Computational protocol: Passage through the mammalian gut triggers a phenotypic switch that promotes Candida albicans commensalism

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Protocol publication

[…] Saturated overnight cultures of wild-type MTLa/α white cells (SN425), MTLa white cells (SN966), MTLa opaque cells (SN967), WOR1OE MTLa/α white cells (SN1044), and WOR1OE MTLa/α GUT cells (SN1045) were inoculated into SC medium (with 100 μg/ml uridine) to OD600=0.1 and incubated with shaking at room temperature for 6–8 hours before harvesting at OD600=0.8–1.0. Two to four biological replicates were performed per strain. RNA isolation, cDNA labeling, and hybridization to custom Agilent C. albicans ORF microarrays were performed as described.Equal amounts of cDNA from each sample were pooled to prepare a mixed reference. Cy5-labeled cDNAs from each strain were directly hybridized against the pooled reference (Cy3). Arrays were scanned using a GenePix 4000A Axon scanner, and spots were filtered using GenePix Pro software. Data were normalized using Goulphar (LOWESS normalization) and subjected to Bayesian Analysis of Gene Expression Levels (BAGEL). Significant changes in expression were defined as ones with BAGEL p-values <0.05, after implementing a correction for comparison of multiple variables (i.e. multiplication by the number of ORFs on the microarray [6168]). [...] The t-test (two-tailed, comparison of unpaired samples) was used for evaluation of competitive fitness, with significance defined as p<0.05. Bayesian Analysis of Gene Expression Levels (BAGEL) was used for analysis of C. albicans transcriptomes. The Bonferroni Correction for Multiple Hypothesis Testing (multiplication by 6168 or the total number of ORFs) was applied, and significance was defined as p<0.05. The GO Term Finder tool on the Candida Genome Database website (http://www.candidagenome.org/) was used to identify functional groups with altered gene expression. This algorithm utilizes a hypergeometric distribution with Bonferroni correction, and significance was defined as p<0.05. […]

Pipeline specifications

Software tools GenePix Pro, Goulphar, BAGEL, GO::TermFinder
Databases CGD
Application Gene expression microarray analysis
Organisms Candida albicans, Mus musculus, Homo sapiens
Diseases Opportunistic Infections