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
<ol>
	<li>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).
	<ol>
		<li>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.</li>
		<li>Check cages daily to ensure food and water levels are enough, and to examine mice for signs of distress.</li>
	</ol>
	</li>
	<li>Replace the water with cefoperazone every 48 h to ensure fresh antibiotic is being provided regardless of remaining water in the cage feeding bottles.</li>
	<li>After 5&#8210;7 days of cefoperazone treatment, euthanize mice via CO2 asphyxiation observing established IACUC protocol. Confirm death via cervical dislocation.</li>
	<li>Dissect mice using autoclave-sterilized sharp ended scissors and autoclave-sterilized forceps.
	<ol>
		<li>After euthanasia, secure the animal&#160;to a dissection surface by pinning all limbs such that the abdomen is exposed.</li>
		<li>Spray the abdominal region with 70% ethanol to prevent fur from sticking to forceps, scissors, or gut sections during dissection.</li>
		<li>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.</li>
		<li>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.</li>
		<li>Pull these flaps laterally and pin to the dissecting surface to fully expose the peritoneal cavity.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Store gut contents at -80 &#176;C for ex vivo assays.</li>
</ol>
2. Preparation of yeast extract-peptone-dextrose (YPD) agar plates
<ol>
	<li>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.</li>
	<li>Autoclave at 121 &#176;C for 30 min on a liquid cycle to sterilize the media.</li>
	<li>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.</li>
	<li>Store plates at 4 &#176;C until ready for use.</li>
</ol>
3. Ex vivo prep for hyphal morphogenesis assay
<ol>
	<li>Streak a fresh culture of C. albicans SC5314 onto a YPD agar plate and incubate overnight at 30 &#176;C.</li>
	<li>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).</li>
	<li>Retrieve frozen gut contents from the -80 &#176;C freezer and thaw at 25 &#176;C.</li>
	<li>Weigh about 150 mg of gut contents into a new 1.5 mL tube.</li>
	<li>Re-suspend the gut contents with 150 &#181;L of PBS (gut content and PBS at a 1:1 weight to volume ratio).</li>
	<li>Vortex at high speed for 30 s to homogenize the gut contents and allow to sit at room temperature for about a minute.</li>
	<li>Centrifuge the homogenates at 1000 x g for 3 min.</li>
	<li>Transfer the supernatant to a new 1.5 mL tube.</li>
	<li>Repeat steps 3.7 and 3.8 to remove all debris in the supernatant.</li>
	<li>Add 10 &#181;L of the C. albicans SC5314 inoculum prepared above to this supernatant</li>
	<li>Mix well and incubate at 37 &#176;C for 4 to 5 h.</li>
</ol>
4. Exogenous addition of metabolites to the gut homogenate extracts for the hyphal morphogenesis assay
<ol>
	<li>Retrieve frozen gut contents from the -80 &#176;C freezer and re-suspended in PBS at 1:1 ratio (weight: volume).</li>
	<li>Add desired concentration of gut metabolites to the gut content and PBS mixture.</li>
	<li>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.</li>
	<li>Centrifuge the homogenates at 1000 x g for 3 min.</li>
	<li>Transfer the supernatant to a new 1.5 mL tube. Repeat steps 4.4 and 4.5 to remove all debris in the supernatant.</li>
	<li>Add 10 &#181;L of the C. albicans SC5314 inoculum prepared above to this supernatant. Mix well and incubate at 37 &#176;C for 4 to 5 h.</li>
</ol>
5. C. albicans morphogenesis assay (immunostaining and imaging)
<ol>
	<li>Centrifuge the samples at 1000 x g for 2 min and discard the supernatant via pipetting.</li>
	<li>Fix the samples in 100 &#181;L of 2% paraformaldehyde (PFA) for 15 min.</li>
	<li>Centrifuge at 1000 x g for 2 min and discard supernatant via pipetting.</li>
	<li>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.</li>
	<li>Incubate the samples at room temperature in 100 &#181;L of PBS containing polyclonal C. albicans antibody (1:100 dilution) for 30 min.</li>
	<li>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.</li>
	<li>Incubate the samples at room temperature for 15 min in 100 &#181;L 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.</li>
	<li>Wash the samples three times with 1 mL of PBS.</li>
	<li>Re-suspend the samples in 100 &#181;L 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.</li>
	<li>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.</li>
</ol>

The authors have no competing financial interests or other conflicts of interest.

