This work was supported by NIH grant 1R01AI33409 and the Department of Pediatrics, Carver College of Medicine, University of Iowa.

<ol>
	<li>Lopes, J. P., Lionakis, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+and+virulence+of+Candida+albicans.">Pathogenesis and virulence of Candida albicans.</a> Virulence. 13 (1), 89-121 (2022).</li><li>Saville, S. P., Lazzell, A. L., Monteagudo, C., Lopez-Ribot, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+control+of+cell+morphology+in+vivo+reveals+distinct+roles+for+yeast+and+filamentous+forms+of+Candida+albicans+during+infection.">Engineered control of cell morphology in vivo reveals distinct roles for yeast and filamentous forms of Candida albicans during infection.</a> Eukaryotic Cell. 2 (5), 1053-1060 (2003).</li><li>Arita, G. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+associated+proteins+involved+in+filamentation+with+impact+on+the+virulence+of+Candida+albicans.">Cell wall associated proteins involved in filamentation with impact on the virulence of Candida albicans.</a> Microbiological Research. 258, 126996 (2022).</li><li>Rai, L. S., Wijlick, L. V., Bougnoux, M. E., Bachellier-Bassi, S., d'Enfert, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulators+of+commensal+and+pathogenic+life-styles+of+an+opportunistic+fungus-Candida+albicans.">Regulators of commensal and pathogenic life-styles of an opportunistic fungus-Candida albicans.</a> Yeast. 38 (4), 243-250 (2021).</li><li>Sudbery, P. 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H., Wellington, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+morphogenesis+of+Candida+albicans+during+infection+in+a+live+animal.">Imaging morphogenesis of Candida albicans during infection in a live animal.</a> Journal of Biomedical Optics. 15 (1), 010504 (2010).</li><li>Wakade, R. S., Huang, M., Mitchell, A. P., Wellington, M., Krysan, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intravital+imaging+of+Candida+albicans+identifies+differential+in+vitro+and+in+vivo+filamentation+phenotypes+for+transcription+factor+deletion+mutants.">Intravital imaging of Candida albicans identifies differential in vitro and in vivo filamentation phenotypes for transcription factor deletion mutants.</a> mSphere. 6 (3), 0043621 (2021).</li><li>Wakade, R. S., Kramara, J., Wellington, M., Krysan, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+filamentation+does+not+require+the+cAMP-PKA+pathway+in+vivo.">Candida albicans filamentation does not require the cAMP-PKA pathway in vivo.</a> mBio. 13 (3), 0085122 (2022).</li><li>Bergeron, A. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Candida+albicans+and+Pseudomonas+aeruginosa+interact+to+enhance+virulence+of+mucosal+infection+in+transparent+zebrafish.">Candida albicans and Pseudomonas aeruginosa interact to enhance virulence of mucosal infection in transparent zebrafish.</a> Infection and Immunity. 85 (11), 00475 (2017).</li><li>Seman, B. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+phenotypic+profile+of+the+Candida+albicans+regulatory+network.">A phenotypic profile of the Candida albicans regulatory network.</a> Plos Genetics. 5 (12), 1000783 (2009).</li><li>Cullen, P. J., Sprague, G. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+regulation+of+filamentous+growth+in+yeast.">The regulation of filamentous growth in yeast.</a> Genetics. 190 (1), 23-49 (2012).</li><li>Herrero, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+filament+formation+in+Candida+albicans+by+polyamine+levels.">Control of filament formation in Candida albicans by polyamine levels.</a> Infection and Immunity. 67 (9), 4870-4878 (1999).</li><li>Ahmad Hussin, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biotin+auxotrophy+and+biotin+enhanced+germ+tube+formation+in+Candida+albicans.">Biotin auxotrophy and biotin enhanced germ tube formation in Candida albicans.</a> Microorganisms. 4 (3), 37 (2016).</li><li>Nantel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcription+profiling+of+Candida+albicans+cells+undergoing+the+yeast-to-hyphal+transition.">Transcription profiling of Candida albicans cells undergoing the yeast-to-hyphal transition.</a> Molecular Biology of the Cell. 13 (10), 3452-3465 (2002).</li><li>Noble, S. M., Johnson, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strains+and+strategies+for+large-scale+gene+deletion+studies+of+the+diploid+human+fungal+pathogen+Candida+albicans.">Strains and strategies for large-scale gene deletion studies of the diploid human fungal pathogen Candida albicans.</a> Eukaryotic Cell. 4 (2), 298-309 (2005).</li><li>Glazier, V. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EFG1,+Everyone's+favorite+gene+in+Candida+albicans:+A+comprehensive+literature+review.">EFG1, Everyone's favorite gene in Candida albicans: A comprehensive literature review.</a> Frontiers in Cellular Infection and Microbiology. 12, 855229 (2022).</li><li>Huang, G., Huang, Q., Wei, Y., Wang, Y., Du, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+roles+and+diverse+regulation+of+the+Ras/cAMP/protein+kinase+A+pathway+in+Candida+albicans.">Multiple roles and diverse regulation of the Ras/cAMP/protein kinase A pathway in Candida albicans.</a> Molecular Microbiology. 111 (1), 6-16 (2019).</li><li>Saville, S. P., Lazzell, A. L., Chaturvedi, A. K., Monteagudo, C., Lopez-Ribot, J. 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T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+disseminated+candidiasis+in+zebrafish+reveals+role+of+phagocyte+oxidase+in+limiting+filamentous+growth.">Live imaging of disseminated candidiasis in zebrafish reveals role of phagocyte oxidase in limiting filamentous growth.</a> Eukaryotic Cell. 10 (7), 932-944 (2011).</li><li>Brothers, K. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase-driven+phagocyte+recruitment+controls+Candida+albicans+filamentous+growth+and+prevents+mortality.">NADPH oxidase-driven phagocyte recruitment controls Candida albicans filamentous growth and prevents mortality.</a> PLoS Pathogens. 9 (10), 1003634 (2013).</li><li>Holmes, H., Kennedy, J. 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The authors have no conflicts of interest to disclose.

