spp. infect medical devices by attaching to the surface and proliferating as a biofilm. Cells in this environment are embedded in a protective extracellular matrix and exhibit profound resistance to antifungal drugs (11
). Because of the lack of effective antifungal therapy against biofilm infections, the recommended treatment for Candida
biofilm infections is removal of the infected device (42
). Defining the pathways and mechanisms of resistance during the biofilm mode of growth is valuable for the design of innovative drug therapies targeted to treat these recalcitrant infections.
The first Candida
biofilm resistance studies tested mechanisms of resistance known to be important in planktonic systems (6
). Although several of these mechanisms were found to be involved in biofilm drug resistance, much of the biofilm resistant phenotype remained an enigma. Subsequent studies have suggested that biofilm drug resistance is multifactorial, with contributions from biofilm-specific processes as well (6
). Models have demonstrated phenotypic variability among the cells in heterogeneous biofilms and have identified subsets of exquisitely resistant cells deep in the biofilm (18
). These studies suggest that cells throughout the biofilm may even employ different mechanisms of resistance.
Previous investigations postulating a contribution of the biofilm matrix to drug resistance measured antifungal diffusion through Candida
). Using variable-flow conditions to alter matrix production, the Douglas group identified a correlation between drug resistance and the extent of biofilm matrix (2
). Further studies with filter disk diffusion assays have also revealed a slowing of antifungal transit through Candida
). Our previous investigations linked a biofilm matrix carbohydrate, glucan, to a biofilm-specific drug resistance mechanism in C. albicans
). By producing matrix β-glucan capable of sequestering an antifungal drug, biofilm cells survive extraordinarily high drug concentrations during biofilm growth.
In the current studies, we use a candidate gene approach to explore a role for the PKC pathway in C. albicans
matrix β-1,3-glucan production and biofilm resistance. This pathway has previously been described to be a positive regulator of β-1,3-glucan synthesis in S. cerevisiae
, but similar studies have not been performed with C. albicans
to our knowledge (17
). Here we show that SMI1
are required for production of the characteristic drug-resistant phenotype of the biofilm lifestyle. Disruption of SMI1
affects the manufacture of C. albicans
β-1,3 matrix and cell wall glucan during biofilm growth. The importance of these genes in S. cerevisiae
β-1,3-glucan cell wall synthesis has been well characterized, but their role in C. albicans
β-1,3-glucan production and biofilm resistance is a novel finding (16
). Surprisingly, the upstream yeast homologs of the PKC pathway were not found to contribute to the biofilm glucan matrix resistance mechanism. Disruption of each of the four kinases in the pathway did not affect biofilm drug resistance or glucan matrix.
Consistent with previous investigations, the current studies support a role for glucan sequestration of antifungals in C. albicans
biofilm drug resistance (34
). Using a radioactive-fluconazole assay, we were able to track the drug accumulation and demonstrate biofilm sequestration in the matrix material. The finding that modulation of SMI1
affects the amount of matrix-sequestered drug indicates a role for the gene product in this biofilm resistance mechanism. This mechanism is specific to the biofilm mode of growth because planktonic drug resistance is not affected. The ability of biofilm matrix to act as a drug sponge, occupying the drug and preventing its activity, has been described for both bacterial and fungal biofilms (24
). In C. albicans
biofilms, this activity is mediated, at least in part, through glucan synthase Fks1p.
These studies show that the action of Smi1p includes β-1,3-glucan matrix synthesis upstream of Fks1p. Transcription of FKS1
is modulated by SMI1
expression, and the β-1,3-glucan changes observed with disruption of SMI1
are similar to those described for FKS1/fks1
Δ mutant biofilm (34
). Furthermore, we show that overexpression of FKS1
in the smi1
Δ mutant restores the biofilm drug-resistant phenotype. In S. cerevisiae
, a link between SMI1/KNR4
upon activation of the cell wall integrity pathway and transcription factor Rlmlp has been described (26
). As predicted from S. cerevisiae
, disruption of RLM1
in C. albicans
produces a biofilm phenotypically similar to the SMI1
mutant biofilms. Together, the findings suggest that the relationship between these gene products is conserved in C. albicans
Phenotypic studies and transcriptional profiling examining the cell wall integrity pathway in the smi1
Δ mutant suggest a partial link to biofilm matrix production. Disruption of SMI1
affected expression of several cell wall damage response genes, suggesting altered cell wall integrity for the mutant strain in the absence of exogenous stressors. In addition, the smi1
Δ mutant was significantly more susceptible to cell wall perturbation by calcofluor white. However, significant differences between the strains were not observed upon treatment of planktonic or biofilm cells with other cell stressors. We hypothesize that the differential susceptibility to calcofluor white is related to the increase in cell wall chitin in the smi1
Δ mutant (data not shown). Disruption of SMI1
did not affect growth at 37°C or planktonic susceptibility to additional antifungals, including amphotericin B, flucytosine, and anidulafungin. An intact cell wall integrity pathway is required for echinocandin resistance in both C. albicans
and S. cerevisiae
). Therefore, similar planktonic susceptibilities to anidulafungin for the C. albicans smi1
Δ mutant and the reference strain do not suggest an altered cell wall integrity pathway.
Taken together, the data suggest that matrix glucan production and biofilm resistance are modulated by Smi1p and networked to the cell wall integrity pathway. However, regulation of this pathway is distinct from that of the PKC pathway. Further defining the genetic regulation of this Candida biofilm pathway may provide insight into how the organism transforms to this lifestyle and resists antifungal treatment.