Name: deep dermal injection as a model of candida albicans skin infection for histological analyses
Uploaded: 2018-06-13T13:00:00.000+00:00
Duration: 10 min 45 s
Description: Watch this Scientific Journal Video about Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses at JoVE.com

FG is supported by Associazione Italiana per la Ricerca sul Cancro (IG 2016Id.18842), Cariplo Foundation (Grant 2014-0655), and Fondazione Regionale per la Ricerca Biomedica (FRRB). 
IZ is supported by NIH grant 1R01AI121066-01A1, 1R01DK115217, HDDC P30 DK034854 grant, Harvard Medical School Milton Found, CCFA Senior Research Awards (412708), the Eleanor and Miles Shore 50th Anniversary Fellowship Program, and the Cariplo Foundation (2014-0859).

All the procedures were approved under the Institutional Animal Care and Use Committee (IACUC) and operated under the supervision of the department of Animal Resources at Children&#39;s Hospital (ARCH) at Boston Children&#39;s Hospital or were approved by the Italian Ministry of Health and performed under the Institutional Animal Care Committee at the University of Milano-Bicocca.
1. C. albicans Preparation
<ol>
	<li>Culture C. albicans, strain CAF3-113, in tubes containing rich medium (yeast extract peptone dextrose (YEPD), 1% (w/v) yeast extract, 2% (w/v) of Bacto Peptone, 2% (w/v) glucose) supplemented with uridine (50 mg/L) at 25 &#176;C. Under these conditions, C. albicans grows as a yeast. 
	NOTE: Other C. albicans strains can be used, such as the clinical isolate C. albicans strain SC531413. Since we have recently demonstrated that the ulcerative process is induced by the activation of the innate immune system14, theoretically every C. albicans strain that maintains both its cell wall components and structure, as well as the ability to originate hyphal projections, could be used.</li>
	<li>Monitor the culture by counting the cellular concentration using a cell counter analyzer.</li>
	<li>Harvest the culture when it reaches a concentration of about 8 x 106 cells/mL. Alternatively, use a spectrophotometer to measure the OD600. 
	NOTE: Usually, a value of OD600 = 1 corresponds to a yeast concentration of 3 x 107 cells/mL, but titration is recommended.</li>
	<li>Resuspend the culture in YEPD-uridine medium, using HEPES (50 mM, pH 7.5) as a buffer agent and incubate the culture at 37 &#176;C to induce hyphae formation. Check hyphal formation under a microscope at 100X magnification. Hyphal formation is visible 5 h after culturing at 37 &#176;C; however, use the cells 16 h after incubation at 37 &#176;C when the hyphal percentage reaches almost 95% of the total culture.</li>
	<li>To further enrich the preparation in hyphal concentration, centrifuge aliquots of the culture at 3,300 x g for 5 min. Discard the supernatant and resuspend the pellet in sterile PBS at a concentration of 1 x 108 hyphae/mL.</li>
</ol>
2. Deep Dermal Injection
<ol>
	<li>24 h prior the infection procedure, anesthetize the mice with an intraperitoneal injection of ketamine (80 - 100 mg/kg) and xylazine (10 mg/kg), and then shave the flank of the mice using an electric clipper. 
	NOTE: After any procedure involving anesthesia, place the mice under a heating lamp until they have efficiently recovered.</li>
	<li>Rest the mice for 24 h to avoid any inflammation due to the shaving process.</li>
	<li>24 h after the shaving procedure, anesthetize the mice with an intraperitoneal injection of ketamine (80 - 100 mg/kg) and xylazine (10 mg/kg).</li>
	<li>Check the paw reflex to ensure that the mice are deeply anesthetized. Assess the paw reflex by firmly pinching the paw. If the anesthesia is achieved, the animal does not feel pain and does not move.</li>
	<li>Inject C. albicans hyphae in the deep derma using a 0.3 mL insulin syringe with a 30 G x 8 mm needle in a final volume of 50 &#181;L/injection, corresponding to 5 x 106 hyphae/injection.
	<ol>
		<li>Pull the skin of the shaved flank taut using two fingers in order to inject the volume directly in the deep derma. Insert the needle with an angle of 10 - 15&#176; with the bevel of the needle facing up.</li>
		<li>With this angle, the pathogen will be injected with a depth of approximately 300 - 500 &#181;m. To ensure the injection is well performed, check that the injected volume forms a bump that is absorbed a few min after the injection. 
		NOTE: The bump formed after the injection will be of approximately 1 cm2. This is the area that will be removed at the designated time point for either molecular or histological analysis.</li>
		<li>For time points of less than 24 h (recommended) mark the area of infection with a marker pen, since the bump formed with the injection persists for a few minutes, after which it is absorbed. For time points of 24 h or more, this step is not required, because either an ulcer or a cyst will be clearly visible.</li>
