The authors would like to thank Carolina Tropini, Katharine Ng, Justin Sonnenburg, and KC Huang for guidance in developing the FISH technique. Teresa O&rsquo;Meara provided helpful comments on the manuscript, and Miriam Levy assisted with photography. This work was supported by NIH grants R01AI108992, R01DK113788, and a Burroughs Welcome Award in the Pathogenesis of Infectious Disease.

<ol>
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The authors have nothing to disclose.

The method described here allows for visualization of C. albicans yeasts and hyphae in the GI tracts of commensally colonized mice of any sex or strain. The FISH probe hybridizes to 23S rRNA, which is distributed throughout the fungal cytoplasm. Our method was adapted from a previously reported protocol for visualizing gut bacteria20. Because C. albicans changes its morphology within the host, the method is useful to monitor fungal cell shape as well as localization. For example, we have used this method to disprove the hypothesis that yeasts predominate throughout the digestive tract, and to expose discrepancies between the in vivo and in vitro phenotypes of certain &ldquo;filamentation-defective&rdquo; mutants12.
Several mouse models exist for C. albicans commensal colonization. The microbiota of laboratory mice and humans are distinct and, in mice, the use of antibiotics or a specialized diet is required to establish stable colonization. Treatment with antibiotics also enhances C. albicans colonization of humans and is a major risk factor for disseminated disease10. The antibiotics used in this study are relatively inexpensive and yield reliable decreases in the bacterial microbiota. Note that antibiotics are used to decrease the burden of antagonistic bacterial species, not to eliminate all bacteria from the animals. If researchers wish to study C. albicans-host interactions in the absence of bacteria or to avoid the use of antibiotics or a special diet, germ-free animals can be substituted for conventionally reared animals; however, gnotobiotic mice exhibit certain immune and anatomic abnormalities and therefore may not be suitable for all purposes.
Several steps are important for successful FISH staining: The recommended fixation method is critical to preserve the structural integrity of GI tissues, particularly the fragile contents of the gut lumen, such as the mucus layer and the three-dimensional organization of fungi and bacteria16. Please note that many commonly used fixation solutions that contain water are highly damaging to the luminal architecture. The fixatives and solutions for post-fixation washes recommended in this protocol do not contain water, and it is important to avoid contamination with water. Another critical step is the hybridization step, where it is important to avoid evaporation of the hybridization solution during incubation of slides in the hybridization oven. We suggest placing the slides in a watertight container to avoid this problem. Alternatively, one can use watertight hybridization chambers such as those originally used for hybridization of microarrays.
A C. albicans-specific FISH probe is described in this protocol. However, because many laboratory-reared mice do not contain C. albicans or other fungi as part of their natural gut microbiota, a panfungal probe may specifically stain C. albicans in experimentally colonized animals. Substitution of a panfungal probe may be desirable because of its superior hybridization characteristics and higher signal-to-noise ratio. If the panfungal probe is used as a pseudospecific probe for C. albicans, it is important to document a lack of staining in uninfected animals (i.e., treated with antibiotics but not C. albicans). Staining of a noncolonized control animal is also useful to assess background staining that may result from adherence of FISH probes to food particulates. Overall, the use of FISH probes offers enhanced specificity over most other methods of staining fungi, such as Calcofluor white (which stains chitin, a cell wall component that may vary between cell types), GMS, or commercially available antifungal antibodies. Moreover, FISH staining allows for costaining of multiple organisms using differentially labeled FISH probes to species-specific rRNAs.
One caveat of this technique is that certain C. albicans yeast cell types appear very similar by FISH (for example, the opaque and GUT cell types). To distinguish among these cell types, it would be useful to develop hybridization probes to cell type-specific fungal mRNAs. Nevertheless, in its current form, the FISH technique has already revealed surprising discrepancies between the in vivo and in vitro behaviors of C. albicans mutants evaluated under both conditions12, indicating complex interactions in the natural commensal environment that are not adequately mimicked by existing in vitro assays. Further studies of different C. albicans stains in additional wild type and mutant hosts are likely to yield additional insights into fungal-host interactions. In particular, it will be instructive to profile C. albicans in models of inflammatory bowel disease, which is associated with high titers of C. albicans in humans21, and other models of C. albicans overgrowth and disease. The FISH technique may also be combined with immunohistochemistry to stain specific host cells. Overall, the method presented here provides a reasonably quick and reliable means to determine C. albicans localization and morphology within the mammalian GI tract.

