An Ex vivo Assay to Study <em>Candida albicans</em> Hyphal Morphogenesis in the Gastrointestinal Tract

Ross Monasky; Sonia Villa; Shankar Thangamani

doi:10.3791/61488

JoVE Journal > Immunology and Infection

Immunologia e infezioni

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

Published: July 01, 2020

doi:

10.3791/61488

Ross Monasky¹, Sonia Villa², Shankar Thangamani³

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

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.

Abstract

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.

Introduction

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 40‒60% 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.

Protocol

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

Use male and female C57BL/6J mice at least six weeks old. Supplement them with sterile water with or without cefoperazone (0.5 mg/mL).
1. Co-house mice in groups of 5, with each cage containing either all male or all female mice. Provide the mice standard mouse chow and water (via a 400 mL bottle) at all times.
2. Check cages daily to ensure food and water levels are enough, and to examine mice for signs of distress.
Replace the water with cefoperazone every 48 h to ensure fresh antibiotic is being provided regardless of remaining water in the cage feeding bottles.
After 5‒7 days of cefoperazone treatment, euthanize mice via CO₂ asphyxiation observing established IACUC protocol. Confirm death via cervical dislocation.
Dissect mice using autoclave-sterilized sharp ended scissors and autoclave-sterilized forceps.
1. After euthanasia, secure the animal to a dissection surface by pinning all limbs such that the abdomen is exposed.
2. Spray the abdominal region with 70% ethanol to prevent fur from sticking to forceps, scissors, or gut sections during dissection.
3. Use forceps to pinch and lift a section of skin at the base of the abdomen and create a small incision through the skin and underlying fascia using scissors. Take great care when making this incision to avoid puncturing the cecum or intestinal wall.
4. Extend this cut to the rib cage, partially exposing the peritoneal cavity. Make a cut starting at the point of the initial incision on either side extending upward and laterally.
5. Pull these flaps laterally and pin to the dissecting surface to fully expose the peritoneal cavity.
Extract the GI tract using forceps, while using scissors to make cuts superior to the stomach and at the distal region of the large intestine to ensure collection of the greatest amount of gut content from each section.
When removing the GI tract, take care to avoid rupturing the individual components. Separate the stomach, small intestine, cecum, and large intestine individually using scissors at their proximal and distal ends.
For collection of each gut contents from each section, make a single incision at the distal end of each section using scissors, followed by manually expelling the gut content into a 1.5 mL microcentrifuge tube using forceps.
Store gut contents at -80 °C for ex vivo assays.

2. Preparation of yeast extract-peptone-dextrose (YPD) agar plates

To a 1 L glass bottle add 25 g of yeast extract peptone-dextrose broth powder, 10 g of agar, and ultrapure water to a final volume of 500 mL.
Autoclave at 121 °C for 30 min on a liquid cycle to sterilize the media.
Under a laminar flow hood, pour approximately 20 mL of agar media into a sterile Petri plate. 500 mL of agar media should yield approximately 25 plates.
Store plates at 4 °C until ready for use.

3. Ex vivo prep for hyphal morphogenesis assay

Streak a fresh culture of C. albicans SC5314 onto a YPD agar plate and incubate overnight at 30 °C.
Pick two to three medium-sized individual colonies from overnight grown C. albicans SC5314 culture and re-suspend in 1 mL of phosphate buffered saline (PBS).
Retrieve frozen gut contents from the -80 °C freezer and thaw at 25 °C.
Weigh about 150 mg of gut contents into a new 1.5 mL tube.
Re-suspend the gut contents with 150 µL of PBS (gut content and PBS at a 1:1 weight to volume ratio).
Vortex at high speed for 30 s to homogenize the gut contents and allow to sit at room temperature for about a minute.
Centrifuge the homogenates at 1000 x g for 3 min.
Transfer the supernatant to a new 1.5 mL tube.
Repeat steps 3.7 and 3.8 to remove all debris in the supernatant.
Add 10 µL of the C. albicans SC5314 inoculum prepared above to this supernatant
Mix well and incubate at 37 °C for 4 to 5 h.

4. Exogenous addition of metabolites to the gut homogenate extracts for the hyphal morphogenesis assay

Retrieve frozen gut contents from the -80 °C freezer and re-suspended in PBS at 1:1 ratio (weight: volume).
Add desired concentration of gut metabolites to the gut content and PBS mixture.
Vortex at high speed for 30 s to homogenize the gut contents containing metabolites and allow to sit at room temperature for about 10 min.
Centrifuge the homogenates at 1000 x g for 3 min.
Transfer the supernatant to a new 1.5 mL tube. Repeat steps 4.4 and 4.5 to remove all debris in the supernatant.
Add 10 µL of the C. albicans SC5314 inoculum prepared above to this supernatant. Mix well and incubate at 37 °C for 4 to 5 h.