	</ol>
	</li>
</ol>
3. Sample Collection and Preparation for Histological Staining
<ol>
	<li>Euthanize the mice according to institutional procedures.</li>
	<li>Using surgical forceps, pull the skin at the border of the injected site, previously marked with the marker pen. Using surgical scissors, excise the infected site by cutting the skin following the marked line. 
	NOTE: Be careful to separate the skin from the peritoneum using round tip scissors.</li>
	<li>Immerse the skin in a disposable base mold filled with optimal cutting temperature (OCT) compound, with the internal side of the skin facing up. Freeze the sample in liquid nitrogen until the compound becomes white. Do not let the liquid nitrogen cover the sample before it is completely frozen as this would damage the sample. 
	NOTE: Caution! During step 3.3, wear a face shield for protection from liquid nitrogen.</li>
	<li>If the samples are not used immediately for slide preparation, rapidly store them at -80 &#176;C. 
	NOTE: Samples can be stored at -80 &#176;C indefinitely.</li>
</ol>
4. Preparation of Slides
<ol>
	<li>Cut the sample in half to prepare the slices for histology starting from the center of the sample.</li>
	<li>Cut 5-&#181;m thick slices with a cryostat. 
	NOTE: For skin samples, the temperature of the cryostat should not be higher than -25 &#176;C. A higher temperature would cause a partial melting of the H-OCT and therefore the samples would not be maintained in the right position during the cutting procedure.</li>
	<li>Use positively charged microscope slides to collect the slices.</li>
	<li>If the collected slices are not used soon after the preparation, store them at -20 &#176;C. At this point, slices can be stored at -20 &#176;C indefinitely.</li>
</ol>
5. Hematoxylin &#38; Eosin Staining
<ol>
	<li>Stain the slides by immersion in Gill&#39;s hematoxylin for 4 min. Wash the slides in running tap water for 5 min.</li>
	<li>Stain the slides by immersion in Eosin Y solution for 1 min. Wash the slides in running tap water for 5 min. Rinse the slides using distilled water before continuing the protocol.</li>
	<li>Dehydrate by immersion in serially increasing concentrations of ethanol solutions (50%, 70%, 80%, 95%, 100%). Immerse the slides for 15 s in each ethanol solution.</li>
	<li>Clear the slides for at least 2 min by immersion in a histological clearing agent.</li>
	<li>Mount the slides using mounting medium and cover slips.</li>
	<li>Acquire images of the slides with a microscope slide scanner at a 40X magnification.</li>
</ol>
6. Periodic Acid-Schiff (PAS) Staining
<ol>
	<li>Fix the slides with &#8805;99.5% acetone at room temperature for 1 min. Wash the slides in running tap water for 1 min.</li>
	<li>Stain the slides by immersion in periodic acid solution (1 g/dL) for 5 min. Wash the slides in 3 changes of distilled water.</li>
	<li>Stain the slides by immersion in Schiff&#39;s reagent for 15 min. Wash the slides in running tap water for 5 min.</li>
	<li>Stain the slides by immersion in Gill&#39;s hematoxylin for 5 min. Wash the slides in running tap water for 30 s.</li>
	<li>Dehydrate the slides by immersion in 3 changes of 100% ethanol. Immerse the slides for 15 s each time.</li>
	<li>Clear the slides for at least 2 min by immersion in a histological clearing agent.</li>
	<li>Mount the slides using mounting medium and cover slips.</li>
	<li>Acquire images of the slides with a microscope slide scanner.</li>
</ol>
7. Sample Collection and Preparation for RNA Extraction
<ol>
	<li>Euthanize the mice according to institutional procedures. Remove the skin samples as in step 3.2.</li>
	<li>Immerse the samples in a 2 mL snap-cap tube with 1 mL of reagent for acid guanidinium thiocyanate-phenol-chloroform extraction. 
	NOTE: The protocol can be paused here and the samples can be stored at -80 &#176;C.</li>
	<li>Cut the sample into pieces of approximately 0.2 cm2 using surgical forceps. 
	NOTE: Caution! Most of the reagents for RNA isolation are toxic and mutagenic. Steps 7.3 and 7.4 must be done under a fume hood.</li>
	<li>Smash the sample with a bead mill at a speed of 20 oscillations/s for 20 min. Alternatively, a mechanical potter can be used.</li>
	<li>Check the effective homogenization of the samples by looking for the presence of intact pieces of skin. Repeat this step if the samples are not completely homogenized.</li>
	<li>Centrifuge the samples at 16,000 x g for 1 min. Keep the supernatants for RNA extraction and discard the pellet. This step is performed to eliminate hair and debris that could block the extraction columns.</li>
	<li>Proceed with RNA extraction using RNA purification columns14.</li>
	<li>Use a spectrophotometer to assess RNA purification and concentration. If RNA extracts are not used immediately for cDNA preparation, store the samples at -80 &#176;C.</li>
</ol>

