Name: use of in vivo imaging to screen for morphogenesis phenotypes in candida albicans mutant strains during active infection in a mammalian host
Uploaded: 2022-10-12T15:00:00
Duration: 9 min 24 s
Description: 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

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

<ol>
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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. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+Candida+albicans+hyphae.">Growth of Candida albicans hyphae.</a> Nature Reviews Microbiology. 9 (10), 737-748 (2011).</li><li>Basso, V., d'Enfert, C., Znaidi, S., Bachellier-Bassi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=From+genes+to+networks:+The+regulatory+circuitry+controlling+candida+albicans+morphogenesis.">From genes to networks: The regulatory circuitry controlling candida albicans morphogenesis.</a> Current Topics in Microbiology and Immunology. 422, 61-99 (2019).</li><li>Mancera, E., 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=Evolution+of+the+complex+transcription+network+controlling+biofilm+formation+in+Candida+species.">Evolution of the complex transcription network controlling biofilm formation in Candida species.</a> Elife. 10, 64682 (2021).</li><li>Mitra, S., Dolan, K., Foster, T. 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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. G., 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=Yeast+and+filaments+have+specialized,+independent+activities+in+a+zebrafish+model+of+Candida+albicans+infection.">Yeast and filaments have specialized, independent activities in a zebrafish model of Candida albicans infection.</a> Infection and Immunity. 86 (10), 00415-00418 (2018).</li><li>. Biosafety in Microbiological and Biomedical Laboratories (BMBL). 6th edition Available from: <a target="_blank" href="https://www.cdc.gov/labs/BMBL.html">https://www.cdc.gov/labs/BMBL.html</a> (2020)</li><li>Homann, O. R., Dea, J., 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=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. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+a+genetically+engineered+strain+to+evaluate+the+pathogenic+potential+of+yeast+cell+and+filamentous+forms+during+Candida+albicans+systemic+infection+in+immunodeficient+mice.">Use of a genetically engineered strain to evaluate the pathogenic potential of yeast cell and filamentous forms during Candida albicans systemic infection in immunodeficient mice.</a> Infection and Immunity. 76 (1), 97-102 (2008).</li><li>Brothers, K. M., Newman, Z. R., Wheeler, R. 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. C., Pottier, R., Rossi, R., Weagle, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+recipe+for+the+preparation+of+a+rodent+food+that+eliminates+chlorophyll-based+tissue+fluorescence.">A recipe for the preparation of a rodent food that eliminates chlorophyll-based tissue fluorescence.</a> Journal of Photochemistry and Photobiology. B: Biology. 29 (2-3), 199 (1995).</li></ol>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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&#34;&#8211;1/2&#34; long and 29&#8211;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>C.albicans reference strain and experimental strains</td>
			<td>SN250</td>
			<td>FGSC Online Catalog</td>
			<td>The specific C. albicans 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.&#160; 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.&#160; This is typically available over the counter in pharmacies and variety stores.&#160; It is important to use this type of tape as it is designed for gentle adherence to skin.&#160; 
			Examples:&#160; 
			https://www.amazon.com/Womens-Fashion-Clothing-Transparent-Suitable/dp/B08S3TWR3H/ref=sr_1_40?crid=2UWFL8FMFAKGM&#38;keywords 
			=fashion+tape&#38;qid=1649174406&#38;sprefix= 
			fashion+tape%2Caps%2C70&#38;sr=8-40 
			https://www.amazon.com/Fearless-Tape-Sensitive-Clothing-Transparent/dp/B07QY8V5XT/ref=sr_1_26?crid=2UWFL8FMFAKGM&#38;keywords 
			=fashion+tape&#38;qid=1649174320&#38;sprefix= 
			fashion+tape%2Caps%2C70&#38;sr=8-26 
			https://www.amazon.com/Hollywood-Fashion-Secrets-Tape-Floral/dp/B009RX77MK/ref=sr_1_29?crid=2UWFL8FMFAKGM&#38;keywords 
			=fashion+tape&#38;qid=1649174406&#38;sprefix= 
			fashion+tape%2Caps%2C70&#38;sr=8-29</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;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&#160; (Leica 11506375) used here&#160; 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. 
			The microscope (Leica DMi8 (SP8 platform) has 488 nm and 638 nm diode laser lines and is equipped with filter-free&#160; spectral detection with computer controlled adjustable bandwidth for detection of emission light. 
			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&#160; 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 &#38; 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 ...

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