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

Integrated Analysis of Clinical and Microbiome Risk Factors Associated with the Development of Oral Candidiasis during Cancer Chemotherapy.

Oral candidiasis is a common side effect of cancer chemotherapy. To better understand predisposing factors, we followed forty-five subjects who received 5-fluorouracil- or doxorubicin-based treatment, during one chemotherapy cycle. Subjects were evaluated at baseline, prior to the first infusion, and at three additional visits within a two-week window. We assessed the demographic, medical and oral health parameters, neutrophil surveillance, and characterized the salivary bacteriome and mycobiome communities through amplicon high throughput sequencing. Twenty percent of all subjects developed oral candidiasis. Using multivariate statistics, we identified smoking, amount of dental plaque, low bacteriome and mycobiome alpha-diversity, and the proportions of specific bacterial and fungal taxa as baseline predictors of oral candidiasis development during the treatment cycle. All subjects who developed oral candidiasis had baseline microbiome communities dominated by Candida and enriched in aciduric bacteria. Longitudinally, oral candidiasis was associated with a decrease in salivary flow prior to lesion development, and occurred simultaneously or before oral mucositis. Candidiasis was also longitudinally associated with a decrease in peripheral neutrophils but increased the neutrophil killing capacity of Candida albicans. Oral candidiasis was not found to be associated with mycobiome structure shifts during the cycle but was the result of an increase in Candida load, with C. albicans and Candida dubliniensis being the most abundant species comprising the salivary mycobiome of the affected subjects. In conclusion, we identified a set of clinical and microbiome baseline factors associated with susceptibility to oral candidiasis, which might be useful tools in identifying at risk individuals, prior to chemotherapy

Chemotherapy-induced oral mucositis is associated with detrimental bacterial dysbiosis.

BACKGROUND: Gastrointestinal mucosal injury (mucositis), commonly affecting the oral cavity, is a clinically significant yet incompletely understood complication of cancer chemotherapy. Although antineoplastic cytotoxicity constitutes the primary injury trigger, the interaction of oral microbial commensals with mucosal tissues could modify the response. It is not clear, however, whether chemotherapy and its associated treatments affect oral microbial communities disrupting the homeostatic balance between resident microorganisms and the adjacent mucosa and if such alterations are associated with mucositis. To gain knowledge on the pathophysiology of oral mucositis, 49 subjects receiving 5-fluorouracil (5-FU) or doxorubicin-based chemotherapy were evaluated longitudinally during one cycle, assessing clinical outcomes, bacterial and fungal oral microbiome changes, and epithelial transcriptome responses. As a control for microbiome stability, 30 non-cancer subjects were longitudinally assessed. Through complementary in vitro assays, we also evaluated the antibacterial potential of 5-FU on oral microorganisms and the interaction of commensals with oral epithelial tissues. RESULTS: Oral mucositis severity was associated with 5-FU, increased salivary flow, and higher oral granulocyte counts. The oral bacteriome was disrupted during chemotherapy and while antibiotic and acid inhibitor intake contributed to these changes, bacteriome disruptions were also correlated with antineoplastics and independently and strongly associated with oral mucositis severity. Mucositis-associated bacteriome shifts included depletion of common health-associated commensals from the genera Streptococcus, Actinomyces, Gemella, Granulicatella, and Veillonella and enrichment of Gram-negative bacteria such as Fusobacterium nucleatum and Prevotella oris. Shifts could not be explained by a direct antibacterial effect of 5-FU, but rather resembled the inflammation-associated dysbiotic shifts seen in other oral conditions. Epithelial transcriptional responses during chemotherapy included upregulation of genes involved in innate immunity and apoptosis. Using a multilayer epithelial construct, we show mucositis-associated dysbiotic shifts may contribute to aggravate mucosal damage since the mucositis-depleted Streptococcus salivarius was tolerated as a commensal, while the mucositis-enriched F. nucleatum displayed pro-inflammatory and pro-apoptotic capacity. CONCLUSIONS: Altogether, our work reveals that chemotherapy-induced oral mucositis is associated with bacterial dysbiosis and demonstrates the potential for dysbiotic shifts to aggravate antineoplastic-induced epithelial injury. These findings suggest that control of oral bacterial dysbiosis could represent a novel preventive approach to ameliorate oral mucositis