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P., Edmond, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nosocomial+Bloodstream+Infections+in+US+Hospitals:+Analysis+of+24,179+Cases+from+a+Prospective+Nationwide+Surveillance+Study.">Nosocomial Bloodstream Infections in US Hospitals: Analysis of 24,179 Cases from a Prospective Nationwide Surveillance Study.</a> Clin Infect Dis. 39 (3), 309-317 (2004).</li><li>Romani, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunity+to+fungal+infections.">Immunity to fungal infections.</a> Nat Rev Immunol. 4 (1), 11-24 (2004).</li><li>Odds, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+Candida+infections.">Pathogenesis of Candida infections.</a> J Am Acad Dermatol. 31 (3), S2-S5 (1994).</li><li>MacCallum, D. M., Odds, F. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+events+in+the+intravenous+challenge+model+for+experimental+Candida+albicans+infections+in+female+mice.">Temporal events in the intravenous challenge model for experimental Candida albicans infections in female mice.</a> Mycoses. 48 (3), 151-161 (2005).</li><li>Dai, T., Kharkwal, G. B., Tanaka, M., Huang, Y. -. Y., Bil de Arce, V. J., Hamblin, M. 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The authors have nothing to disclose.

Here we described a method of C. albicans infection to study the inflammatory process that is initiated upon fungal entrance in the deep derma.
Even though skin abscess formation is a relatively rare event upon C. albicans infection15, injection of the fungus directly in the deep derma allows not only the study of fungal-driven abscess formation, but also the analysis of specific immune cells that participate to contain fungal spread. With the method d...