5. C. albicans morphogenesis assay (immunostaining and imaging)

Centrifuge the samples at 1000 x g for 2 min and discard the supernatant via pipetting.
Fix the samples in 100 µL of 2% paraformaldehyde (PFA) for 15 min.
Centrifuge at 1000 x g for 2 min and discard supernatant via pipetting.
Wash the samples twice with 1 mL of PBS. To wash samples, re-suspend the pellet in PBS by pipetting gently. Do not vortex the sample as this can damage hyphal structures. After re-suspension, centrifuge at 1000 x g for 2 min and discard the supernatant via pipetting.
Incubate the samples at room temperature in 100 µL of PBS containing polyclonal C. albicans antibody (1:100 dilution) for 30 min.
Wash the samples three times with 1 mL of PBS.
NOTE: When using a fluorescent antibody, it is recommended that all dilution and wash steps be performed in dim light to avoid photo bleaching and improve sample longevity.
Incubate the samples at room temperature for 15 min in 100 µL of PBS containing anti-Rabbit IgG Alexafluor 488 antibody at 1:500 dilution. Perform incubation in a dark drawer or room to avoid photo bleaching.
Wash the samples three times with 1 mL of PBS.
Re-suspend the samples in 100 µL of PBS and transfer to a 96-well plate for imaging.
NOTE: When not being imaged, it is recommended that the 96-well plate be wrapped in aluminum foil to avoid photo bleaching.
Image fungal cells using 20x and 40x objective lenses using a fluorescence imaging microscope. Use a green fluorescent protein (GFP) filter (excitation wavelength 470/40 and emission wavelength 525/50) to detect fluorescence.

Representative Results

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, resulting in samples containing yeast and hyphae forms (Figure 1B); this does not occur in control mice. This supports previous results, which showed a significant increase in hyphae forms in samples grown in antibiotic-treated cecal extracts, but not in any other antibiotic-treated gut extracts⁶⁰. These results suggest that antibiotic treatment causes changes in the cecal environment, which induce hyphal morphogenesis of C. albicans. Additionally, the specific localization of this phenotype noticed only in the cecum also suggests that these hyphae-promoting conditions may not necessarily present throughout the GI tract, but instead are restricted to specific segments of the GI tract depending on the availability of nutrients, metabolites and other unknown molecules.

Since the cecal extract of antibiotic-treated mice promotes the morphogenesis of C. albicans⁵⁷^,⁵⁸^,⁵⁹^,⁶⁰, we examined whether exogenous addition of a selected group of gut metabolites (identified from previous in vitro studies) to the cecal content of cef-treated mice will affect the morphogenesis of C. albicans ex vivo. Previous work performed by the Thangamani laboratory has characterized the metabolomics profile of cecal content homogenate extracted from untreated and antibiotic-treated mice, revealing significant changes in the abundance of various metabolites as a result of antibiotic-treatment—specifically, decreased abundance of secondary bile acids and increased abundance of carbohydrates⁶⁰. Further, this study identified that secondary bile acids and carboxylic acids inhibit hyphae development, whereas carbohydrates including glucose, promote the hyphal morphogenesis of C. albicans in vitro⁶⁰. Results indicate that adding back a pool of inhibitory gut metabolites containing deoxycholic acid (DCA, 0.5 mg/mL), lithocholic acid (LCA, 0.1 mg/mL), palmitic acid (0.1 mg/mL), p-tolylacetic acid (0.1 mg/mL), sebacic acid (0.5 mg/mL), 2-methylbutyric acid (0.5 mg/mL), and lactic acid (5 mg/mL) to the cecal homogenate of cef-treated mice completely inhibited hyphal morphogenesis ex vivo. On the other hand, exogenous addition of glucose (1 mg/mL) to the cecal homogenate of cef-treated mice showed a massive hyphal development ex vivo (Figure 2B). Collectively, these findings indicate that addition of gut metabolites back to the cecal homogenate of the cef-treated mice differentially regulates the morphogenesis of C. albicans, thus confirming previous in vitro findings. These results indicate that gut metabolites play a critical role in hyphal morphogenesis of C. albicans and understanding the gene targets and signaling pathways modulated by these metabolites will aid in the development of new therapeutic approaches to prevent and treat C. albicans infections.

Figure 1: Ex vivo assay to determine the effect of cefoperazone treatment on C. albicans hyphal morphogenesis in the gut contents. (A) Protocol schematic outline. (B) Antibiotic-treated (top panels) and non-treated (bottom panels) gut contents were taken from the stomachs, small intestines, cecums, and large intestines of C57BL/6J mice. Gut contents inoculated with C. albicans SC5314 were incubated at 37 °C for 4‒5 h and stained with C. albicans antibody. Cells were imaged at 40x magnification. Representative images are shown here. Please click here to view a larger version of this figure.

