C albicans Biofilms

C. albicans-associated biofilms can be found on catheter tips and on dentures. As discussed earlier, catheters are one of the major risk factors for systemic candidiasis. A number of reviews of C. albicans biofilms have been recently published (Lopez-Ribot, 2005; Mukherjee et al., 2005; Nett & Andes, 2006; Nobile & Mitchell, 2006; Ramage et al., 2006). Biofilms are composed of C. albicans cells embedded in an extracellular matrix material composed of carbohydrate, protein, hexosamine, phosphorus, and uronic acid, with the major component of the C. albi cans matrix being glucose (32%) (Al-Fattani & Douglas, 2006). Understanding of the behaviour of C. albicans growing as a biofilm has been enhanced by the development of an in vivo rat CVC biofilm model (Andes et al., 2004). The development of a biofilm over time has been characterised, with yeast cells being densely embedded in extracellular matrix on the catheter surface. The outermost surface of the biofilm contained both yeast and hyphal cells in a more fibrous extracellular material. Host cells are also found within the matrix (Ramage et al., 2001; Andes et al., 2004). While the presence of serum and saliva conditioning films increased initial adherence, there was little effect on overall biofilm formation (Ramage et al., 2004).

Biofilms were demonstrated to have very different transcript profiles when compared to planktonic grown cells (Garcia-Sanchez et al., 2004). Transcript profiling demonstrated that transcriptional changes in biofilm formation begin within 30 min of contact with the substrate, and involve genes associated with sulphur metabolism and amino acid biosynthesis being upregulated (Garcia-Sanchez et al., 2004; Murillo et al., 2005). Some gene expression changes are restricted to the earliest stages of biofilm formation (Murillo et al., 2005).

ALS genes were found to be differentially expressed in biofilms (Nailis et al., 2006). An ALS3 mutant was found to produce defective biofilms, weakened structurally and much reduced in biomass (Zhao et al., 2006). GFP-promoter fusion with the ALS3 promoter demonstrated that GFP was produced in hyphal cells throughout the biofilm. Overexpression of ALS3 resulted in biofilms with similar mass to wild-type cells, but the cells had a yeast-like morphology (Zhao et al., 2006). The role in biofilm formation did not appear to be purely an adhesion role. Bcr1p was identified as a protein required for biofilm formation, but not hyphal formation, although several of the regulated genes are induced during hyphal formation (Nobile & Mitchell, 2005). Overexpression of ALS3, one of the targets for Brc1p, rescued the biofilm defect, with overexpression of other Bcr1p targets (ALS1, ECE1, and HWP1) only partially rescuing the biofilm phenotype (Nobile et al., 2006).

Other biofilm-deficient mutants have been identified and include insertions in NUP85, MDS3, KEM1, and SUV3 (Richard et al., 2005). All except kem1 were blocked at early stages of biofilm development, with kem1 at an intermediate stage. The mutants were all defective in hypha formation in several different media, leading to the suggestion that hyphae provide an adherent scaffold to stabilise the structure (Richard et al., 2005). Protein mannosylation (Pmt) mutants were also found to be defective for biofilm formation (Peltroche-Llacsahuanga et al., 2006). A Pmt inhibitor was blocked early stages of biofim formations, suggesting that surface anchoring and adherence to the substrate may be affected (Peltroche-Llacsahuanga et al., 2006).

Proteomics have been applied to biofilm formation, demonstrating that alcohol dehydrogenase (Adh1p) is downregulated in C. albicans biofilms (Mukherjee et al., 2006). Subsequently it was shown that Adh1p restricts biofilm formation through an ethanol-dependent mechanism, and that ethanol treatment of a rabbit model of catheter-associated biofilm actually inhibited biofilm formation (Mukherjee et al., 2006).

Further studies (Thomas et al., 2006) investigated changes in proteins between planktonic and biofilm growing C. albicans. In biofilms the following proteins were upregulated: Hsp70, pyruvate decarboxylase, inositol-1-phosphate synthase (Inolp), enolase (Enolp), and inosine 5' monophosphate dehydrogenase (Imh3p) (Thomas et al., 2006). Many of the upregulated proteins are the same as those found to be upregulated in hyphae, and those that are immunogenic in patients.