The human body is covered with an extremely high number of microorganisms. The skin surface is the habitat of almost one million bacteria per square centimeter1. This number, however, does not reflect the full variety of microorganisms that colonizes the skin. In addition to bacteria, the human body is colonized by many fungal species, including C. albicans, which is able to survive both at the mucosal and the skin level2.
In recent years, the percentage of people diagnosed with fungal infections has enormously increased. This is mainly due to the higher number of immunocompromised persons, i.e., HIV-positive patients and patients that have gone through either chemotherapy or immunosuppressive drugs after transplantation3. In a surveillance study performed in the United States, Wisplinghoff et al. showed that 9.5% of the nosocomial bloodstream infections were caused by Candida species4. Due to the increased occurrence of fungal infections, and especially because of the elevated percentage of Candida species found during bloodstream infections, understanding how this pathogen escapes the control of the immune system is extremely important.
C. albicans is a dimorphic fungus that grows in different morphological forms such as yeast, blastospores, pseudohyphae, and hyphae depending on the environmental conditions5. In its hyphal form, C. albicans shows its highest invasiveness capacity and has the ability to penetrate the epithelium6.
C. albicans infections have been studied using several experimental approaches. The most common model of infection is the intravenous injection of C. albicans yeast7. However, this model does not take into consideration all the processes that happen before the fungus manages to spread into the bloodstream. Another model takes advantage of the ability of C. albicans to invade the epithelium. This method, also known as the sand paper model8, was developed by Gaspari et al. in 19989, and consists of using sand paper to abrade the skin, thus eliminating the stratum corneum before applying C. albicans. This procedure allows the fungus to penetrate the epithelium, thus enabling the analysis of the invasive abilities of this pathogen. Finally, other models of infections for the gastrointestinal10 and respiratory tracts11 have been used in different studies.
The formation of a wound (as in the sand paper model) causes the activation of several pathways, including immune cell recruitment and activation, in order to promote the healing process12. This can either alter or mask the immune response specifically elicited against the pathogen, thus leading to confounding results.
Here we describe a method of skin infection that avoids initial wound formation and the induction of a basal inflammatory environment. To maintain the intact epithelial structure, we directly inject C. albicans in its hyphal form in the deep derma. Even though a single injection can elicit mild inflammation, the amount of inflammation is limited and restricted compared to the formation of an open wound as in the sand paper model. The approach that we describe here allows the study of the immune response to fungal infection and spread while avoiding the excessive and pre-existing inflammatory environment caused by mechanical damage.

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PBS</td><td>Euroclone</td><td>ECB4053L</td><td>warm in 37 &amp;deg;C bath before use</td></tr><tr><td>H-OCT compound</td><td>histo-line laboratories</td><td>R0030</td><td></td></tr><tr><td>Gill&#039;s Hematoxilyn</td><td>histo-line laboratories</td><td>09-178-2</td><td></td></tr><tr><td>Eosin Y solution, alcoholic</td><td>histo-line laboratories</td><td>09-209-05</td><td></td></tr><tr><td>Ethanol absolute</td><td>scharlau</td><td>ET00232500</td><td></td></tr><tr><td>Citro-HISTOCLEAR</td><td>histo-line laboratories</td><td>R0050</td><td></td></tr><tr><td>Eukitt, mounting medium</td><td>bio-optica</td><td></td><td></td></tr><tr><td>Acetone</td><td>sigma-aldrich</td><td>179124</td><td></td></tr><tr><td>PAS staining system</td><td>sigma-aldrich</td><td>395B-1KT</td><td></td></tr><tr><td>Bacto Peptone</td><td>BD</td><td>211677</td><td></td></tr><tr><td>Bacto Yeast Extract</td><td>BD</td><td>212750</td><td></td></tr><tr><td>D(+)-Glucose anhydrous for molecular biology</td><td>Applichem PanReac</td><td>50-99-7</td><td></td></tr><tr><td>Uridine</td><td>Merck Millipore</td><td>8451</td><td></td></tr><tr><td>HEPES</td><td>Applichem PanReac</td><td>A1070,0500</td><td></td></tr><tr><td>Safe-Lock tubes 2 mL</td><td>eppendorf</td><td>30121597</td><td></td></tr><tr><td>TRIzol Reagent</td><td>Life Technologies</td><td>15596018</td><td>Toxic, corrosive and mutagen. Use all precaution needed</td></tr><tr><td>Rneasy Mini Kit</td><td>QIAGEN</td><td>74104</td><td></td></tr><tr><td>liquid nitrogen</td><td></td><td></td><td>Wear eye protection</td></tr><tr><td>&lt;strong&gt;Instrument&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Coulter Counter-Particle Count</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>Centrifuge 5415 R</td><td>eppendorf</td><td></td><td></td></tr><tr><td>MC 3000 Microtome Cryostat</td><td>histo-line laboratories</td><td></td><td></td></tr><tr><td>TissueLiser</td><td>QIAGEN</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Materials&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>0.3 ml Insulin Syringe with a 30G x 8mm needle&amp;nbsp;</td><td>BD</td><td>324826</td><td></td></tr><tr><td>Surgical forceps</td><td></td><td></td><td></td></tr><tr><td>Surgical scissors</td><td></td><td></td><td></td></tr><tr><td>Base mould disposable</td><td>histo-line laboratories</td><td>2781</td><td></td></tr><tr><td>Positively charged bio microscope slides</td><td>bio-optica</td><td>09-2000</td><td></td></tr><tr><td>Cover slips 24 x 50 mm</td><td>thermo scientific</td><td>11911998</td><td></td></tr></tbody></table>