Figure 2: Exogenous addition of gut metabolites to the cecal contents from cef-treated mice on hyphae formation of C. albicans ex vivo. (A) Protocol schematic outline. (B) Inhibitory gut metabolites pool containing DCA (0.5 mg/mL), LCA (0.1 mg/mL), palmitic acid (0.1 mg/mL), p-tolylacetic acid (0.1 mg/mL), sebacic acid (0.5 mg/mL); 2-methylbutyric acid (0.5 mg/mL), and lactic acid (5 mg/mL) or glucose (1 mg/mL) were added back to the cecal content of cef-treated mice, mixed thoroughly and incubated at 37 °C for 15 min to carry out the ex vivo hyphae assay. Cecal contents inoculated with C. albicans SC5314 were incubated at 37 °C for 4‒5 h and stained with C. albicans antibody. Cells were imaged at 40x magnification. Representative images are shown here. Please click here to view a larger version of this figure.

Discussion

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^,²⁸^,²⁹^,³⁰^,³¹^,³²^,³³^,³⁴ and hyphae formation is a critical virulence factor that promotes the dissemination of C. albicans from the GI tract, understanding the factors that controls this morphogenesis in the GI tract will expand the knowledge about pathogenesis mechanisms and identify novel treatment options.

While the method presented here is relatively straightforward, certain steps discussed below were identified as critical and important. (i) The initial inoculum of C. albicans should be optimal to allow for both growth and hyphal morphogenesis of fungi. With the limited availability of nutrients in the gut homogenate extracts, higher volume of inoculum may significantly reduce the fungal growth and morphogenesis process. However, the growth of different clinical isolates and strains are likely to be variable, thus optimizing the inoculum and incubation time for specific C. albicans isolates is essential. (ii) Multiple centrifugation steps when preparing the gut homogenate extract were found to be crucial to remove the debris in gut contents as much as possible. (iii) Due to the relatively low speed of centrifugation (to avoid damaging hyphal structures), care must be taken to avoid cell loss during immunostaining steps in this protocol.

Alternative methods to visualize fungal hyphae in the GI tract have been used in the past, with certain advantages and limitations associated with each method. One relatively notable method using fluorescent in situ hybridization (FISH) to visualize fungal hyphae in the GI tract has been recently demonstrated by Witchley et al.⁶¹^,⁶². This is a promising in vivo method currently available to detect C. albicans hyphae directly in the GI tract, however the complexity of this protocol makes it difficult to adapt it to rapid, large scale initial screening studies. Traditional histopathology methods have also been used in the past to vitalize C. albicans yeast and hyphae forms in the GI tract. However, observation and imaging of fungal cells with basic histopathology, and Hematoxylin and Eosin (H/E) stains remains challenging, as many standard fixation methods have the potential to disrupt the mucosal layer of GI tract samples, often damaging hyphal structures in the process and leading to contradictory reports over the relative abundance of hyphal cell morphology during infection⁶³^,⁶⁴^,⁶⁵^,⁶⁶. This method was developed to avoid damage to hyphae during processing to address this issue. In addition, tissue explants have been used as a way to examine biological conditions ex vivo, however these methods are generally focused and useful for examining the adherence or invasion potential of C. albicans⁶⁷, but also they generally exclude the majority of metabolomics and microbiome components that contribute to in vivo pathogenesis. Although the ex vivo protocol described here does not completely mimic in vivo GI environment as described previously⁶¹^,⁶², it provides the closest possible conditions that C. albicans encounters in the gut environment compared to in vitro methods using artificial growth conditions.

This protocol can be used for basic screening assays to identify the impact of environmental signals in the GI tract on C. albicans hyphal morphogenesis. This method allows for large groups of compounds including small molecule inhibitors, novel antimycotics, and metabolites to be screened rapidly for hyphal development, and could be used in screening therapeutic treatments or identifying risk factors for systemic disease. Since C. albicans colonizes throughout the GI tract, this protocol will further aid in identifying the environmental signals present in the specific segments of GI tract that control hyphal morphogenesis in individuals taking antibiotics, chemotherapeutic agents, and in patients with metabolic disorders including diabetes mellitus. Ultimately the method described here allows for quick characterization of hyphal morphogenesis in C. albicans over a wide range of environmental factors in a manner that is more biologically relevant than current in vitro methods and is substantially faster and more resource efficient than current in vivo methods.

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

The authors acknowledge resources and support from 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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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).