This model utilizes confocal microscopy to obtain images of C. albicans organisms as they grow within the tissue of a mammalian host, thus allowing us to evaluate morphogenesis phenotypes during active infection. The process of morphogenesis is central to C. albicans pathogenesis and has been widely studied using a variety of in vitro assays2,3,4. However, no in vitro assay can fully model the complex biochemical and structural environment of the host.
The protocol described here is focused on the use of this in vivo imaging system to screen a series/library of C. albicans mutants to identify the genes involved in morphogenesis during infection. The use of C. albicans strains expressing different fluorescent proteins allows us to quantify in vivo morphogenesis of C. albicans mutant strains compared to that of a reference strain. Comparing morphogenesis in the mutant to the reference strain within the same area of infection ensures that the organisms are exposed to identical environments. This allows for quantitative measurement of the percentage of cells undergoing filamentation, as well as the extent of filamentation. Normalization of the measurements of the mutant strain(s) to those of the reference strain allows us to better compare the performance of one mutant to another.
The representative results presented here demonstrate the potential for a significant discrepancy between in vitro and in vivo phenotypes. The C. albicans efg1&#916;&#916; mutant strain is often used as a negative control for morphogenesis assays as it fails to filament in almost all in vitro conditions20. Although the in vivo results were very similar to the in vitro results, even this severely hampered strain did occasionally form filaments in the host tissue environment (Figure 3). This emphasizes the strength of the host environment in triggering morphogenesis.
In contrast, the cyr1&#916;&#916; mutant strain demonstrates a substantial discordance between in vitro and in vivo growth; although none of the mutant cells undergo filamentation in vitro, approximately half of the cells grow as filaments in vivo (Figure 4)10,21. Interestingly, these filaments were significantly shorter than those formed by the reference strain, suggesting that CYR1 contributes to either the rate of filament growth or the ability to maintain a filamentous phenotype. To facilitate the analysis of filament length, the curve path length of the filaments was measured using a two-dimensional projection of the images. In two-dimensional projections of filaments growing in three dimensions, any filament growing on an axis that is not parallel to the xy plane will project as shorter than its true length. As this foreshortening also occurs for the reference strain, evaluating the distribution of filament lengths in a two-dimensional projection still allows a quantitative comparison between the reference and mutant strains. Analysis of filament length in two rather than three dimensions requires less intensive image analysis; thus, it can be performed relatively quickly on a typical desktop computer. Using this simpler analysis allows for the inclusion of filament length distribution as part of a screening protocol, giving a more nuanced understanding of the ability of each mutant to undergo morphogenesis without causing substantial delays in throughput.
The representative studies presented here were performed using DBA2/N mice, which have a defect in their complement system causing a failure to recruit neutrophils to the site of C. albicans infection22. The goal of these studies was to investigate mechanisms of the regulation of C. albicans filamentation within the host tissue. Therefore DBA2/N mice were used to avoid confounding the results because of an individual strain&#39;s susceptibility or resistance to neutrophils. As the neutrophil anti-C. albicans response can impact filamentation23, neutrophil recruitment to the site of infection could impact the results of a morphogenesis assay. If a strain is capable of filamenting in vivo but is strongly inhibited from filamentation when neutrophils are present, filamentation would be detected in DBA2/N mice but would be unlikely to be seen when using mice with intact neutrophil chemotaxis. Thus, the strain of the mouse used as a host is an important factor when using this protocol.
