A quantitative real-time RT-PCR assay for mature C. albicans biofilms

<p>Abstract</p> <p>Background</p> <p>Fungal biofilms are more resistant to anti-fungal drugs than organisms in planktonic form. Traditionally, susceptibility of biofilms to anti-fungal agents has been measured using the 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxyanilide (XTT) assay, which measures the ability of metabolically active cells to convert tetrazolium dyes into colored formazan derivatives. However, this assay has limitations when applied to high <it>C. albicans </it>cell densities because substrate concentration and solubility are limiting factors in the reaction. Because mature biofilms are composed of high cell density populations we sought to develop a quantitative real-time RT-PCR assay (qRT-PCR) that could accurately assess mature biofilm changes in response to a wide variety of anti-fungal agents, including host immune cells.</p> <p>Results</p> <p>The XTT and qRT-PCR assays were in good agreement when biofilm changes were measured in planktonic cultures or in early biofilms which contain lower cell densities. However, the real-time qRT-PCR assay could also accurately quantify small-medium size changes in mature biofilms caused by mechanical biomass reduction, antifungal drugs or immune effector cells, that were not accurately quantifiable with the XTT assay.</p> <p>Conclusions</p> <p>We conclude that the qRT-PCR assay is more accurate than the XTT assay when measuring small-medium size effects of anti-fungal agents against mature biofilms. This assay is also more appropriate when mature biofilm susceptibility to anti-fungal agents is tested on complex biological surfaces, such as organotypic cultures.</p

Characterization of Mucosal Candida albicans Biofilms

C. albicans triggers recurrent infections of the alimentary tract mucosa that result from biofilm growth. Although the ability of C. albicans to form a biofilm on abiotic surfaces has been well documented in recent years, no information exists on biofilms that form directly on mucosal surfaces. The objectives of this study were to characterize the structure and composition of Candida biofilms forming on the oral mucosa. We found that oral Candida biofilms consist of yeast, hyphae, and commensal bacteria, with keratin dispersed in the intercellular spaces. Neutrophils migrate through the oral mucosa and form nests within the biofilm mass. The cell wall polysaccharide β-glucan is exposed during mucosal biofilm growth and is more uniformly present on the surface of biofilm organisms invading the oral mucosa. We conclude that C. albicans forms complex mucosal biofilms consisting of both commensal bacterial flora and host components. These discoveries are important since they can prompt a shift of focus for current research in investigating the role of Candida-bacterial interactions in the pathogenesis of mucosal infections as well as the role of β-glucan mediated signaling in the host response

Role of Bcr1-Activated Genes Hwp1 and Hyr1 in Candida Albicans Oral Mucosal Biofilms and Neutrophil Evasion

Candida albicans triggers recurrent infections of the oropharyngeal mucosa that result from biofilm growth. Prior studies have indicated that the transcription factor Bcr1 regulates biofilm formation in a catheter model, both in vitro and in vivo. We thus hypothesized that Bcr1 plays similar roles in the formation of oral mucosal biofilms and tested this hypothesis in a mouse model of oral infection. We found that a bcr1/bcr1 mutant did not form significant biofilm on the tongues of immunocompromised mice, in contrast to reference and reconstituted strains that formed pseudomembranes covering most of the tongue dorsal surface. Overexpression of HWP1, which specifies an epithelial adhesin that is under the transcriptional control of Bcr1, partly but significantly rescued the bcr1/bcr1 biofilm phenotype in vivo. Since HWP1 overexpression only partly reversed the biofilm phenotype, we investigated whether additional mechanisms, besides adhesin down-regulation, were responsible for the reduced virulence of this mutant. We discovered that the bcr1/bcr1 mutant was more susceptible to damage by human leukocytes when grown on plastic or on the surface of a human oral mucosa tissue analogue. Overexpression of HYR1, but not HWP1, significantly rescued this phenotype. Furthermore a hyr1/hyr1 mutant had significantly attenuated virulence in the mouse oral biofilm model of infection. These discoveries show that Bcr1 is critical for mucosal biofilm infection via regulation of epithelial cell adhesin and neutrophil function