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 &amp;micro;L Pipet Tips</td><td>Fisher Scientific</td><td>02-707-454</td><td>Misc</td></tr><tr><td>100 - 1000 &amp;micro;L Pipet Tips</td><td>Fisher Scientific</td><td>02-707-400</td><td>Misc</td></tr><tr><td>20 - 200 &amp;micro;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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Research

JoVE Journal

Immunology and Infection

5.3K Views.  Midwestern University. Het protocol aangetoond in deze video stelt ons in staat om de rol van darmmetabolieten op schimmelhyphal hyphal morfogenese te begrijpen. Deze ex vivo techniek min of meer de dichtstbijzijnde benadering van de darm voor het bestuderen van schimmel hyphal morfogenese op een manier die geen in vitro studie kan repliceren. Deze methode kan worden toegepast om de rol van darmmetabolieten op andere enterische pathogenen te bestuderen.Ontleden muizen met behulp van autoclaved gesteriliseerd...

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

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Genetics

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of phagocytes to the site where pathogens are located; (ii) recognition of pathogen-associated molecular patterns (PAMPs) through pattern recognition receptors (PRRs); (iii) engulfment of microorganisms bound to the phagocyte cell membrane, and (iv) processing of engulfed cells within maturing phagosomes and digestion of the ingested particle. Studies that assess phagocytosis in its entirety are informative1, 2, 3, 4, 5 but are limited in that they do not normally break the process down into migration, engulfment and phagosome maturation, which may be affected differentially. Furthermore, such studies assess uptake as a single event, rather than as a continuous dynamic process. We have recently developed advanced live-cell imaging technologies, and have combined these with genetic functional analysis of both pathogen and host cells to create a cross-disciplinary platform for the analysis of innate immune cell function and fungal pathogenesis. These studies have revealed novel aspects of phagocytosis that could only be observed using systematic temporal analysis of the molecular and cellular interactions between human phagocytes and fungal pathogens and infectious microorganisms more generally. For example, we have begun to define the following: (a) the components of the cell surface required for each stage of the process of recognition, engulfment and killing of fungal cells1, 6, 7, 8; (b) how surface geometry influences the efficiency of macrophage uptake and killing of yeast and hyphal cells7; and (c) how engulfment leads to alteration of the cell cycle and behavior of macrophages 9, 10.
In contrast to single time point snapshots, live-cell video microscopy enables a wide variety of host cells and pathogens to be studied as continuous sequences over lengthy time periods, providing spatial and temporal information on a broad range of dynamic processes, including cell migration, replication and vesicular trafficking. Here we describe in detail how to prepare host and fungal cells, and to conduct the video microscopy experiments. These methods can provide a user-guide for future studies with other phagocytes and microorganisms.

We describe methods for live-cell video microscopy of Candida albicans phagocytosis by macrophages. These methods enable stage-specific analysis of macrophage migration, recognition, engulfment and phagosome maturation and reveal novel aspects of phagocytosis.

Phagocytic clearance of fungal pathogens, and microorganisms more generally, may be considered to consist of four distinct stages: (i) migration of ...

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

The current article outlines a protocol to establish an in vitro cell culture model system to study the interaction of a facultative intracellular human fungal pathogen Candida glabrata with human macrophages which will be a useful tool to advance our knowledge of fungal virulence mechanisms.

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable ...

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic fungal pathogen of humans, is the fourth most common cause of nosocomial infections which results in ~40% mortality. However, other C. albicans infections are less severe and rarely lethal and include vulvovaginal candidiasis, impacting ~75% of women, as well as oropharyngeal candidiasis, predominantly impacting infants, AIDS patients and cancer patients. While murine models are most frequently used to study C. albicans pathogenesis, these models predominantly assess host survival and are costly, time consuming, and limited in replication. Therefore, several mini-model systems, including Drosophila melanogaster, Danio rerio, Galleria mellonella, and Caenorhabditis elegans, have been developed to study C. albicans. These mini-models are well-suited for screening mutant libraries or diverse genetic backgrounds of C. albicans. Here we describe two approaches to study C. albicans infection using C. elegans. The first is a fecundity assay which measures host reproduction and monitors survival of individual hosts. The second is a lineage expansion assay which measures how C. albicans infection affects host population growth over multiple generations. Together, these assays provide a simple, cost-effective way to quickly assess C. albicans virulence.