The observation that the efg1&#916;&#916; mutant strain fails to filament in vivo is unlikely to be related to host neutrophil responses, because this strain also fails to filament in vitro. The filamentation observed in vivo with the cyr1&#916;&#916; strain is discordant with its failure to filamentation in vitro. Data from the zebrafish model of C. albicans infection indicate that responding neutrophils are important in the prevention of morphogenesis24. Therefore, the use of DBA2/N mice, which lack neutrophil responses, is unlikely to account for the increase in filamentation of the cyr1&#916;&#916; in vivo compared to in vitro. Nevertheless, the in vivo environment is clearly impacting the morphogenesis of the cyr1&#916;&#916; strain; thus, further investigation of this strain may provide important information about the regulation of C. albicans morphogenesis during active infections. The protocol described here is designed as a screening assay to identify strains such as the cyr1&#916;&#916; strain to be used in future studies.
The use of a low-flow gas anesthesia system is very helpful for this protocol (Figure 1A,B). During the initial development of this protocol, mice were anesthetized using an injectable anesthetic cocktail of ketamine mixed with xylazine. While it was possible to perform limited imaging with that anesthetic method, the duration of anesthesia was unpredictable, requiring imaging sessions to be ended quickly to avoid the mouse recovering from anesthesia during imaging. Traditional inhaled anesthetic systems are bulky and require high rates of flow of anesthetic gasses, often requiring them to be used within a fume hood. Thus, traditional inhaled anesthetic systems would be very difficult to use with the space constraints of a confocal microscope without inadvertently exposing the investigators to the anesthetic agents. The use of a low-flow inhaled anesthetic system allows consistent anesthesia of the animal while maintaining a safe environment for the investigator. The low-flow nosecone allows easy positioning of the animal for both inoculation and microscopy. The small-caliber, low-volume delivery tubing allows the use of relatively long tubes, which enables the anesthesia machine to be placed at a sufficient distance so as not to interfere with microscopy.
The chlorophyll present in typical mouse chow leads to significant tissue autofluorescence25. This creates substantial noise in the images, making it difficult to obtain high-quality, high-spatial resolution images. When animals were fed chlorophyll-free chow for 7 days prior to imaging, background from autofluorescence was substantially decreased in tissue, but chlorophyll deposited in the hair continued to be problematic. Removing the hair on the pinnae using an over-the-counter chemical depilatory cream is effective at minimizing autofluorescence in the hair (Figure 1C,D). Thus, the combination of chlorophyll-free chow and adequate hair removal substantially decreased autofluorescence and improved image quality dramatically. Because the hair is removed from the ear prior to imaging, the color of the animal&#39;s hair does not impact this system. This protocol has been used successfully to study C. albicans infections in BALB/c (white), C57BL/6 (black), and DBA2/N (brown) mice. The protocol can also be used with C57BL/6 knockout mice that are deficient in various host genes; this will allow future investigations into how the mammalian host immune system impacts filamentation. One feature of this model not discussed in this protocol is that, because this imaging system is non-invasive, the same animal can be imaged repeatedly over several days, allowing to follow the progress of individual infection over time. This feature will likely play a key role in future studies on the host-pathogen interaction.
In summary, this protocol results in high-spatial resolution images of C. albicans growing in the tissue of a live mammalian host, allowing accurate evaluation of morphogenesis in mutant strains8,9,10. The results presented here demonstrate how this protocol can be used to screen a library of C. albicans mutants. Of the C. albicans mutants tested to date, a large portion of mutants with known defects in morphogenesis in vitro readily undergo filamentation in vivo9,10. This highlights the importance of including an in vivo system such as this one in experiments designed to elucidate the mechanisms of C. albicans pathogenesis.

