenobiotiska och terapeutiska effekter på C. albicans hyfalans morphogenesis i mag-tarmkanalen. Eftersom majoriteten av systemiska infektioner härstammar från mag-tarmkanalen21,22,23,24,25,26,<sup class=…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

Författarna erkänner resurser och stöd från Midwestern University Cellular and Molecular Core Research facility.

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

Keywords: 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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