Use of the Porcine Intestinal Epithelium (PIE)‐Assay to analyze early stages of colonization by the human fungal pathogen Candida albicans

Abstract

Virulence of C. albicans strains can be tested using a mouse model of haematogenously disseminated Candida cells. Initial steps of host-pathogen contact such as adhesion and colonization are not taken into account due to the injection of Candida cells into the blood stream. Here we describe an assay, based on the ex vivo usage of porcine intestinal epithelium (PIE), that is useful to monitor the early stages of a C. albicans infection. The ability of C. albicans to undergo morphogenetic switching between yeast and hyphal stages is thought to contribute to its virulence. We found that hyphal formation was required to allow cells to colonize the PIE. The non-filamentous mutant strains efg1/cph1 which lacks two of the central transcription factors that are required to promote hyphal growth and wal1 that carries a deletion of the C. albicans homolog of the human Wiskott-Aldrich Syndrome Protein and is deficient in endocytosis showed only weak adherence. Furthermore, the wal1 mutant was found to be reduced in virulence using the mouse tail vein injection assay. We also analyzed the colonization properties of a variety of other mutant strains carrying deletions of either secreted aspartyl proteinase (SAP)-family genes or amino acid permease encoding genes (GAP1, SSY1, and PUT4). Interestingly, the nag5 strain which lacks an N-acetylglucosamine kinase showed enhanced filamentation and invasive growth as well as increased resistance against farnesol.

N-Acetylglucosamine Kinase, HXK1 Is Involved in Morphogenetic Transition and Metabolic Gene Expression in Candida albicans

“…Such hyperfilamentation has also been reported in an hxk1 mutant by a separate group [22]. Here, we have isolated and characterized in detail, an hxk1 mutant that showed hyperfilamentation similar to that of nag cluster mutant.…”

Molecular Characterization of the N-Acetylglucosamine Catabolic Genes in Candida africana, a Natural N-Acetylglucosamine Kinase (HXK1) Mutant

“…Rao et al [ 29 ] reported that the homozygous HXK1 mutant, after 2 days of incubation at 30°C on YEPD plus serum, started to form wrinkled colonies which were completely surrounded by hyphae after 6 days in such conditions. Similar results were also obtained in a separate study [ 37 ] and are, in part, consistent with our data. In fact we observed that C .…”

“…albicans revealed some interesting phenotypic characteristics including the ability of the triple mutant to hyperfilament under stress-induced conditions [ 36 ]. This intrinsic morphological property has also been reported for HXK1 null mutants [ 29 , 37 ] and further confirmed in this study for C . africana .…”

N-Acetylglucosamine (GlcNAc) Induction of Hyphal Morphogenesis and Transcriptional Responses in Candida albicans Are Not Dependent on Its Metabolism

“…3). Deletion of HXK1 was reported previously to cause filamentous growth as part of a study aimed at defining the mechanisms of epithelial cell colonization (27). Analysis of mutant cells grown in dextrose medium revealed that the filamentous growth was most pronounced in cell cultures grown to high density, whereas the cells maintained at low density were grew primarily as buds (Fig.…”