deep dermal injection as a model of candida albicans skin infection for histological analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily lead to infections in the dermis which can, in turn, result in the dissemination of pathogens into the bloodstream. Understanding how the immune system fights infections at the very early stage and how the host can eliminate the pathogens is an important step to set the base for future therapeutic interventions. Here we describe a model of Candida albicans infection that can visualize the processes that occur early during an infection, including when the pathogen has passed the epithelial barrier, as well as the immune response elicited by the C. albicans invasion. We used this infection model to perform histological analyses that show the immune cells that infiltrate the skin as well as the presence and localization of the pathogen. Samples collected after the infection can be processed for RNA extraction.

Through the injection of the pathogen directly in the deep derma, the structural morphology of the tissue remains intact (Figure 1A).
The maintenance of skin structural integrity allows the detection of immune cells and their localization at the site of infection. The higher magnification shown in Figure 1B reveals that the abscess is mainly composed of polymorphonuclea...

Watch this Scientific Journal Video about Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses at JoVE.com

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

The skin is an extremely extended organ of the body and, due to this large surface, it is continuously exposed to microorganisms. Skin damage can easily ...

deep-dermal-injection-as-model-candida-albicans-skin-infection-for

Research

JoVE Journal

Immunology and Infection

10.7K Views.  University of Milano-Bicocca. Here we describe a protocol that allows histological and molecular analysis of skin samples after Candida albicans intradermal injection. This protocol maintains the structural integrity of the skin and allows for the localization of tissue-resident or newly recruited immune cells as well as the pathogen distribution.

Video: Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their intercommunication, are paramount for the protection against infections. The interactions of epithelial and innate immune cells with a pathogen are best investigated in vivo, where complex behavior unfolds over time and space. However, existing models do not allow for easy spatio-temporal imaging of the battle with pathogens at the mucosal level.
The model developed here creates a mucosal infection by direct injection of the fungal pathogen, Candida albicans, into the swimbladder of juvenile zebrafish. The resulting infection enables high-resolution imaging of epithelial and innate immune cell behavior throughout the development of mucosal disease. The versatility of this method allows for interrogation of the host to probe the detailed sequence of immune events leading to phagocyte recruitment and to examine the roles of particular cell types and molecular pathways in protection. In addition, the behavior of the pathogen as a function of immune attack can be imaged simultaneously by using fluorescent protein-expressing C. albicans. Increased spatial resolution of the host-pathogen interaction is also possible using the described rapid swimbladder dissection technique.
The mucosal infection model described here is straightforward and highly reproducible, making it a valuable tool for the study of mucosal candidiasis. This system may also be broadly translatable to other mucosal pathogens such as mycobacterial, bacterial or viral microbes that normally infect through epithelial surfaces.