Two Infection Assays to Study Non-Lethal Virulence Phenotypes in C. Albicans using C. Elegans

Fungal opportunist pathogens can cause life-threatening as well as minor infections, but non-lethal phenotypes are frequently ignored when studying virulence. Therefore, we developed a nematode model that monitors both the survival and reproduction aspects of host to investigate fungal virulence.

While pathogens can be deadly to humans, many of them cause a range of infection types with non-lethal phenotypes. Candida albicans, an opportunistic ...

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans biofilms have been studied predominantly in vitro but there is a crucial need for better understanding of this dynamic process under in vivo conditions. We developed an in vivo subcutaneous rat model to study C. albicans biofilm formation. In our model, multiple (up to 9) Candida-infected devices are implanted to the back part of the animal. This gives us a major advantage over the central venous catheter model system as it allows us to study several independent biofilms in one animal. Recently, we adapted this model to study C. albicans biofilm development in BALB/c mice. In this model, mature C. albicans biofilms develop within 48 hr and demonstrate the typical three-dimensional biofilm architecture. The quantification of fungal biofilm is traditionally analyzed post mortem and requires host sacrifice. Because this requires the use of many animals to perform kinetic studies, we applied non-invasive bioluminescence imaging (BLI) to longitudinally follow up in vivo mature C. albicans biofilms developing in our subcutaneous model. C. albicans cells were engineered to express the Gaussia princeps luciferase gene (gLuc) attached to the cell wall. The bioluminescence signal is produced by the luciferase that converts the added substrate coelenterazine into light that can be measured. The BLI signal resembled cell counts obtained from explanted catheters. Non-invasive imaging for quantifying in vivo biofilm formation provides immediate applications for the screening and validation of antifungal drugs under in vivo conditions, as well as for studies based on host-pathogen interactions, hereby contributing to a better understanding of the pathogenesis of catheter-associated infections.

Candida albicans Biofilm Development on Medically-relevant Foreign Bodies in a Mouse Subcutaneous Model Followed by Bioluminescence Imaging

We present an experimental procedure of Candida albicans biofilm development in a mouse subcutaneous model. Fungal biofilms were quantified by determining the number of colony forming units and by a non-invasive bioluminescence imaging, where the amount of light that is produced corresponds with the number of viable cells.

Candida albicans biofilm development on biotic and/or abiotic surfaces represents a specific threat for hospitalized patients. So far, C. albicans ...

Medicine

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. albicans is its ability to form biofilms, structured communities of cells attached to biotic and abiotic surfaces. C. albicans biofilms can form on host tissues, such as mucosal layers, and on medical devices, such as catheters, pacemakers, dentures, and joint prostheses. Biofilms pose significant clinical challenges because they are highly resistant to physical and chemical perturbations, and can act as reservoirs to seed disseminated infections. Various in vitro assays have been utilized to study C. albicans biofilm formation, such as microtiter plate assays, dry weight measurements, cell viability assays, and confocal scanning laser microscopy. All of these assays are single end-point assays, where biofilm formation is assessed at a specific time point. Here, we describe a protocol to study biofilm formation in real-time using an automated microfluidic device under laminar flow conditions. This method allows for the observation of biofilm formation as the biofilm develops over time, using customizable conditions that mimic those of the host, such as those encountered in vascular catheters. This protocol can be used to assess the biofilm defects of genetic mutants as well as the inhibitory effects of antimicrobial agents on biofilm development in real-time.

Visualization of Biofilm Formation in Candida albicans Using an Automated Microfluidic Device

This protocol describes the use of a customizable automated microfluidic device to visualize biofilm formation in Candida albicans under host physiological conditions.

Candida albicans is the most common fungal pathogen of humans, causing about 15% of hospital-acquired sepsis cases. A major virulence attribute of C. ...