Candida albicans is a common human fungal pathogen, causing mucocutaneous disease, disseminated disease, and localized tissue infections1. A key feature of C. albicans physiology is its complex polymorphic growth, which is linked to its role as both a commensal and a pathogen2,3,4. Under rich nutrient conditions in vitro at 30 &#176;C, it typically grows as an ovoid budding yeast. A variety of environmental triggers, including nutrient deprivation, pH changes, growth at 37 &#176;C, exposure to serum, and growth when embedded in agar, result in the transition to a polarized growth pattern, resulting in the formation of true hyphae and/or pseudohyphae5. The initiation of polarized growth and the resulting growth of filamentous organisms is referred to as morphogenesis.
Because of the importance of morphogenesis in the organism&#39;s virulence, the regulation of hyphal formation has been extensively studied6,7. There is a complex network of signaling pathways and transcriptional regulation that triggers morphogenesis. Despite the relationship of C. albicans morphogenesis with pathogenesis, most studies investigating morphogenesis have used in vitro stimuli to trigger hyphal formation. It is becoming increasingly clear that the various in vitro models of filamentation are not identical in terms of the individual regulatory pathways stimulated. Furthermore, no in vitro growth conditions correspond tightly with the complex environment of the host. Given the importance of C. albicans as a human pathogen, the goal of this protocol is to investigate its morphogenesis during active infection in a mammalian host using a system with moderate throughput, thus allowing an investigator to screen C. albicans mutant libraries.
To facilitate these investigations, an in vivo imaging system was developed that allows us to obtain high spatial resolution images of C. albicans cells during infection of the pinna of an anesthetized mouse using an inverted confocal microscope8,9,10. Because the skin of the pinna is quite thin, these images can be obtained without the need for tissue dissection. Thus, quantitative phenotype data can be measured at the site of active infections within the host tissue. The protocol described here involves the transformation of a reference strain and one or more mutant strains with different fluorescent protein expression cassettes11,12. The fluorescent protein-expressing strains are then mixed and co-injected intradermally. After the infection is established, confocal imaging is used to quantify both the frequency of filamentation and the length of the formed filaments. The data obtained from the mutant strains is normalized to that obtained from the reference strain, which is present in the same tissue region, thus providing an internal control. This system has allowed us to successfully screen several series of C. albicans mutant strains, many of which have morphogenesis defects in vitro9,10. Many of these strains readily filament in vivo, highlighting the importance of in vivo models for the investigation of morphogenesis.