Candida albicans is a fungal commensal as well as an opportunistic human pathogen. This yeast lacks a defined environmental niche and instead propagates within the gastrointestinal (GI) tract, skin, and genitourinary tract of humans and other mammals1. Whereas early research on C. albicans focused primarily on its virulence potential, several recent reports suggest that commensally propagating organisms within the gut may play important roles in normal health, including the immune development of the host2,3,4. To facilitate investigations of C. albicans commensalism within the mammalian gut, we developed a mouse model of stable GI colonization and a fluorescence in situ hybridization (FISH)-based method to visualize fungal yeast cells and hyphae within the intestinal lumen.
With some exceptions5, laboratory-bred mice typically exhibit resistance to fungal colonization of the digestive tract. Colonization resistance is thought to be mediated by specific bacterial species; however, this can be overcome by treatment of the animals with antibiotics6,7 or use of a chemically-defined diet that presumably alters bacterial species composition8,9. Similarly, in humans, the use of broad-spectrum antibiotics has been associated with C. albicans overgrowth and dissemination10. Our murine colonization model uses broad-spectrum antibiotics to establish C. albicans colonization of immunocompetent, conventionally reared mice. Penicillin and streptomycin are provided in the animals&#8217; drinking water for one week prior to gavage with 108 colony forming units (CFUs) of C. albicans. As long as the antibiotic-infused water is continued, C. albicans will propagate through the GI tract, reaching fecal titers of 106&#8722;108 CFUs/g. Despite the high level of fungal colonization, animals remain healthy and gain weight at the same rate as uninfected controls. This model has been used successfully to screen for and characterize multiple C. albicans commensalism factors11,12.
Like other members of the fungal kingdom, C. albicans is capable of enormous morphological plasticity13. Under in vitro conditions, it has been shown to transition among at least six unicellular yeast cell types, as well as multicellular hyphae and pseudohyphae. The yeast-to-hypha transition is one of its best-characterized virulence attributes, and hyphae and pseudohyphae predominate in most mammalian disease models, as well as in infected human tissues. To determine C. albicans localization and cell morphology within the murine digestive tract, we developed a FISH technique for staining yeasts and hyphae in fixed histological sections. The probes consist of fluorescently labeled DNA oligonucleotides that hybridize to fungal 23S ribosomal RNA (rRNA), which is distributed throughout the fungal cytoplasm. Because host tissues are fixed in a manner that preserves the three-dimensional architecture of the gut, including the host mucosa, digestive material, the bacterial microbiota, and mucus within the gut lumen, this technique permits the localization of fungal cells with respect to these landmarks when stained. The FISH technique compares favorably to traditional histological stains for fungi, such as Periodic Acid Schiff (PAS) or Gomori&#39;s Methenamine-Silver (GMS), as well as commercially available antifungal antibodies, because these reagents are not specific for C. albicans. Moreover, standard fixatives remove the mucus layer and disrupt other contents of the gut lumen14,15.
In this article, we provide detailed instructions for establishing high-grade C. albicans colonization of the mouse GI tract, for dissection of the digestive tract from euthanized animals, for tissue fixation in a manner that preserves luminal architecture, and for detection of C. albicans within host tissues using FISH. In addition to wild type and mutant strains of C. albicans, the gavage technique can be used to deliver other microorganisms. The fixation technique would be useful for any study in which preservation of gut contents is desired. The FISH procedure can be completed within a day and can be used to localize multiple fungal species by using multiple, differentially labeled probes.