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most colonized hosts, the commensal reservoir does serve as a repository for infectious disease, and the presence of high fungal titers in the gut is associated with inflammatory bowel disease. Here, we describe a method to visualize C. albicans cell morphology and localization in a mouse model of stable gastrointestinal colonization. Colonization is established using a single dose of C. albicans in animals that have been treated with oral antibiotics. Segments of gut tissue are fixed in a manner that preserves the architecture of luminal contents (microorganisms and mucus) as well as the host mucosa. Finally, fluorescent in situ hybridization is performed using probes against fungal rRNA to stain for C. albicans and hyphae. A key advantage of this protocol is that it allows for simultaneous observation of C. albicans cell morphology and its spatial association with host structures during gastrointestinal colonization.

Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization

The purpose of this protocol is to visualize Candida albicans cell shape and localization in the mammalian gastrointestinal tract.

Candida albicans is a fungal component of the gut microbiota in humans and many other mammals. Although C. albicans does not cause symptoms in most ...

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Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular respiration, oxidative metabolism, signal transduction, and apoptosis. Consequently, mitochondrial dysfunction is reported to play an important role in the antifungal drug tolerance and virulence of pathogenic fungi. Recent data have also led to the recognition of the importance of the mitochondria as an important contributor to fungal pathogenesis. Despite the importance of the mitochondria in fungal biology, standardized methods to understand its function are poorly developed. Here, we present a procedure to study the basal oxygen consumption rate (OCR), a measure of mitochondrial respiration, and extracellular acidification rates (ECAR), a measure of glycolytic function in C. albicans strains. The method described herein can be applied to any Candidaspp. strains without the need to purify mitochondria from the intact fungal cells. Furthermore, this protocol can also be customized to screen for inhibitors of mitochondrial function in C. albicans strains.

Bio-energetics Investigation of Candida albicans Using Real-time Extracellular Flux Analysis

Here, we present a stepwise protocol to investigate the mitochondrial respiration and glycolytic function in Candida Albicans using an extra flux analyzer.

Mitochondria are essential organelles for the cellular metabolism and survival. A variety of key events take place in mitochondria, such as cellular ...

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting cells. When the skin encounters invading pathogens, different cutaneous DCs initiate a distinct T cell immune response to protect the body. Among the invading pathogens, fungal infection specifically drives a protective interleukin-17-producing Th17 immune response. A protocol was developed to efficiently differentiate Th17 cells by intradermal Candida albicans infection to investigate a subset of cutaneous DCs responsible for inducing Th17 immunity. Flow cytometry and gene expression analyses revealed a prominent induction of Th17 immune response in skin-draining lymph nodes and infected skin. Using diphtheria toxin-induced DC subset-depleting mouse strains, CD301b+ dermal DCs were found to be responsible for mounting optimal Th17 differentiation in this model. Thus, this protocol provides a valuable method to study in vivo function of differential subsets of cutaneous DCs to determine Th17 immunity against deep skin fungal infection.

In vivo Function of Differential Subsets of Cutaneous Dendritic Cells to Induce Th17 Immunity in Intradermal Candida albicans Infection

Here, we demonstrate the in vivo function of cutaneous dendritic cell subsets in Th17 immunity of deep dermal Candida albicans infection.

The skin is the outermost barrier organ in the body, which contains several types of dendritic cells (DCs), a group of professional antigen-presenting ...

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic changes are regulated by a complex signaling network controlled in response to environmental stimuli. These regulatory components have been highly studied, but almost all studies use a variety of in vitro stimuli to trigger filamentation. To determine how morphogenesis is regulated during the pathogenesis process, we developed an in vivo microscopy system to obtain high spatial resolution images of organisms undergoing hyphal formation within the mammalian host. The protocol presented here describes the use of this system to screen small collections of C. albicans mutant strains, allowing us to identify key regulators of morphogenesis as it occurs at the site of infection. Representative results are presented, demonstrating that some regulators of morphogenesis, such as the transcriptional regulator Efg1, have consistent phenotypes in vitro and in vivo, whereas other regulators, such as adenyl cyclase (Cyr1), have significantly different phenotypes in vivo compared to in vitro.

Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host

This manuscript describes a method for screening moderately sized Candida albicans mutant libraries for morphogenesis phenotypes during active infection in a mammalian host using non-invasive confocal microscopy.

Candida albicans is an important human pathogen. Its ability to switch between morphologic forms is central to&#160;its pathogenesis; these morphologic ...

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

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