<table><tbody><tr><td>#1.5 coverslips</td><td>Thermo-Fisher</td><td>20811</td><td>large enough to cover the universal stage opening</td></tr><tr><td>0.1 mL Insulin syringes</td><td>EXELint</td><td>26018</td><td>Can use syringes that are 5/16&quot;&amp;ndash;1/2&quot; long and 29&amp;ndash;32 G</td></tr><tr><td>3.7% formaldehyde in dPBS</td><td>Sigma-Aldrich</td><td>SHBJ5734</td><td></td></tr><tr><td>70% Ethanol/30% water</td><td>Decon Laboratories</td><td>A05061001A</td><td></td></tr><tr><td>Alcohol prep pads</td><td>Covidien</td><td>5110</td><td>Alternative: gauze pads soaked in 70% isopropyl alcohol</td></tr><tr><td>&lt;em&gt;C.albicans&lt;/em&gt; reference strain and experimental strains</td><td>SN250</td><td>FGSC Online Catalog</td><td>The specific &lt;em&gt;C. albicans&lt;/em&gt; strain varies with experiment and the investigators goals. We have used strains derived from SC5314 as well as other clinical isolates.</td></tr><tr><td>Chlorophyl free mouse chow</td><td>Envigo</td><td>2920x</td><td></td></tr><tr><td>Computer</td><td>Dell</td><td>Optiplex 7050</td><td>Computer that can run imaging software for acquisition and for analysis of images. A variety of imaging software is available and varies with the specific microscope and user system.</td></tr><tr><td>Cotton tip applicator</td><td>Pro Advantage</td><td>76200</td><td></td></tr><tr><td>DBA2/N (6-12 week old mice)</td><td></td><td></td><td>BALB/c and C57/BL6 mice can also be used.&amp;nbsp; The latter allow for the use of widely available knockout mouse models as well as mouse models in which individual cell types, such as phagocytes, are identified by their expression of fluorescent proteins.</td></tr><tr><td>Double sided tape designed to hold fabric to skin (fashion tape)</td><td>local pharmacy or grocery store</td><td></td><td>Double sided adhesive tape designed for keeping clothing in place over human skin.&amp;nbsp; This is typically available over the counter in pharmacies and variety stores.&amp;nbsp; It is important to use this type of tape as it is designed for gentle adherence to skin.&amp;nbsp;&lt;br/&gt; Examples:&amp;nbsp;&lt;br/&gt; https://www.amazon.com/Womens-Fashion-Clothing-Transparent-Suitable/dp/B08S3TWR3H/ref=sr_1_40?crid=2UWFL8FMFAKGM&amp;amp;keywords&lt;br/&gt; =fashion+tape&amp;amp;qid=1649174406&amp;amp;sprefix=&lt;br/&gt; fashion+tape%2Caps%2C70&amp;amp;sr=8-40&lt;br/&gt; https://www.amazon.com/Fearless-Tape-Sensitive-Clothing-Transparent/dp/B07QY8V5XT/ref=sr_1_26?crid=2UWFL8FMFAKGM&amp;amp;keywords&lt;br/&gt; =fashion+tape&amp;amp;qid=1649174320&amp;amp;sprefix=&lt;br/&gt; fashion+tape%2Caps%2C70&amp;amp;sr=8-26&lt;br/&gt; https://www.amazon.com/Hollywood-Fashion-Secrets-Tape-Floral/dp/B009RX77MK/ref=sr_1_29?crid=2UWFL8FMFAKGM&amp;amp;keywords&lt;br/&gt; =fashion+tape&amp;amp;qid=1649174406&amp;amp;sprefix=&lt;br/&gt; fashion+tape%2Caps%2C70&amp;amp;sr=8-29</td></tr><tr><td>Dulbecco&#039;s phosphate buffered saline</td><td>Gibco / Thermo-Fisher</td><td>14190-144</td><td>Must be sterile; open a new container for every experiment</td></tr><tr><td>Fetal bovine serum</td><td>Gibco / Thermo-Fisher</td><td>26140-079</td><td></td></tr><tr><td>Gauze pad</td><td>Pro Advantage</td><td>P157112</td><td></td></tr><tr><td>Gel eye lurbicant</td><td>local pharmacy or grocery store</td><td></td><td></td></tr><tr><td>ImageJ or FIJI analysis software</td><td>NIH</td><td>ImageJ (FIJI)</td><td></td></tr><tr><td>Isoflurane</td><td>Akorn</td><td>J119005</td><td></td></tr><tr><td>Leica DMi8 (SP8 platform) with Leica 11506375 objective lens</td><td>Leica</td><td>DMi8 (SP8)</td><td>The objective lens&amp;nbsp; (Leica 11506375) used here&amp;nbsp; is a 25x water immersion lens to allow us to have a high NA (0.95) while approximating the refractive index of the ear tissue.&lt;br/&gt; The microscope (Leica DMi8 (SP8 platform) has 488 nm and 638 nm diode laser lines and is equipped with filter-free&amp;nbsp; spectral detection with computer controlled adjustable bandwidth for detection of emission light.&lt;br/&gt; The stage must have enough clearance to allow the objective to reach the bottom coverslip without hitting the stage.</td></tr><tr><td>Low-flow anesthesia system or traditional anesthesia vaporizer</td><td>Kent Scientific International</td><td>SomnoSuite</td><td></td></tr><tr><td>Nair hair remover lotion</td><td>local pharmacy or grocery store</td><td></td><td>Over the counter depilatory cream</td></tr><tr><td>Nourseothricin</td><td>Jena Bioscience</td><td>AB-101L</td><td></td></tr><tr><td>pENO1-NEON-NATR pENO1-iRFP-NATR plasmids</td><td></td><td></td><td>Fluorescent protein expression transformation constructs&amp;nbsp; generously given to us by Dr. Robert Wheeler (Seman, et al., 2018, Infection and Immunity; Bergeron, et al., 2017, Infection and Immunity)</td></tr><tr><td>Pressure sensitive laboratory tape</td><td>Tape &amp;amp; Label Graphic Systems Inc</td><td>1007910</td><td></td></tr><tr><td>RPMI1640 cell culture medium</td><td>Gibco / Thermo-Fisher</td><td>11875-093</td><td></td></tr><tr><td>Thimble, plastic 15 mL conical tube, or Falcon 5 mL round bottom polystyrene tubes</td><td>Falcon</td><td>352196</td><td>To safely hold the animals ear during injectinos</td></tr></tbody></table>