<table><tbody><tr><td>1 mL syringe</td><td>BD</td><td>309659</td><td>can be substituted from any vendor</td></tr><tr><td>BHI blood agar</td><td></td><td></td><td>can be substituted from any vendor</td></tr><tr><td>&lt;em&gt;C. albicans&lt;/em&gt; FISH probe</td><td>IDT DNA Technologies</td><td>custom order</td><td></td></tr><tr><td>Chamber for hybridization incubation</td><td></td><td></td><td>watertight chamber meant to reduce evaporation</td></tr><tr><td>Chloroform</td><td>Sigma</td><td>C2432</td><td>&gt;=99.5%</td></tr><tr><td>DAPI</td><td>Roche</td><td>10236276001</td><td>can be substituted from any vendor</td></tr><tr><td>Delicate task wiper tissues</td><td>Kimberley-Clark</td><td>34256CT</td><td>Kimwipes</td></tr><tr><td>D-glucose</td><td>Sigma</td><td>G7021-5KG</td><td>can be substituted from any vendor</td></tr><tr><td>Ethanol</td><td>Sigma</td><td>E7023</td><td>molecular biology grade</td></tr><tr><td>Feeding needles</td><td>Cadence, Inc.</td><td>7910</td><td>metal; 20 X1-1/2&quot; W/2-1/4; can be autoclaved to sterilize</td></tr><tr><td>FITC-UEA-1</td><td>Sigma</td><td>L9006-1MG</td><td>other fluorophores available</td></tr><tr><td>FITC-WGA</td><td>Sigma</td><td>L4895-2MG</td><td>other fluorophores available</td></tr><tr><td>Foam pads</td><td>Fisher</td><td>22038221</td><td>Order foam pads that will fit within cassettes</td></tr><tr><td>Formamide</td><td>Sigma</td><td>47671</td><td>molecular biology grade</td></tr><tr><td>Glacial acetic acid</td><td>Macron Fine Chemicals</td><td>MK881746</td><td>ACS reagent, &gt;=99.5%</td></tr><tr><td>Glass Coplin jar</td><td>Fisher</td><td>08-815</td><td>hold up to 10 slides back to back</td></tr><tr><td>Histology cassettes</td><td>Simport</td><td>M512</td><td>Deep cassettes so cecum is not squished</td></tr><tr><td>Hybridization coverslips</td><td>Sigma</td><td>GBL712222</td><td>RNase-free</td></tr><tr><td>Hybridization oven</td><td></td><td></td><td>can be substituted from any vendor</td></tr><tr><td>LB</td><td></td><td></td><td>can be substituted from any vendor</td></tr><tr><td>Lee&#039;s media</td><td></td><td></td><td>prepared as described in Lee et al. 1975</td></tr><tr><td>Methanol</td><td>Sigma</td><td>179337</td><td>ACS reagent, &gt;=99.8%</td></tr><tr><td>Mice</td><td>Charles River Laboratories</td><td>028</td><td>adult BALB/c; 18-21 grams (8-10 weeks)</td></tr><tr><td>Paraformaldehyde</td><td>Fisher</td><td>50-980-487</td><td>16% solution</td></tr><tr><td>Parrafin wax</td><td>Sigma</td><td>P3558-1KG</td><td>Paraplast for tissue embedding</td></tr><tr><td>PBS, pH 7.4</td><td>UCSF Cell Culture Facility Media Production Unit</td><td>CCFAL003</td><td>calcium, magnesium-free; can be substituted from any vendor</td></tr><tr><td>Penicillin G</td><td>Sigma</td><td>PENNA-100MU</td><td></td></tr><tr><td>Sabouraud dextrose agar</td><td></td><td></td><td>can be substituted from any vendor</td></tr><tr><td>Saline</td><td>Baxter</td><td>2F7123</td><td>sterile, can be substituted from any vendor</td></tr><tr><td>Sodium chloride (NaCl)</td><td>Sigma</td><td>S3014</td><td>can be substituted from any vendor; maintain RNase-free</td></tr><tr><td>Sodium dodecyl sulfate (SDS)</td><td>Sigma</td><td>L3771</td><td>can be substituted from any vendor; maintain RNase-free</td></tr><tr><td>Streptomycin</td><td>Sigma</td><td>S9137-100G</td><td></td></tr><tr><td>Super PAP pen</td><td>Life Technologies</td><td>8899</td><td>can be substituted from any vendor</td></tr><tr><td>Tris-HCl</td><td>Sigma</td><td>T3253</td><td>can be substituted from any vendor; maintain RNase-free</td></tr><tr><td>Vectashield</td><td>Vector Laboratories</td><td>H-1000</td><td>does not contain DAPI</td></tr><tr><td>Xylenes</td><td>Sigma</td><td>214736</td><td>reagent grade</td></tr><tr><td>YEPD</td><td></td><td></td><td>can be substituted from any vendor</td></tr></tbody></table>

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.