In vivo spatio-temporal interactions of pathogen and immune defenses at the mucosal level are not easily imaged in existing vertebrate hosts. The method presented here describes a versatile platform to study mucosal candidiasis in live vertebrates using the swimbladder of the juvenile zebrafish as an infection site.

Early defense against mucosal pathogens consists of both an epithelial barrier and innate immune cells. The immunocompetency of both, and their ...

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 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 ...

Clarifying and Imaging Candida albicans Biofilms

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch from yeast-form growth to hyphal growth. Cells initiating this process become adherent to surfaces as well as to each other, with the resulting development of a biofilm colony. This commonly occurs not only on mucosal tissue surfaces in yeast infections, but also on medical implants such as catheters. It is well known that biofilm cells are resistant to antifungal drugs, and that cells that shed from the biofilm can lead to dangerous systemic infections. Biofilms range from heavily translucent to opaque due to refractive heterogeneity. Therefore, fungal biofilms are difficult to study by optical microscopy. To visualize internal structural, cellular, and subcellular features, we clarify fixed intact biofilms by stepwise solvent exchange to a point of optimal refractive index matching. For C. albicans biofilms, sufficient clarification is attained with methyl salicylate (n = 1.537) to enable confocal microscopy from apex to base in 600 &#181;m biofilms with little attenuation. In this visualization protocol we outline phase contrast refractometry, the growth of laboratory biofilms, fixation, staining, solvent exchange, the setup for confocal fluorescence microscopy, and representative results.

Clarifying and Imaging Candida albicans Biofilms

To view and quantify the internal features of Candida albicans biofilms, we prepare fixed intact specimens that are clarified by refractive index matching. Then, optical sectioning microscopy can be used to obtain three-dimensional image data though the full thickness of the biofilm.

The microbial fungus Candida albicans can undergo a change from commensal colonization to virulence that is strongly correlated with its ability to switch ...

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.

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.

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

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 ...

Effective aPDT Using Curcuminoids for Oral Candidiasis in Mice

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for evaluating alternative treatments prior to clinical trials. This study describes the efficacy of aPDT in a murine model of oral candidiasis. Forty mice were immunosuppressed with subcutaneous injections of prednisolone, and their tongues were inoculated using an oral swab previously soaked in a C. albicans cell suspension. Tetracycline was administered via drinking water during the course of the experiment. Five days after fungal inoculation, mice were randomly distributed into eight groups; a ninth group of untreated uninfected mice was included as a negative control (n = 5). Three concentrations (20 &#181;M, 40 &#181;M, and 80 &#181;M) of a mixture of curcuminoids were tested with a blue LED light (89.2 mW/cm2; ~455 nm) and without light (C+L+ and C+L- groups, respectively). Light alone (C-L+), no treatment (C-L-), and animals without infection were evaluated as controls. Data were analyzed using Welch&#39;s ANOVA and Games-Howell tests (&#945; = 0.05). Oral candidiasis was established in all infected animals and visualized macroscopically through the presence of characteristic white patches or pseudomembranes on the dorsum of the tongues. Histopathological sections confirmed a large presence of yeast and filaments limited to the keratinized layer of the epithelium in the C-L- group, and the presence of fungal cells was visually decreased in the images obtained from mice subjected to aPDT with either 40 &#181;M or 80 &#181;M curcuminoids. aPDT mediated by 80 &#181;M curcuminoids promoted a 2.47 log10 reduction in colony counts in comparison to those in the C-L- group (p = 0.008). All other groups showed no statistically significant reduction in the number of colonies, including photosensitizer (C+L-) or light alone (C-L+) groups. Curcuminoid-mediated aPDT reduced the fungal load from the tongues of mice.

Author Spotlight: Exploring Photodynamic Therapy with Curcumin in a Murine Model for Oral Candidiasis

This protocol describes the application of antimicrobial Photodynamic Therapy (aPDT) in a murine model of oral candidiasis. aPDT was performed using a water-soluble mixture of curcuminoids and blue LED light.