Host Cell Invasion and Virulence Mediated by Candida albicans Ssa1

Candida albicans Ssa1 and Ssa2 are members of the HSP70 family of heat shock proteins that are expressed on the cell surface and function as receptors for antimicrobial peptides such as histatins. We investigated the role of Ssa1 and Ssa2 in mediating pathogenic host cell interactions and virulence. A C. albicans ssa1Δ/Δ mutant had attenuated virulence in murine models of disseminated and oropharyngeal candidiasis, whereas an ssa2Δ/Δ mutant did not. In vitro studies revealed that the ssa1Δ/Δ mutant caused markedly less damage to endothelial cells and oral epithelial cell lines. Also, the ssa1Δ/Δ mutant had defective binding to endothelial cell N-cadherin and epithelial cell E-cadherin, receptors that mediate host cell endocytosis of C. albicans. As a result, this mutant had impaired capacity to induce its own endocytosis by endothelial cells and oral epithelial cells. Latex beads coated with recombinant Ssa1 were avidly endocytosed by both endothelial cells and oral epithelial cells, demonstrating that Ssa1 is sufficient to induce host cell endocytosis. These results indicate that Ssa1 is a novel invasin that binds to host cell cadherins, induces host cell endocytosis, and is critical for C. albicans to cause maximal damage to host cells and induce disseminated and oropharyngeal disease

Cytokeratin presence in <i>C. albicans</i> biofilms formed on the tongue of mice with oropharyngeal candidiasis.

<p>Tissue sections in panels A, B and C were stained with anti-cytokeratin mAb (red) and anti-<i>Candida</i> pAb (green). Host cell nuclei were visualized with TO-PRO-3 (blue). Panel D represents an isotype control (IgG) stain. Arrows indicate areas where fungal cells are surrounded by keratin. Scale bar = 20 µm.</p

Presence of bacteria in mucosal biofilms of mice with oropharyngeal candidiasis.

<p>Panels A and B depict tissue sections stained with an anti-<i>Candida</i> pAb (green) and the nucleic acid stain Syto59 (red). Panel B is a 3.5× zoom image of the marked area in Panel A. Notice the close association of <i>C. albicans</i> and bacterial cells (arrows). Panel C depicts a tissue section stained with an anti-<i>Candida</i> antibody (green), processed for fluorescence in situ hybridization (FISH) with the all bacteria-specific oligonucleotide probe EUB388 (red) and counterstained with the nucleic acid stain Hoechst 33258 (blue). Notice the presence of bacteria (pink) throughout the mucosal biofilms (arrows). Scale bar = 20 µm.</p

β-glucan and extracellular material staining during <i>C. albicans</i> SC5314 in vitro biofilm growth on glass.

<p>Panels depict 3D reconstructions of confocal stacks of images of 24h (A), 48h (B) and 72h (C) biofilms of <i>C. albicans</i> grown on cover slips and stained for β-glucan with BFDiv mAb (red) and ConA-Alexa 488 (green). Notice that regardless of biofilm thickness β-glucan is localized in the growing end of the biofilm (arrows).</p

β-glucan and extracellular material staining in <i>C. albicans</i> biofilms forming on glass.

<p>Panel A depicts a 2h biofilm and panel B depicts a 48h biofilm. Biofilms of the GFP-expressing <i>C. albicans</i> strain (green) were stained for β-glucan with a BFDiv monoclonal antibody (red) and the extracellular material was stained with ConA-Alexa 350 (blue). In 2h biofilms there is partial co-localization of the BFDiv mAb and ConA (pink). BFDiv stains parts of the fungal cell, but not the germinating buds, and ConA stains the entire fungal cell surface (3A). In 48h biofilms deposits of cell-dissociated ECM stained with ConA but not with BFDiv (3B, arrows). Scale bar = 20 µm.</p