Following the instructions provided, the outcome of this technique will be fluorescent labelling of nuclei in the host epithelium, mucus, and C. albicans in sectioned GI tissues. Wild type Candida albicans cells should appear as either round, unicellular yeast cells or highly elongated, sometimes branching, multicellular hyphae (cell-cell divisions will not be apparent in the hyphae). Mutants of C. albicans may additionally appear as smaller, slightly elongated &#8220;gray&#8221; yeasts or as larger, slightly elongated &#8220;GUT&#8221; (gastrointestinal-induced transition) or &#8220;opaque&#8221; yeasts.
Figure 1 depicts the feeding needle used for oral gavage, as well as key positions of the needle during the gavage procedure. Figure 2 provides a typical setup used for organ dissection and the appearance of GI segments of the mouse.
Figure 3 displays FISH staining of different C. albicans cell types following propagation in vitro and within the mouse model. Figure 3A shows fluorescence and phase images of fixed, permeabilized, in vitro-propagated hyphae. Hypha formation was induced with liquid Lee&#8217;s medium18 with 2% N-acetylglucosamine, pH 6.8 at 37 &#176;C. Figure 3B shows fluorescence and phase images of fixed, permeabilized, in vitro-propagated yeast cells. Figure 3C,D shows fixed, permeabilized, in vitro-propagated GUT cells and opaque cells, respectively. GUT and opaque cell types are morphologically indistinguishable except when visualized by scanning electron microscopy. Please note that FISH staining of in vitro-propagated cells will be suboptimal if the cells are not well permeabilized. An example of weak and diffuse staining is shown in Figure 3C. For best results, cell fixation and permeabilization conditions should be determined empirically for each cell type and species to be investigated. Fortunately, the recommended protocol for visualizing C. albicans in sectioned tissues produces more consistent permeabilization.
Figure 3E-G depict C. albicans in mouse large intestines, stained with the C. albicans-specific probe (Figure 3E) or the panfungal probe (Figure 3F,G). C. albicans appears red in these images, host cell nuclei are blue, and the mucus layer is green. Both round yeasts and highly elongated hyphae (white arrowheads) occur in the large intestines. Please note that staining is brighter with the panfungal probe. Figure 3G depicts a ume6 mutant in the same compartment, stained with the panfungal probe. Ume6 encodes a transcription factor that is required for hypha formation under in vitro conditions19. Interestingly, the yeast-locked phenotype displayed by this mutant under in vitro conditions is not recapitulated within the gut, suggesting that a redundant factor must be activated within the host12. Figure 4 depicts the appearance of wild type C. albicans in different segments of the murine GI tract, including the regions immediately adjacent to the host mucosa and more central regions of the gut lumen.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/60283/60283fig01.jpg" /> 
Figure 1: Key feeding needle positions during gavage. (A) Feeding needle. (B) Feeding needle ball inserted to the side of the tongue. (C) Feeding needle in the upright position with insertion in the esophagus. (D) Feeding needle inserted into the stomach with a few millimeters visible above the nose. <a href="https://www.jove.com/files/ftp_upload/60283/60283fig01large.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/60283/60283fig02.jpg" /> 
Figure 2: Example dissection layout. (A) Dissection pad and tools. (B) Excised GI tract. Proximal (esophagus) to distal (anus) sections: I. Stomach. II. Proximal small intestines. III. Medial small intestines. IV. Distal small intestines. V. Cecum. VI. Large intestine. Pink dashed border boxes indicate the tissues to be fixed. (C) Tissues positioned in cassettes. Roman numerals are the same as in panel B. <a href="https://www.jove.com/files/ftp_upload/60283/60283fig02large.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/60283/60283fig03.jpg" /> 