Antimicrobial Photodynamic Therapy (aPDT) has been extensively investigated in vitro, and preclinical animal models of infections are suitable for ...

Establishing a Mouse Model for Candida albicans Biofilm-Related PJI

A Periprosthetic Joint Candida albicans Infection Model in Mouse

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and scientists. Generally, biofilms that can shield C. albicans from antibiotics and immune clearance are formed at the infection site. Surgery involving the removal of the infected implant, debridement, antimicrobial treatment, and reimplantation is the gold standard for the treatment of PJI. Thus, establishing animal PJI models is of great significance for the research and development of new drugs or therapeutics for PJI. In this study, a smooth nickel-titanium alloy wire, a widely used implant in orthopedic clinics, was inserted into the femoral joint of a C57BL/6 mouse before the C. albicans were inoculated into the articular cavity along the wire. After 14 days, mature and thick biofilms were observed on the surface of implants under a scanning electronic microscope (SEM). A significantly reduced bone trabecula was found in the H&amp;E staining of the infected joint specimens. To sum up, a mouse PJI model with the advantages of easy operation, high successful rate, high repeatability, and high clinical correlation was established. This is expected to be an important model for clinical studies of C. albicans biofilm-related PJI prevention.

Author Spotlight: Advancing Research on Candida albicans Biofilm-Associated Prosthetic Joint Infections 

Periprosthetic joint infection (PJI) caused by dangerous pathogens is common in clinical orthopedics. Existing animal models cannot accurately simulate the actual situation of PJI. Here, we established a Candida albicans biofilm-associated PJI mouse model to research and develop new therapeutics for PJI.

Periprosthetic joint infection (PJI) is one of the common infections caused by Candida albicans (C. albicans), which increasingly concerns surgeons and ...

Establishing a Mouse Model for Candida albicans-Associated Catheter-Related Infection

A Catheter-Related Candida albicans Infection Model in Mouse

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed on the outer surface of the catheter and lead to disseminated infections, which are fatal to patients. There are no effective prevention and treatment management in clinics. Therefore, it is urgent to establish an animal model of CRI for the preclinical screening of new strategies for its prevention and treatment. In this study, a polyethylene catheter, a widely used medical catheter, was inserted into the back of the BALB/c mice after hair removal. Candida albicans ATCC MYA-2876 (SC5314) expressing enhanced green fluorescent protein was subsequently inoculated on the skin's surface along the catheter. Intense fluorescence was observed on the surface of the catheter under a fluorescent microscope 3 days later. Mature and thick biofilms were found on the surface of the catheter via scanning electron microscopy. These results indicated the adhesion, colonization, and biofilm formation of candida albicans on the surface of the catheter. The hyperplasia of the epidermis and the infiltration of inflammatory cells in the skin specimens indicated the histopathological changes of the CRI-associated skin. To sum up, a mouse CRI model was successfully established. This model is expected to be helpful in the research and development of therapeutic management for candida albicans associated CRI.

Author Spotlight: Enhancing Candida albicans Detection in Catheter Infections Using Fluorescent Protein Tagging 

We establish a mouse model of C.albicans-associated catheter-related infection (CRI), in which biofilm forms on the catheter, and the interaction between C.albicans and host correlates well with the clinical CRI. This model helps screen therapies for C.albicans biofilm-associated CRI, laying a foundation for clinical transformation.

Catheter-related infection (CRI) is a common nosocomial infection caused by candida albicans during catheter implantation. Typically, biofilms are formed ...

Deep Dermal Injection As a Model of Candida albicans Skin Infection for Histological Analyses

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Chapters in this video

Modeling Mucosal Candidiasis in Larval Zebrafish by Swimbladder Injection

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

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

Clarifying and Imaging Candida albicans Biofilms

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

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

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

Curcuminoid-Mediated Antimicrobial Photodynamic Therapy on a Murine Model of Oral Candidiasis

A Periprosthetic Joint Candida albicans Infection Model in Mouse

A Catheter-Related Candida albicans Infection Model in Mouse