Human Epithelial Model Systems for the Study of Candida infections In Vitro: Part II. Histologic Methods for Studying Fungal Invasion

Abstract

Although the role of invasion in the virulence of Candida albicans has been demonstrated, the mechanism that governs fungal invasion is not fully understood. Among the tools that exist to fill these gaps in knowledge, in vitro tissue models based on reconstituted human epithelia (RHE) have already been developed. Such models are designed to study more reproducably the fungus-host relationship, as they eliminate the complexity and variability found in vivo. Herein we describe the preparation of these RHE and their application in study of the invasion properties of C. albicans by further histologic processing and microscopic observation. For this purpose, different epithelial cell lines are grown on a collagen gel to build up models of intestinal (Caco-2 cell line), vaginal (A431 cell line), and oral (TR146 cell line) mucosa. The use of these in vitro models applied to test the invasiveness of C. albicans strains (clinical isolates or gene deleted mutants) and to identify changes in gene expression during the invasion of the RHE will help to advance our knowledge of pathogenesis and to study specific mechanisms used by C. albicans to adapt to changing environments present in different epithelia. Furthermore, because these models are useful to study the host response during the challenge with the pathogen, they will also offer important new insights into host cell biology and identify new targets for treatment.

A Screening Assay Based on Host-Pathogen Interaction Models Identifies a Set of Novel Antifungal Benzimidazole Derivatives

“…Each tissue model was infected with approximately 250 cells for up to 48 h. At the same time, EMC120B12 was added at a concentration of 2 M (2 g/ml) to the medium basolaterally supporting the tissue model. The tissue models were fixed after 48 h of incubation at 37°C and processed for histological staining as described previously (17).…”

Adaptation, adhesion and invasion during interaction of Candida albicans with the host – Focus on the function of cell wall proteins

“…Zakikhany et al (2007), using oral epithelia as infection models as well as samples from patients with pseudomembranous oropharyngeal candidiasis, identified both an overlapping as well as a distinct set The start of invasion can be clearly seen at the time point 4 h, whereas in the preceding panels only adhesion of C. albicans to the Caco-2 cell surface is visible. To visualize deep invasive infections, histological staining was performed (Hernandez and Rupp, 2009). Penetration of the cell layer and invasion of the collagen matrix beneath was observable already after 18 h. of genes in their experiments, consistent with the hypothesis that C. albicans reacts differently to distinct epithelia.…”

“…Nevertheless these models have been tremendously useful in delineating mechanisms of host-pathogen interaction. The enterocytic Caco-2 (colon adenocarcinoma derived cell line), A-431 (derived form vulvo-vaginal carcinoma), FaDu (oral epithelial cell line from a pharyngeal carcinoma) and the TR-146 cell line derived from squamous carcinoma of buccal mucosa are among the most frequently used cell lines for setting up simple infection models, representing the respective body site (Dalle et al, 2010;Dieterich et al, 2002;Hernandez and Rupp, 2009;Park et al, 2009;Sohn and Rupp, 2009;Sohn et al, 2006b;Spiering et al, 2010;Zhu and Filler, 2010). Some of the cell line-based models are commercially available and have been termed reconstituted human epithelia (RHE), by the manufacturer (e.g.…”

Biofilm formation and adhesive/invasive properties of Candida dubliniensis in comparison with Candida albicans

“…Cells were cultivated at 37°C under 5% CO 2 and at saturated humidity up to 80% confluence. Cell cultures were then split 1:5 using standard methods [23,24] and distributed into the 24-well plates for the adhesion assay, as well as plating onto collagen-coated surfaces for the adhesion assay and invasion experiment, as described below.…”

“…The invasion assay was performed on an ex vivo model designed as a three dimensional model employing reconstituted human epithelium as described previously by Hernandez & Rupp [24]. Firstly, polystyrene inserts (ThinCertTM, Greiner Bio-One) with a bottom of microporous polyethylene terephthalate membrane (pore diameter 0.4 μm) were placed in polystyrene 24-well plates (ThinCertTM-24well-Multiwell-Platte, Greiner Bio-One).…”