Figure 3: Representative images of FISH-stained C. albicans. (A) FISH of C. albicans hyphae grown in liquid Lee&#8217;s media18 with 2% N-acetylglucosamine, pH 6.8. (B) FISH of C. albicans round yeast cells grown on YEPD + 2% agar plates. (C) FISH of C. albicans GUT cells (ySN1045) grown on YEPD + 2% agar plates. (D) FISH of C. albicans opaque cells grown on YEPD + 2% agar plates. (E) Wild type C. albicans (ySN250) in large intestines, stained with the C. albicans-specific probe. (F) Wild type C. albicans in large intestines, stained with the panfungal probe. (G) Ume6 mutant (ySN1479) in large intestines, stained with the panfungal probe: red is C. albicans, blue is host epithelial nuclei, and green is mucin. Arrowheads indicate hyphae. Arrows indicate yeast. Scale bars = 20 &#181;m. Images F and G are adapted from Witchley et al.12. <a href="https://www.jove.com/files/ftp_upload/60283/60283fig03large.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/60283/60283fig04.jpg" /> 
Figure 4: Appearance of FISH-stained C. albicans in different gut compartments. Wild type C. albicans is shown in the indicated segments of the mouse GI tract. Within each compartment, images depict the area adjacent to the host mucosa and the central region of the gut lumen. Staining was performed with the panfungal probe.&#160;Scale bar = 20 &#181;m. Images are adapted from Witchley et al.12. <a href="https://www.jove.com/files/ftp_upload/60283/60283fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Antibiotic stocks (200x)</td>
		</tr>
		<tr>
			<td>Penicillin G</td>
			<td>181 mg/mL</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin</td>
			<td>400 mg/&#181;L</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Methacarn</td>
		</tr>
		<tr>
			<td>Stock</td>
			<td>Final concentration</td>
			<td>Volume</td>
			<td>Units</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>60%</td>
			<td>300</td>
			<td>mL</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>30%</td>
			<td>150</td>
			<td>mL</td>
		</tr>
		<tr>
			<td>Glacial acetic acid</td>
			<td>10%</td>
			<td>50</td>
			<td>mL</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Hybridization solution</td>
		</tr>
		<tr>
			<td>Stock</td>
			<td>Final concentration</td>
			<td>Volume</td>
			<td>Units</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH 7.4</td>
			<td>20 mM</td>
			<td>20</td>
			<td>&#181;L</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>0.9 M</td>
			<td>180</td>
			<td>&#181;L</td>
		</tr>
		<tr>
			<td>10% SDS</td>
			<td>0.10%</td>
			<td>10</td>
			<td>&#181;L</td>
		</tr>
		<tr>
			<td>100% formamide</td>
			<td>1.00%</td>
			<td>10</td>
			<td>&#181;L (kept in small aliquots at -20 &#176;C between uses)</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td></td>
			<td>780</td>
			<td>&#181;L</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">FISH washing solution</td>
		</tr>
		<tr>
			<td>1 M Tris-HCl, pH 7.4</td>
			<td>20 mM</td>
			<td>1</td>
			<td>mL</td>
		</tr>
		<tr>
			<td>5 M NaCl</td>
			<td>0.9 M</td>
			<td>9</td>
			<td>mL</td>
		</tr>
		<tr>
			<td>RNase-free water</td>
			<td></td>
			<td>40</td>
			<td>mL</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4">Mucin-nuclei staining solution</td>
		</tr>
		<tr>
			<td>DAPI, 10 &#181;g/mL</td>
			<td>20 ng/mL</td>
			<td>2</td>
			<td>&#181;L</td>
		</tr>
		<tr>
			<td>lectin, 40 &#181;g/mL</td>
			<td>1.6 &#181;g/mL</td>
			<td>40</td>
			<td>&#181;L</td>
		</tr>
		<tr>
			<td>PBS, pH 7.4</td>
			<td></td>
			<td>958</td>
			<td>&#181;L</td>
		</tr>
	</tbody>
</table>
Table 1: Typical volumes and concentrations of solutions used in this protocol.