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.

The results presented here are based on previously published reports9,10. The goal of this analysis is to quantitatively evaluate the ability of mutant C. albicans strains to undergo morphogenesis during active infections. The typical parameters that distinguish pseudohyphae from hyphae can be difficult to evaluate in organisms growing in three dimensions in a complex in vivo environment. This is particularly true when looking at the two-dimensional cross-sections created by confocal imaging. Therefore, this screening analysis is focused on identifying organisms growing as filamentous versus yeast. For follow-up studies using a more in-depth analysis, including three-dimensional reconstructions, this method could be adapted to discriminate yeast, hyphae, and pseudohyphae.
The expression of a fluorescent protein in a reference or mutant strain of C. albicans allows straightforward detection of strain morphology in vivo (Figure 3 and Figure 4). In general, quantitative light microscopy analysis is best performed when few to none of the pixels in the image are saturated. For this protocol, however, a saturation of the image often simplifies the analysis. Fluorescent protein localization is not uniform throughout the cell and is often higher in the mother yeast than in filaments. Fortunately, for the investigation of morphogenesis, the spatial distribution of the signal, rather than its intensity, defines the outcome. Therefore, obtaining images in which many pixels are saturated improves the ability to quantify morphogenesis in this assay.
To illustrate the importance of evaluating morphogenesis in vivo, representative results are presented for the reference strain (SN250) and two mutants: one lacking the transcription factor Efg1 and the other lacking adenylyl cyclase Cyr1. In vitro, neither of these strains grow as filaments20,21.&#160;When grown in vitro in RPMI medium supplemented with 10% serum, the reference strain rapidly forms filaments, whereas the efg1&#916;&#916; and cyr1&#916;&#916; strains do not (Figure 3 and Figure 4). Under these conditions, the efg1&#916;&#916; mutant exhibits somewhat polarized growth, with the daughter cells being slightly elongated compared to the mother cells. This emphasizes the importance of using a clear definition of filamentation. Any such definition is by default arbitrary, but it is necessary for consistent evaluation of the phenotype. For these studies, a filamentous pattern of growth is defined as an organism with the longest dimension of a daughter cell more than twice that of the mother cell. Using this definition, the elongated efg1&#916;&#916; cells are not filamentous.
Consistent with its in vitro phenotype, the efg1&#916;&#916; exhibits a significant filamentation defect in vivo: approximately 9% of efg1&#916;&#916; cells grew as filaments in vivo (Figure 3). In contrast, 53% of the cyr1&#916;&#916; mutant cells grew as filaments in vivo (Figure 4). Although the number of cyr1&#916;&#916; mutant cells undergoing filamentation in vivo was significantly lower than the reference strain, the ability of the cyr1&#916;&#916; mutant to form filaments in vivo represents a substantial change from its complete lack of morphogenesis in vitro. Visually, the filaments formed by the cyr1&#916;&#916; mutant appeared to be shorter than the reference strain. To evaluate this quantitatively, the curve path length of the filamentous cells was measured using a two-dimensional projection of the in vivo images (Figure 4E). The median length of cyr1&#916;&#916; filaments was 29% shorter than filaments of the reference strain.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64258/64258fig01.jpg" /> 
Figure 1: Anesthesia and inoculation. (A) Induction of anesthesia using an induction chamber. (B) Anesthesia is maintained using a nose cone, allowing the mouse to be repositioned as needed. (C) Hair removal cream is applied using a cotton-tipped applicator. Eye lubrication gel has been applied to protect the eyes during anesthesia. (D) Effective hair removal of the right ear. Compare to the untreated left ear. (E) Intradermal injection of C. albicans into the mouse ear. The ear is held in place using the tip of a conical tube wrapped with double-sided skin tape (fashion tape). (F) Close up of intradermal injection. A pale bubble is formed in the skin, which is a sign of a successful intradermal placement. <a href="https://www.jove.com/files/ftp_upload/64258/64258fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64258/64258fig02.jpg" /> 