The quorum-sensing molecules tyrosol and farnesol are produced by C. albi cans, accelerating and blocking yeast-hypha transition, respectively. Both appear to have roles to play in biofilm formation, with tyrosol's action most significant in the earlier stages of biofilm formation. Biofilms secrete more tyrosol compared to planktonic cells relative to dry weight, with addition of farnesol inhibiting biofilm formation (Alem et al., 2006). DNA microarrays have been carried out for biofilms exposed to farnesol, demonstrating that hypha formation-associated genes (TUP1, CRK1, and PDE2), genes associated with drug resistance (FCR1 and PDR16), cell wall maintenance genes (CHT2 and CHT3), and several heat-shock protein (HSP70s and 90) genes were differentially regulated (Cao et al., 2005).

A surprising finding was that C. albicans opaque cells can influence the overall structure of a biofilm (Daniels et al., 2006). Pheromone produced by opaque cells actually selectively upregulates mating-associated genes in white cells, and produces more cohesive white cells (Daniels et al., 2006). The resulting biofilm formed, when there are only occasional opaque cells in a population, tends to be thicker than those produced with white cell only biofilms (Daniels et al., 2006). However, for this to occur would require a host to be infected by both MTL a and a simultaneously, or for strains to become MTL homozygous within the host.

One of the major problems with C. albicans biofilms associated with patients is that they are inherently more resistant to antifungal agents (Ramage et al., 2001; Kuhn et al., 2002; Lewis et al., 2002; Andes et al., 2004; Ramage et al., 2004; Cocuaud et al., 2005; Al-Fattani & Douglas, 2006; Khot et al., 2006; Seidler et al., 2006; Shuford et al., 2006a). Biofilm have been shown to be more resistant to amphotericin B (Ramage et al., 2001; Kuhn et al., 2002; Lewis et al., 2002; Al- Fattani & Douglas, 2006; Khot et al., 2006; Shuford et al., 2006a), azoles (Ramage et al., 2001; Kuhn et al., 2002; Lewis et al., 2002; Al-Fattani & Douglas, 2006; Shuford et al., 2006a), and echinocandins (Cocuaud et al., 2005; Seidler et al., 2006; Shuford et al., 2006a). However, at therapeutic levels echinocandins and lipid-formulation amphotericin B were shown to significantly reduce biofilm metabolism (Kuhn et al., 2002; Lewis et al., 2002; Cocuaud et al., 2005; Seidler et al., 2006; Shuford et al., 2006b). Fluconazole and voriconazole were also shown to have some antifungal effects against biofilms, but never completely eradicated colonisation (Lewis et al., 2002). Antifungal agents do experience some problems in penetrating the biofilm (Samaranayake et al., 2005), but this does not appear to a major mechanism of drug resistance (Al-Fattani & Douglas, 2004). It has been suggested however, that P-glucans found within the matrix may have a role in biofilm drug resistance (Nett et al., 2006).

The biofilm layer of filamentous cells and yeasts were only slightly more antifungal resistant compared to planktonic cells (Khot et al., 2006). However, the substratum layer of yeasts was much more resistant to amphotericin B, which was associated with differential regulation of ergosterol and ß-1,6-glucan synthesis pathways, again implicating ß-glucans (Khot et al., 2006). It has been suggested that within this layer there is a subpopulation of persister cells usually associated with attachment (LaFleur et al., 2006). These cells are switch variants, rather than mutants as detachment of the cells leads to the cells eventually becoming susceptible again (Andes et al., 2004). Within 2 h of attaching to a silicone surface, cells had increased tolerance to fluconazole (Mateus et al., 2004). GFP-promoter fusions demonstrated that enhanced tolerance of attached cells was partially due to increased expression of the drug pump genes CDR1 and MDR1 (Mateus et al., 2004). CDR1 and CDR2 expression was also shown to be upregulated in biofilms (Andes et al., 2004). Two proteins previously associated with drug resistance, Grp2p and orf19.822p, were also found in greater abundance when C. albicans cells formed a biofilm on catheter material (Vediyappan & Chaffin, 2006). These studies suggest that attachment to a substrate induce changes in C. albicans which incidentally also increase their resistance to antifungal agents. This also suggests that genes involved in drug resistance also have roles in normal C. albicans biology, including adaptation to being adhered to a surface.

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