Watch this Scientific Journal Video about Visualization of Candida albicans in the Murine Gastrointestinal Tract Using Fluorescent In Situ Hybridization at JoVE.com

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.

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 ...

visualization-candida-albicans-murine-gastrointestinal-tract-using

Research

JoVE Journal

Immunology and Infection

11.8K Views.  University of California, San Francisco. Dit protocol heeft een aantal van de beste inzichten die we tot nu toe hebben opgeleverd in hoe Candida albicans het zoogdierarmkanaal bewoont. Bijvoorbeeld, tot voor kort, we wisten niet echt waar Candida is gelokaliseerd in de darm of welke van de vele morfologische vormen het aanneemt binnen deze niche. Het belangrijkste voordeel van deze techniek is dat het mogelijk maakt om Candida te visualiseren in zijn natuurlijke omgeving zonder het verstoren van de driedimensionale interacties van d...

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

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 ...

Live-cell Video Microscopy of Fungal Pathogen Phagocytosis

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

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

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

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

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 ...

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota and the vertebrate gut. The spatial landscape of the gut microbiota is tightly controlled by physical features - intestinal mucus, crypts, and folds - and is affected by host-controlled properties such as pH, oxygen availability, and immune factors. These properties limit the ability of commensal microbes and pathogens alike to colonize the gut stably. At the micron-scale, microbial organization determines the close-range interactions between different microbes as well as the interactions between microbes and their host. These interactions then affect large-scale organ function and host health.
This protocol enables the visualization of the gut microbiota spatial organization from distances between cells to organ-wide scales. The method is based on fixing gut tissues while preserving intestinal structure and mucus properties. The fixed samples are then embedded, sectioned, and stained to highlight specific bacterial species through fluorescence in situ hybridization (FISH). Host features, such as mucus and host cell components, are labeled with fluorescently labeled lectins. Finally, the stained sections are imaged using a confocal microscope utilizing tile-scan imaging at high magnification to bridge the micron to centimeter length scales. This type of imaging can be applied to intestinal sections from animal models and biopsies from human tissues to determine the biogeography of the microbiota in the gut in health and disease.

Visualization of Gut Microbiota-host Interactions via Fluorescence In Situ Hybridization, Lectin Staining, and Imaging

This streamlined protocol details a workflow to detect and image bacteria in complex tissue samples, from fixing the tissue to staining microbes with fluorescent in situ hybridization.

Measuring the localization of microbes within their in vivo context is an essential step in revealing the functional relationships between the microbiota ...

RNA Fluorescence in situ Hybridization (FISH) to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as microsporidia and viruses. Because of the similarities between Caenorhabditis elegans and mammalian intestinal cells, as well as the power of the C. elegans system, this host has emerged as a model system to study host intestine-microbe interactions in vivo. While it is possible to observe some aspects of these interactions with bright-field microscopy, it is difficult to accurately classify microbes and characterize the extent of colonization or infection without more precise tools. RNA fluorescence in situ hybridization (FISH) can be used as a tool to identify and visualize microbes in nematodes from the wild or to experimentally characterize and quantify infection in nematodes infected with microbes in the lab. FISH probes, labeling the highly abundant small subunit ribosomal RNA, produce a bright signal for bacteria and microsporidian cells. Probes designed to target conserved regions of ribosomal RNA common to many species can detect a broad range of microbes, whereas targeting divergent regions of the ribosomal RNA is useful for narrower detection. Similarly, probes can be designed to label viral RNA. A protocol for RNA FISH staining with either paraformaldehyde (PFA) or acetone fixation is presented. PFA fixation is ideal for nematodes associated with bacteria, microsporidia, and viruses, whereas acetone fixation is necessary for the visualization of microsporida spores. Animals were first washed and fixed in paraformaldehyde or acetone. After fixation, FISH probes were incubated with samples to allow for the hybridization of probes to the desired target. The animals were again washed and then examined on microscope slides or using automated approaches. Overall, this FISH protocol enables detection, identification, and quantification of the microbes that inhabit the C. elegans intestine, including microbes for which there are no genetic tools available.

RNA Fluorescence in situ Hybridization FISH to Visualize Microbial Colonization and Infection in Caenorhabditis elegans Intestines

Intestinal microbes, including extracellular bacteria and intracellular pathogens like the Orsay virus and microsporidia (fungi), are often associated with wild Caenorhabditis nematodes. This article presents a protocol for detecting and quantifying microbes that colonize and/or infect C. elegans nematodes, and for measuring pathogen load after controlled infections in the lab.

The intestines of wild Caenorhabditis nematodes are inhabited by a variety of microorganisms, including gut microbiome bacteria and pathogens, such as ...

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

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

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

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

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

Effective aPDT Using Curcuminoids for Oral Candidiasis in Mice

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

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

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

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

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

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

A Catheter-Related Candida albicans Infection Model in Mouse

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

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

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

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

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

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