Name: an ex vivo assay to study candida albicans hyphal morphogenesis in the gastrointestinal tract
Uploaded: 2020-07-01T14:30:00
Description: Watch this Scientific Journal Video about An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract at JoVE.com

The authors acknowledge resources and support from Midwestern University Cellular and Molecular Core Research facility.

All animal protocols were approved by Midwestern University Institutional Animal Care and Use Committee (IACUC) as described before57. The Institutional Animal Care and Use Committee at Midwestern University approved this study under MWU IACUC Protocol #2894. The MWU animal care policies follow the Public Health Service (PHS) Policy on Humane Care and Use of Laboratory Animals and the policies laid out in the Animal Welfare Act (AWA).
1. Mice study standard protocol
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The method described here presents a novel way to investigate the effect of antibiotic, dietary, xenobiotic and therapeutic impacts on C. albicans hyphal morphogenesis in the GI tract. Since the majority of systemic infections originate from the GI tract21,22,23,24,25,26,27,</s...

Candida albicans is an opportunistic, polymorphic fungal pathogen that is normally commensal, but can undergo a morphological change into a virulent form capable of causing life-threatening infections in immunocompromised individuals1,2,3,4,5,6,7,8,9,10,11,12,13. C. albicans is a leading cause of systemic nosocomial infections, with a 40&#8210;60% mortality rate even with antifungal treatment2,14,15. Though C. albicans resides in different host niches including the female reproductive system16,17, the oral cavity of healthy individuals18 and the gastrointestinal (GI) tract19,20, the majority of the systemic infections originate from the GI tract and furthermore, the source of systemic infection is often confirmed to be the GI tract21,22,23,24,25,26,27,28,29,30,31,32,33,34. C. albicans pathogenicity in the GI tract is influenced by a wide range of factors; however, a major characteristic necessary for virulence is the transition from a yeast cell morphology into a virulent hyphal cell morphology35,36,37,38,39,40,41,42,43,44. C. albicans attachment and dissemination from the GI tract during infection is highly associated with its capacity to transition from a commensal yeast into virulent hyphae, allowing the fungi to cause invasive disease44,45,46,47,48,49,50,51,52,53.
A variety of factors in the gut, including n-acetylglucosamine, regulate hyphal formation by C. albicans. Therefore, it is crucial to narrow the gap in knowledge regarding the hyphal morphogenesis of this fungal pathogen in the GI tract54,55,56. Recent evidence indicates that various gut metabolites differentially control the hyphal morphogenesis of C. albicans in vitro57,58,59,60. However, technical constraints present issues when attempting to study C. albicans hyphae formation in in vivo gut samples, especially staining yeast and hyphae cells and quantitative analysis of hyphal development. To understand C. albicans hyphal morphogenesis in the GI tract, an ex vivo method was developed using soluble extracts of homogenized gut content from mice to study the effect of metabolites on fungal hyphal morphogenesis. Utilizing gut samples from mice that are resistant and susceptible to C. albicans GI infection, this method will help to identify and study the effect of metabolites, antibiotics and xenobiotics on fungal hyphal morphogenesis in the GI tract.

