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Immunology and Infection

Un ensayo ex vivo para estudiar Candida albicans Morphogenesis hifal en el tracto gastrointestinal

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

El ensayo ex vivo descrito en este estudio utilizando extractos de homogeneización intestinal y tinción de inmunofluorescencia representa un método novedoso para examinar la morfogénesis hifágica de Candida albicans en el tracto gastrointestinal. Este método se puede utilizar para investigar las señales ambientales que regulan la transición morfogenética en el intestino.

Abstract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) está estrechamente controlada por varias señales ambientales, y desempeña un papel importante en la difusión y patogénesis de este patógeno fúngico oportunista. Sin embargo, los métodos para visualizar las hifas fúngicas en el tracto gastrointestinal in vivo son desafiantes, lo que limita la comprensión de las señales ambientales en el control de este proceso de morfogénesis. El protocolo descrito aquí demuestra un novedoso método ex vivo para la visualización de la morfogénesis hifal en extractos de homogeneización intestinal. Utilizando un ensayo ex vivo, este estudio demuestra que el contenido de cecal de ratones tratados con antibióticos, pero no de ratones de control no tratados, promueven la morfogénesis hiphal de C. albicans en el contenido intestinal. Además, la adición de grupos específicos de metabolitos intestinales al contenido de cecal de ratones tratados con antibióticos regula diferencialmente la morfogénesis hifílo ex vivo. En conjunto, este protocolo representa un método novedoso para identificar e investigar las señales ambientales que controlan la morfogénesis hipófrica de C. albicans en el tracto gastrointestinal.

Introduction

Candida albicans es un patógeno fúngico oportunista y polimórfico que normalmente es commensal, pero puede sufrir un cambio morfológico en una forma virulenta capaz de causar infecciones potencialmente mortales en individuos inmunocomprometidos1,2,3,4,5,6,7,8,9,10,11,12,13. C. albicans es una de las principales causas de infecciones nosocomiales sistémicas, con una tasa de mortalidad de 40-u201260% incluso con tratamiento antifúngico2,14,15. Aunque C. albicans reside en diferentes nichos de acogida, incluido el sistema reproductivo femenino16,17, la cavidad oral de individuos sanos18 y el tracto gastrointestinal (GI)19,20, la mayoría de las infecciones sistémicas se originan en el tracto gastrointestinal y, además, la fuente de infección sistémica se confirma a menudo como el tracto gastrointestinal21,22,23,24,25,26,27,28,29,30,31,32,33,34. C. patogenicidad albicans en el tracto gastrointestinal está influenciado por una amplia gama de factores; sin embargo, una característica importante necesaria para la virulencia es la transición de una morfología celular de levadura a una morfología celular hiphal virulenta35,36,37,38,39,40,41,42,43,44. C. El apego y la difusión de los hongos del tracto gastrointestinal durante la infección está altamente asociado con su capacidad de transición de una levadura commensal a hifas virulentas, permitiendo que los hongos causen enfermedades invasivas44,45,46,47,48,49,50,51,52,53.

Una variedad de factores en el intestino, incluyendo n-acetilglucosamina, regular la formación de hiphal por C. albicans. Por lo tanto, es crucial reducir la brecha en el conocimiento con respecto a la morfogénesis hifal de este patógeno fúngico en el tracto gastrointestinal54,55,56. La evidencia reciente indica que varios metabolitos intestinales controlan diferencialmente la morfogénesis hifral de C. albicans in vitro57,58,59,60. Sin embargo, las limitaciones técnicas presentan problemas al intentar estudiar la formación de las hifas de C. albicans en muestras intestinales in vivo, especialmente la tinción de células de levadura e hifas y el análisis cuantitativo del desarrollo hiphal. Para entender la morfogénesis hifrética de C. albicans en el tracto gastrointestinal, se desarrolló un método ex vivo utilizando extractos solubles de contenido intestinal homogeneizado de ratones para estudiar el efecto de los metabolitos en la morfogénesis hipófológica fúngica. Utilizando muestras intestinales de ratones que son resistentes y susceptibles a la infección por C. albicans GI, este método ayudará a identificar y estudiar el efecto de metabolitos, antibióticos y xenobióticos en la morfogénesis hipófológica fúngica en el tracto gastrointestinal.

Protocol

Todos los protocolos de animales fueron aprobados por el Comité Institucional de Cuidado y Uso de Animales de la Universidad del Medio Oeste (IACUC) como se describió antesdel 57. El Comité Institucional de Cuidado y Uso de Animales de la Universidad del Medio Oeste aprobó este estudio bajo el Protocolo MWU IACUC #2894. Las políticas de cuidado animal de MWU siguen la Política del Servicio de Salud Pública (PHS) sobre cuidado y uso humano de animales de laboratorio y las políticas establec…

Representative Results

Estos resultados junto con los hallazgos anteriores del laboratorio Thangamani60 indican que cuando C. albicans se cultiva ex vivo en extractos de homogeneato intestinal tomados del estómago, intestino delgado e intestino grueso de control no tratado y ratones tratados con antibióticos, C. albicans generalmente se desarrolla con una morf levaduraología (Figura 1B). Sin embargo, cuando se cultiva en el extracto de cecal de ratones tratados con …

Discussion

El método descrito aquí presenta una forma novedosa de investigar el efecto de los impactos antibiótico, dietético, xenobiótico y terapéutico en la morfogénesis hiphal de C. albicans en el tracto gastrointestinal. Dado que la mayoría de las infecciones sistémicas se originan en el tracto gastrointestinal21,22,23,24,25,<sup class="x…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Los autores reconocen los recursos y el apoyo de la central de investigación de núcleos celulares y moleculares de la Universidad del Medio Oeste.

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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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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