Correlation between biofilm formation and the hypoxic response in Candida parapsilosis

Abstract

The ability of Candida parapsilosis to form biofilms on indwelling medical devices is correlated with virulence. To identify genes that are important for biofilm formation, we used arrays representing approximately 4,000 open reading frames (ORFs) to compare the transcriptional profile of biofilm cells growing in a microfermentor under continuous flow conditions with that of cells in planktonic culture. The expression of genes involved in fatty acid and ergosterol metabolism and in glycolysis, is upregulated in biofilms. The transcriptional profile of C. parapsilosis biofilm cells resembles that of Candida albicans cells grown under hypoxic conditions. We therefore subsequently used whole-genome arrays (representing 5,900 ORFs) to determine the hypoxic response of C. parapsilosis and showed that the levels of expression of genes involved in the ergosterol and glycolytic pathways, together with several cell wall genes, are increased. Our results indicate that there is substantial overlap between the hypoxic responses of C. parapsilosis and C. albicans and that this may be important for biofilm development. Knocking out an ortholog of the cell wall gene RBT1, whose expression is induced both in biofilms and under conditions of hypoxia in C. parapsilosis, reduces biofilm development.

FIG. 1.

Transcriptional profile of C. parapsilosis biofilm cells. (A) The expression of several genes was confirmed by qRT-PCR by comparing expression levels in planktonic cells in exponential-phase growth (Exp) and in stationary-phase growth (Stat) with 50-h biofilm samples. At least three biological replicates were used. (B) The effect of pH was determined by comparing the level of gene expression in unbuffered planktonic cultures (pH 3.8) with that in buffered cultures (pH 5.4), which more closely resembles the pH of biofilm cells. The scale is broken to allow the inclusion of the high level of expression of PHR1.

FIG. 2.

Transcriptional response of C. parapsilosis to hypoxic conditions. (A) C. parapsilosis (CLIB214, 74/046, and 81/041) and C. albicans (SC5314) isolates were grown overnight at 30°C in liquid YPD medium. Approximately 50 cells were plated onto YPD agar plates and incubated for 5 days under either normoxic (21% O₂ in a standard laboratory incubator) (top) or hypoxic (1% O in a hypoxia chamber) (bottom) conditions at 30°C. All plates were photographed on the same day and at the same magnification. The same effect was observed during growth at 37°C. (B) The expression levels of several potential genes under hypoxic conditions were confirmed using qRT-PCR. Ergosterol genes (ERG1 and ERG11) and glycolytic genes (PFK2 and PGK1) were induced four- to sixfold and are shown in one panel. The data for expression of a cell wall gene (RBT1) and a gene unique to C. parapsilosis (CPAG_05061), which are induced 30- to 45-fold, are shown separately. Three biological replicates were used, and each sample was analyzed in duplicate. (C) Distribution of genes with differential expression in biofilms and under hypoxic conditions. Sixty genes are differentially regulated under both conditions; the full list is available in Table S6 in the supplemental material.

FIG. 3.

Gene expression changes in biofilms and hypoxia. C. albicans orthologs of C. parapsilosis genes with altered expression in biofilms or under hypoxic conditions were identified as described in Materials and Methods. GO terms with significant enrichment were determined using GeneSet enrichment analysis. The figure shows selected GO processes and the associated genes that are enriched in the biofilm and hypoxia arrays. C. albicans gene names are used, and genes highlighted in gray are common to both experiments. Several genes are associated with more than one process. CoA, coenzyme A. (The structure of the figure is based on a similar diagram in reference .)

FIG. 4.

Construction of an rbt1 deletion of C. parapsilosis. (A) The RBT1 gene was disrupted as described previously (15). Briefly, upstream and downstream sequences from the RBT1 gene were amplified using primer pairs Rbt1kpn/Rbt1apa and Rbt1sII/Rbt1sI and inserted at either side of the SAT1 flipper cassette in plasmid pCD8, which contains a nourseothricin resistance gene and an FLP (flippase recombination enzyme), surrounded by two FRT (flippase recognition target) sites. The entire fragment was used to replace one RBT1 allele in C. parapsilosis CLIB214 by homologous recombination. The SAT1 cassette was removed by inducing the expression of the recombinase, used again to delete the second RBT1 allele, and the cassette was finally recycled. A reconstituted strain was generated by cloning a wild-type copy of RBT1 upstream from the SAT1 flipper cassette. The entire fragment was used to replace one rbt1 allele by homologous recombination, and the cassette was recycled by inducing recombination between the FRT sites. (B) All steps in the construction were confirmed using PCR (top and middle) and by Southern hybridization (bottom). The order of the strains for each reaction shown at the top is as follows: lane 1, CLIB214; lane 2, CDR1 (integration at the first allele); lane 3, CDR14 (first recycle); lane 4, CDR210 (integration at the second allele); lane five, CDR212 (second recycle). In the middle, lanes 1 and 2 are CDR311 (reconstituted strain), and lanes 3 and 4 are CDRbt8 (reconstituted strain following recycling of the cassette). M, marker. (C) Fluorescence microscopy of biofilms on Thermanox slides. Biofilms were grown for 50 h and stained with a fluorescent conjugate of concanavalin A. z-stack images were acquired at intervals of 1 μm, and a three-dimensional reconstruction was generated using Imais software. (CLIB214, wild type; CDR212, rbt1Δ/rbt1Δ.) (D) Biofilm quantification in 96-well plates. Cells were first inoculated at and A₆₀₀ of 0.05 for 1 h, wells were washed two times with PBS, and fresh RPMI 1640 with Glutamax medium (Invitrogen) buffered with 50 mM HEPES was added. The plates were then incubated for 24 h at 37°C. Cells were washed with PBS, and biofilms were quantified using the FDA assay. (CLIB214, wild type; CDR14, RBT1/rbt1Δ; CDR212, rbt1Δ/rbt1Δ; CDRbt8, RBT1/rbt1Δ::RBT1.)

FIG. 5.

Expression of the ALS gene family in C. parapsilosis. (A) RNA samples were extracted from planktonic cultures and from 24-h and 50-h biofilm samples. The levels of expression of five ALS family members and RBT1 were measured using qRT-PCR. At least three biological replicates were used. (B) RNA samples grown under normoxic conditions (21% O₂) and under hypoxic conditions (1% O₂) were extracted. The expression levels of five ALS family members were measured using qRT-PCR. At least three biological replicates were used.