<table>
	<tbody>
		<tr>
			<td>1 - 10 &#181;L Pipet Tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-454</td>
			<td>Misc</td>
		</tr>
		<tr>
			<td>100 - 1000 &#181;L Pipet Tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-400</td>
			<td>Misc</td>
		</tr>
		<tr>
			<td>20 - 200 &#181;L Pipet Tips</td>
			<td>Fisher Scientific</td>
			<td>02-707-451</td>
			<td>Misc</td>
		</tr>
		<tr>
			<td>2-methylbutyric acid</td>
			<td>Sigma</td>
			<td>193070-25G</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>488 goat anti-rabbit IgG</td>
			<td>Invitrogen (Fisher)</td>
			<td>A11008</td>
			<td>IF Staining secondary ab</td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Fisher</td>
			<td>BP1423-500</td>
			<td>YPD agar component</td>
		</tr>
		<tr>
			<td>Automated Imaging Microscope</td>
			<td>Keyence</td>
			<td>BZX700</td>
			<td></td>
		</tr>
		<tr>
			<td>Candida Albicans Antibody</td>
			<td>Invitrogen (Fisher)</td>
			<td>PA1-27158</td>
			<td>IF Staining primary ab</td>
		</tr>
		<tr>
			<td>cefoperazone</td>
			<td>Cayman</td>
			<td>16113</td>
			<td>antibiotic</td>
		</tr>
		<tr>
			<td>deoxycholic acid</td>
			<td>Sigma</td>
			<td>30960</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Fisher</td>
			<td>D16-500</td>
			<td>hyphal-promoting compound</td>
		</tr>
		<tr>
			<td>forceps</td>
			<td>Fisher</td>
			<td>08-885</td>
			<td></td>
		</tr>
		<tr>
			<td>lactic acid</td>
			<td>Alfa Aesar</td>
			<td>AAAL13242-06</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>lithocholic acid</td>
			<td>Sigma</td>
			<td>L6250-10G</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>palmitic acid</td>
			<td>Sigma</td>
			<td>P5585-10G</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Alfa Aesar</td>
			<td>A11313</td>
			<td>IF Staining fixative</td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS), 10x</td>
			<td>Alfa Aesar</td>
			<td>J62692</td>
			<td>PBS component</td>
		</tr>
		<tr>
			<td>p-tolylacetic acid</td>
			<td>SCBT</td>
			<td>sc-257959</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>sebacic acid</td>
			<td>Sigma</td>
			<td>283258-250G</td>
			<td>hyphal-inhibitory compound</td>
		</tr>
		<tr>
			<td>sharp ended scissors</td>
			<td>Fisher</td>
			<td>28301</td>
			<td></td>
		</tr>
		<tr>
			<td>sterile Milli-Q water</td>
			<td>N/A</td>
			<td>N/A</td>
			<td>Misc</td>
		</tr>
		<tr>
			<td>YPD Broth</td>
			<td>BD Biosciences</td>
			<td>242810</td>
			<td>YPD agar component</td>
		</tr>
	</tbody>
</table>

an ex vivo assay to study candida albicans hyphal morphogenesis in the gastrointestinal tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important role in the dissemination and pathogenesis of this opportunistic fungal pathogen. However, methods to visualize fungal hyphae in the GI tract in vivo are challenging which limits the understanding of environmental signals in controlling this morphogenesis process. The protocol described here demonstrates a novel ex vivo method for visualization of hyphal morphogenesis in gut homogenate extracts. Using an ex vivo assay, this study demonstrates that cecal contents from antibiotic treated mice, but not from untreated control mice, promote C. albicans hyphal morphogenesis in the gut content. Further, adding back specific groups of gut metabolites to the cecal contents from antibiotic-treated mice differentially regulates hyphal morphogenesis ex vivo. Taken together, this protocol represents a novel method to identify and investigate the environmental signals that control C. albicans hyphal morphogenesis in the GI tract.

These results along with previous findings from the Thangamani laboratory60 indicate that when C. albicans is grown ex vivo in gut homogenate extracts taken from the stomach, small intestines and large intestines of untreated control and antibiotic-treated mice, C. albicans generally develops with a yeast morphology (Figure 1B). However, when grown in the cecal extract from antibiotic-treated mice, C. albicans readily undergoes morphogenesis...

Watch this Scientific Journal Video about An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract at JoVE.com

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

The ex vivo assay described in this study using gut homogenate extracts and immunofluorescence staining represents a novel method to examine the hyphal morphogenesis of Candida albicans in the GI tract. This method can be utilized to investigate the environmental signals regulating morphogenetic transition in the gut.

An Ex vivo Assay to Study Candida albicans Hyphal Morphogenesis in the Gastrointestinal Tract

Candida albicans hyphal morphogenesis in the gastrointestinal (GI) tract is tightly controlled by various environmental signals, and plays an important ...

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