Figure 2: Preparation for imaging. (A) Microscope stage prepared for imaging. The anesthesia nose cone is secured in place. A coverslip is taped to the stage covering the lens opening. Additional pieces of tape are available. The heated stage is pre-warmed to 37 &#176;C (not shown). (B) Placement of the anesthetized mouse into the anesthesia nose cone. (C) The mouse is rotated slightly so that the side of the ear that was inoculated is facing toward the bottom coverslip/objective lens. The ear is then flattened against the bottom coverslip and secured in place by placing a second coverslip on top of the ear. (D) The top coverslip is taped to the stage to secure the ear in place relative to the microscope stage. <a href="https://www.jove.com/files/ftp_upload/64258/64258fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64258/64258fig3v2.jpg" /> 
Figure 3: In vitro and in vivo morphology of the efg1&#916;&#916; mutant strain. (A) Widefield image of WT and efg1&#916;&#916; mutant strains after in vitro induction of filamentation by growing cells in RPMI + 10% serum at 37&#160;&#176;C for 4 h. Scale bars represent 10 &#181;m. Image contrast was adjusted using photo editing software to facilitate viewing. (B) Percentage of filamentation in vitro observed in the WT and efg1&#916;&#916; mutant strains. Und = undetectable (no filaments were detected). Bar height represents the median percentage of filamentous cells from three independent experiments wherein at least 100 cells were quantified. The error bars indicate standard deviation (results compared by student&#39;s t-test, p &#60; 0.001). (C) Confocal micrograph of WT (green) and the efg1&#916;&#916; mutant (red) growing in vivo 24 h post-inoculation. Arrows indicate examples of efg1&#916;&#916; mutant cells which meet our definition of &#34;filamentous&#34;. Scale bar represents 50 &#181;m. (D) Percentage of filamentation in vivo observed in the WT and efg1&#916;&#916; mutant strains. Bar height represents the median percentage of filamentous cells from two independent experiments. The error bars indicate standard deviation (results compared by student&#39;s t-test, p &#60; 0.001). <a href="https://www.jove.com/files/ftp_upload/64258/64258fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/64258/64258fig04v2.jpg" /> 
Figure 4: In vitro and in vivo morphology of the cyr1&#916;&#916; mutant strain. (A) Widefield image of the cyr1&#916;&#916;&#160;mutant strain after in vitro induction of filamentation by growing cells in RPMI + 10% serum at 37&#160;&#176;C for 4 h. Scale bar represents 10 &#181;m. Image contrast was adjusted using photo editing software to facilitate viewing. (B) Percentage of filamentation in vitro observed in the WT and cyr1&#916;&#916; mutant strains. Und = undetectable (no filaments were detected). Bar height represents the median percentage of filamentous cells from three independent experiments wherein at least 100 cells were quantified. The error bars indicate standard deviation (results compared by student&#39;s t-test, p &#60; 0.001). (C) Confocal micrograph of WT (green) and the cyr1&#916;&#916; mutant (red) growing in vivo 24 h post-inoculation. Scale bar represents 50 &#181;m. (D) Percentage of filamentation in vivo observed in the WT and cyr1&#916;&#916;&#160;mutant strains. Bar height represents the median percentage of filamentous cells from two independent experiments. The error bars indicate standard deviation (results compared by student&#39;s t-test, p &#60; 0.001). (E) Distribution of filament length in vivo, as measured by curve path length in a two-dimensional projection of the z-stack. Each dot represents the length of one filament. Cells growing as yeast were not included in this analysis. Bar indicates median filament length. The distribution of lengths is significantly different when analyzed using a Mann-Whitney U test (p &#60; 0.001). <a href="https://www.jove.com/files/ftp_upload/64258/64258fig04largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Use of In Vivo Imaging to Screen for Morphogenesis Phenotypes in Candida albicans Mutant Strains During Active Infection in a Mammalian Host at JoVE.com

