In most natural environments, microorganisms follow a sessile way of life in which they remain surface attached in close contact with each other. As a result, such populations exhibit a number of features that are very different from those of planktonic communities. Many forms of candidiasis develop on organ or implant surfaces. Consequently, C. albicans
populations can follow a surface-attached, biofilm-like way of life that confers, among other characteristics, an increased resistance to antifungal drugs (7
In this study we used transcript profiling to perform a global analysis of the biofilm-specific features of C. albicans. We produced biofilms in three laboratory models that involved very different environmental conditions. Transcript profile comparisons of these biofilms with planktonic populations led us to our initial observation that biofilm transcriptomes are highly correlated (Fig. ) and that nutrient flow, aerobiosis, and other factors that strongly influence the transcriptome of planktonic populations do not have a noticeable effect on biofilm-growing communities. Hence, by comparison to planktonic populations, transcriptome invariance is proposed as a specific feature of biofilm populations. This observation is unlikely to reflect a slower growth of all biofilm populations than of planktonic populations, since both actively growing biofilms and stationary planktonic populations were included in the study.
The second observation is the identification of genes encoding biofilm-associated traits. As expected from the low correlation between biofilm and planktonic populations, the number of differentially expressed genes is considerable (Table ). This is also true when both populations arise from a cph1/cph1 efg1/efg1 strain unable to develop hyphae (Table ), indicating that the differential expression is not due only to differences in the hyphal content of planktonic and biofilm populations. We identified three significant sets of genes: the 325-gene set contains biofilm-related genes whose expression is independent of extrinsic conditions, the 317-gene set contains biofilm-related genes that are expressed independently of the intrinsic ability of the cell to form mycelia, and the 86-gene set identifies genes whose expression varies between hypha-containing and hypha-noncontaining biofilms. The classification of these genes into functional categories and the study of their representation in each set revealed that expression changes specifically affect some cellular functions (Fig. ; Table ).
Genes involved in protein synthesis are the most highly represented in the three sets (Fig. ; Table ), and they are exclusively overexpressed. One reason for this activation of the protein synthesis machinery could be that biofilms grow faster than planktonic populations. However, genes related to cell-cycle and DNA processing are underrepresented. Thus, the increase of protein synthesis is independent of cell division and unlikely to be caused by differences in growth rate. On the other hand, this excess protein might be used for the production of extracellular matrix, which is not present in planktonic cells. This is in keeping with the inclusion in the 325-gene set of several components of the secretion machinery, such as SEC14 or SYS3, and the dramatic increase in the expression of the extracellular agglutinin gene ALS1. Enhanced production and secretion of certain proteins with agglutination properties may facilitate the cohesion of cells within the biofilm. In addition to ALS1, several new genes that encode proteins with secretion signals and adhesin-like domains, such as IPF20161, IFP5185, or IPF20008, were found to be significantly overexpressed.
Among metabolic activities, amino acid biosynthetic pathways are strongly represented. We have demonstrated that GCN4
is required for efficient biofilm-like growth. In C. albicans
, this transcription factor induces two different processes in response to amino acid starvation: the activation of amino acid biosynthesis and the triggering of morphogenesis (30
). The latter is unlikely to be related to the biofilm-forming defect exhibited by the gcn4
strain, since the proportion of yeast and hyphal cells was roughly the same in wild-type and gcn4
biofilms (data not shown). Therefore, the effect of GCN4
on biofilm formation resembles a GCN-like response, although it was not observed under conditions that are known to impose amino acid starvation on planktonic populations. The situation might be different in biofilms that seem to have an increased need for protein synthesis, and amino acids might therefore become limiting faster. Under these conditions, the regulatory role of GCN4
could be decisive for biofilm progression.
A more specific role for the sulfur amino acid biosynthesis/salvage pathway is revealed by the 317- and 86-gene subsets, which identify overexpression of 9 of the 11 key genes in this route (Fig. ). The route leads to the production of S
-adenosylmethionine (SAM), with a separate branch for the production of cysteine (29
). Since SAM is the precursor of polyamines and since ODC1
are also included in the 325-gene set, all of these changes could be associated with cell wall rearrangements that allow closer interaction of cells within the biofilm (13
). Alternatively, biofilms may have a special requirement for methionine- and cysteine-rich proteins, an idea supported by the overexpression of genes in the cysteine biosynthesis branch. Finally, activation of the genes for SAM biosynthesis might be related to the production of a quorum-sensing molecule associated with biofilm formation. Indeed, the bacterial autoinducer AI-2 is produced from SAM, which has been proposed to serve as a “universal signal” for interspecies communication (34
The activation of glycolytic flux and catabolic repression is underscored by the 317-gene set. In the wild-type-versus-cph1
experiment, biofilms were produced under a continuous flow of nutrients, in contrast to planktonic cultures (Table ). Thus, activation of glycolysis could be merely a reflection of unlimited sugar availability in the microfermentor. However, activation of MIG1
in biofilms is revealed by the 325-gene set as well and is therefore independent of whether biofilms were produced under continuous flow. Overexpression of MIG1
in S. cerevisiae
is associated with flocculation (28
). Whether overexpression of MIG1
in C. albicans
biofilms could promote flocculation and hence increase the aggregation of the cells in biofilms remains to be investigated.
Biofilm resistance to antifungals has been associated with increased expression of the MDR
). Expression of these genes was studied previously in a single microtiter plate model, whereas we report a multiple-model comparison. Here, changes in the expression of these genes are not considered significant. However, CDR1
is included in the 86-gene set arising from the biofilm produced in the microfermenter model. Thus, expression of this gene could be significant under certain conditions of biofilm production, and it is also predicted to be influenced by the triggering of hyphal development concomitant with biofilm formation. Instead, we observed differential expression of ERG25
(in the 325-gene set) and ERG6 (in the 317-gene set). ERG16
mRNA levels correlate with azole resistance in clinical isolates C. albicans
). An enrichment or redistribution of sterols in biofilm membranes could explain their resistance to azole-derived antifungal agents. This is consistent with the recent data of Mukherjee et al. (18
) showing that expression of MDR
genes in biofilms is phase specific and contributes to azole resistance only during the early phase of biofilm development whereas changes in sterol composition are involved in the resistance of the mature phase. Hence, our laboratory models for biofilm production and transcript profiling analysis converge with previous approaches to the identification of differentially expressed genes related to pathogenesis and underline the importance of the study of biofilm-like growth for a full understanding of virulence in C. albicans