Real-time PCR expression profiling of genes encoding potential virulence factors in Candida albicans biofilms: identification of model-dependent and -independent gene expression

Abstract

Background: Candida albicans infections are often associated with biofilm formation. Previous work demonstrated that the expression of HWP1 (hyphal wall protein) and of genes belonging to the ALS (agglutinin-like sequence), SAP (secreted aspartyl protease), PLB (phospholipase B) and LIP (lipase) gene families is associated with biofilm growth on mucosal surfaces. We investigated using real-time PCR whether genes encoding potential virulence factors are also highly expressed in biofilms associated with abiotic surfaces. For this, C. albicans biofilms were grown on silicone in microtiter plates (MTP) or in the Centres for Disease Control (CDC) reactor, on polyurethane in an in vivo subcutaneous catheter rat (SCR) model, and on mucosal surfaces in the reconstituted human epithelium (RHE) model.

Results: HWP1 and genes belonging to the ALS, SAP, PLB and LIP gene families were constitutively expressed in C. albicans biofilms. ALS1-5 were upregulated in all model systems, while ALS9 was mostly downregulated. ALS6 and HWP1 were overexpressed in all models except in the RHE and MTP, respectively. The expression levels of SAP1 were more pronounced in both in vitro models, while those of SAP2, SAP4 and SAP6 were higher in the in vivo model. Furthermore, SAP5 was highly upregulated in the in vivo and RHE models. For SAP9 and SAP10 similar gene expression levels were observed in all model systems. PLB genes were not considerably upregulated in biofilms, while LIP1-3, LIP5-7 and LIP9-10 were highly overexpressed in both in vitro models. Furthermore, an elevated lipase activity was detected in supernatans of biofilms grown in the MTP and RHE model.

Conclusions: Our findings show that HWP1 and most of the genes belonging to the ALS, SAP and LIP gene families are upregulated in C. albicans biofilms. Comparison of the fold expression between the various model systems revealed similar expression levels for some genes, while for others model-dependent expression levels were observed. This suggests that data obtained in one biofilm model cannot be extrapolated to other model systems. Therefore, the need to use multiple model systems when studying the expression of genes encoding potential virulence factors in C. albicans biofilms is highlighted.

Figure 1

Number of sessile C. albicans cells in biofilms grown in the various model systems. Average number of culturable sessile cells (mean log₁₀ CFU/cm² ± SD) at selected time points during biofilm growth of C. albicans strain SC5314 in the various biofilm model systems. Biofilm growth was monitored on silicone in two in vitro models (MTP and CDC reactor), on polyurethane in an in vivo SCR model and on oral mucosal epithelium in the RHE model.

Figure 2

LDH activity in the supernatant of sessile C. albicans cells. LDH activity (IU/l at 37°C) at selected time points during biofilm growth of C. albicans strain SC5314 in the RHE model. Epithelial cell damage in the RHE model was correlated with release of the LDH marker.

Figure 3

Percentage of filaments in C. albicans biofilms. Percentage (%) of filaments (with corresponding SD) at selected time points during biofilm growth of C. albicans strain SC5314 in the MTP, the CDC and the RHE model.

Figure 4

Extracellular lipase activity of sessile C. albicans cells. Extracellular lipase activity in the supernatant of sessile and planktonic C. albicans cells was determined using 4-MU palmitate. Relative slopes (%) of biofilms versus start cultures (derived from fluorescence-time curves) are shown for biofilms grown at selected time points in the MTP and RHE model.