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.

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 ...

use-vivo-imaging-to-screen-for-morphogenesis-phenotypes-candida

Research

JoVE Journal

Immunology and Infection

1.7K Views.  University of Iowa. Candida albicans morfogenese wordt veroorzaakt door een groot aantal omgevingssignalen. Door morfogenese in vivo te bestuderen, bepalen we hoe het organisme al deze omgevingssignalen integreert en erop reageert. Het belangrijkste voordeel van deze techniek is dat het ons in staat stelt om morfogenese te bestuderen in afwezigheid van bias of artefacten die kunnen worden geïntroduceerd met behulp van in vitro modelsystemen.Zeer weinig mensen hebben ervaring met intradermale injecties. I...

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

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

Non-invasive Imaging of Disseminated Candidiasis in Zebrafish Larvae

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 to 40% attributable mortality6. Systemic candidiasis is normally controlled by innate immunity, and individuals with genetic defects in innate immune cell components such as phagocyte NADPH oxidase are more susceptible to candidemia7-9. Very little is known about the dynamics of C. albicans interaction with innate immune cells in vivo. Extensive in vitro studies have established that outside of the host C. albicans germinates inside of macrophages, and is quickly destroyed by neutrophils10-14. In vitro studies, though useful, cannot recapitulate the complex in vivo environment, which includes time-dependent dynamics of cytokine levels, extracellular matrix attachments, and intercellular contacts10, 15-18. To probe the contribution of these factors in host-pathogen interaction, it is critical to find a model organism to visualize these aspects of infection non-invasively in a live intact host. 
The zebrafish larva offers a unique and versatile vertebrate host for the study of infection. For the first 30 days of development zebrafish larvae have only innate immune defenses2, 19-21, simplifying the study of diseases such as disseminated candidiasis that are highly dependent on innate immunity. The small size and transparency of zebrafish larvae enable imaging of infection dynamics at the cellular level for both host and pathogen. Transgenic larvae with fluorescing innate immune cells can be used to identify specific cells types involved in infection22-24. Modified anti-sense oligonucleotides (Morpholinos) can be used to knock down various immune components such as phagocyte NADPH oxidase and study the changes in response to fungal infection5. In addition to the ethical and practical advantages of using a small lower vertebrate, the zebrafish larvae offers the unique possibility to image the pitched battle between pathogen and host both intravitally and in color. 
The zebrafish has been used to model infection for a number of human pathogenic bacteria, and has been instrumental in major advances in our understanding of mycobacterial infection3, 25. However, only recently have much larger pathogens such as fungi been used to infect larva5, 23, 26, and to date there has not been a detailed visual description of the infection methodology. Here we present our techniques for hindbrain ventricle microinjection of prim25 zebrafish, including our modifications to previous protocols. Our findings using the larval zebrafish model for fungal infection diverge from in vitro studies and reinforce the need to examine the host-pathogen interaction in the complex environment of the host rather than the simplified system of the Petri dish5.

The rapid development, small size and transparency of zebrafish are tremendous advantages for the study of innate immune control of infection1-4. Here we demonstrate techniques for infecting zebrafish larvae using the fungal pathogen Candida albicans by microinjection, methodology recently used to implicate phagocyte NADPH oxidase activity in control of fungal dimorphism5.

Disseminated candidiasis caused by the pathogen Candida albicans is a clinically important problem in hospitalized individuals and is associated with a 30 ...

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening pathogen in immune-compromised individuals. Upon congenital or neonatal infection, the virus can infect and replicate in the developing brain, which may induce severe neurological damage, including deafness and mental retardation. Despite the potential severity of the symptoms, the therapeutic options are limited by the unavailability of a vaccine and the absence of a specific antiviral therapy. Furthermore, a precise description of the molecular events occurring during infection of the central nervous system (CNS) is still lacking since observations mostly derive from the autopsy of infected children. Several animal models, such as rhesus macaque CMV, have been developed and provided important insights into CMV pathogenesis in the CNS. However, despite its evolutionary proximity with humans, this model was limited by the intracranial inoculation procedure used to infect the animals and consistently induce CNS infection. Furthermore, ethical considerations have promoted the development of alternative models, among which neonatal infection of newborn mice with mouse cytomegalovirus (MCMV) has recently led to significant advances. For instance, it was reported that intraperitoneal injection of MCMV to Balb/c neonates leads to infection of neurons and glial cells in specific areas of the brain. These findings suggested that experimental inoculation of mice might recapitulate the deficits induced by HCMV infection in children. Nevertheless, a dynamic analysis of MCMV infection of neonates is difficult to perform because classical methodology requires the sacrifice of a significant number of animals at different time points to analyze the viral burden and/or immune-related parameters. To circumvent this bottleneck and to enable future investigations of rare mutant animals, we applied in vivo imaging technology to perform a time-course analysis of the viral dissemination in the brain upon peripheral injection of a recombinant MCMV expressing luciferase to C57Bl/6 neonates.

Use of In vivo Imaging to Monitor the Progression of Experimental Mouse Cytomegalovirus Infection in Neonates

Human Cytomegalovirus (HCMV) infection of neonates represents an important cause of mental retardation, yet the molecular events leading to virus-induced pathogenesis are still poorly understood. To investigate the dynamics of brain infection, we adapted whole-animal in vivo imaging to perform time-course analysis of neonates infected with a luciferase-recombinant virus.

Human Cytomegalovirus (HCMV or HHV-5) is a life-threatening  pathogen in immune-compromised individuals. Upon congenital or neonatal  infection, the virus ...

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 ...

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 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 ...

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 ...

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

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