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

C. albicans Colonization of Human Mucosal Surfaces

Background: Candida albicans is a low level commensal organism in normal human populations with the continuous potential to expand and cause a spectrum of clinical conditions. Methodology/Principal Findings: Using ex vivo human organ cultures and populations of primary human cells, we have developed several related experimental systems to examine early-stage interactions between C. albicans and mucosal surfaces. Experiments have been conducted both with exogenously added C. albicans and with overtly normal human mucosal surfaces supporting pre-existing infections with natural isolates of Candida. Under different culture conditions, we have demonstrated the formation of C. albicans colonies on human target cells and filament formation, equivalent to tissue invasion. Conclusions/Significance: These organ culture systems provide a valuable new resource to examine the molecular and cellular basis for Candida colonization of human mucosal surfaces

Candida albicans forms biofilms on the vaginal mucosa

Current understanding of resistance and susceptibility to vulvovaginal candidiasis challenges existing paradigms of host defence against fungal infection. While abiotic biofilm formation has a clearly established role during systemic Candida infections, it is not known whether C. albicans forms biofilms on the vaginal mucosa and the possible role of biofilms in disease. In vivo and ex vivo murine vaginitis models were employed to examine biofilm formation by scanning electron and confocal microscopy. C. albicans strains included 3153A (lab strain), DAY185 (parental control strain), and mutants defective in morphogenesis and/or biofilm formation in vitro (efg1/efg1 and bcr1/bcr1). Both 3153A and DAY815 formed biofilms on the vaginal mucosa in vivo and ex vivo as indicated by high fungal burden and microscopic analysis demonstrating typical biofilm architecture and presence of extracellular matrix (ECM) co-localized with the presence of fungi. In contrast, efg1/efg1 and bcr1/bcr1 mutant strains exhibited weak or no biofilm formation/ECM production in both models compared to wild-type strains and complemented mutants despite comparable colonization levels. These data show for the first time that C. albicans forms biofilms in vivo on vaginal epithelium, and that in vivo biotic biofilm formation requires regulators of biofilm formation (BCR1) and morphogenesis (EFG1)

Candida albicans promotes invasion and colonisation of Candida glabrata in a reconstituted human vaginal epithelium

The principal aim of this study was to investigate the in vitro co-infection of a reconstituted human vaginal epithelium (RHVE) by Candida albicans and Candida glabrata. Methods The ability of both species to invade and colonise the RHVE was examined using species-specific peptide nucleic acid (PNA) probe hybridisation, confocal laser scanning microscopy (CLSM) and a novel qRT-PCR protocol for Candida quantification in the tissues. RHVE damage was evaluated by measuring lactate dehydrogenase (LDH) activity. Candida virulence gene expression (HWP1, ALS, EPA, PLB, PLD and SAP) was evaluated by quantitative RT-PCR. Results The results showed that whilst both species induced damage to the RHVE, this was notably less with C. glabrata. Interestingly, there was a significant increase in C. glabrata RHVE colonisation and invasiveness when it was added to the tissue with C. albicans. The extent of RHVE damage caused by the two species appeared to be primarily dependent on the process of invasion. Of the virulence genes assayed, HWP1, PLD1 and ALS3 were deemed to be most associated with pathogenicity in the model. Conclusions For the first time, we have demonstrated that the RHVE model coupled with specific tools of analysis, allows assessment of Candida colonisation and invasion in single and co-infection. Using this model we have demonstrated that C. albicans enhanced C. glabrata colonisation, invasion and tissue damage, which was also evidenced by the expression of virulence genes.We would like to thank Mrs Kath Allsopp for processing and sectioning tissue samples. This work was supported by the research grant SFRH/BD/72742/2010 from "Fundacao para a Ciencia e Tecnologia (FCT)", Portugal. This work was supported by the Programa Operacional, Fatores de competitividade - COMPETE and by national funds through FCT - Fundacao para a Ciencia e a Tecnologia on the scope of the projects FCT PTDC/EBB-EBI/120495/2010, PTDC/SAU-MIC/119069/2010, RECI/EBB-EBI/0179/2012 and PEst-OE/EQB/LA0023/2013. The authors thank the Project "Bio-Health - Biotechnology and Bioengineering approaches to improve health quality", Ref. NORTE-07-0124-FEDER-000027, co-funded by the Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER