Project description:Biofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. Large-scale genetics strategies aimed at identifying genes involved in biofilm development are hampered by lack of a complete sexual cycle in this diploid yeast in addition to the tedious generation of homozygous gene-deletion mutants. Gene overexpression is an attractive alternative strategy for large-scale phenotypic analyses and gene-function studies. We combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (~10% of the genome) for genes affecting i) planktonic cell fitness and ii) biofilm development in mixed-population experiments. We found 5 genes whose overexpression affects planktonic strain fitness, including RAD53, RAD51, PIN4, orf19.2781, all encoding (or predicted to encode) regulators of DNA-damage response or cell-cycle progression and SFL2, involved in filamentous growth. We identified 16 and 4 genes (out of 531) whose overexpression respectively increases and decreases strain occupancy within the multi-strain biofilm. Strikingly, strains with increased abundance in the multi-strain biofilm were significantly enriched for genes encoding cell wall proteins (10 genes), including the glycosylphosphatidylinositol (GPI)-anchored proteins Pga15, Pga19, Pga22, Pga59, Pga32 and Pga41. Data validation experiments using either individually- or competitively-grown overexpression and/or the respective knock-out strains revealed that the identified genes differently contribute to biofilm formation during specific stages of biofilm development, including increased or decreased substrate adherence (IHD1, PGA32, PGA37, PGA15, PGA22, PGA59 and PGA19) or biofilm biomass growth and cohesion (PGA15, PGA59 and PGA22). In line with the hypothesis that cell wall genes contribute to biofilm development, we show that strains overexpressing PGA15 or PGA22 display altered cell wall structures. Our study reveals that cell wall proteins are important actors during C. albicans biofilm formation and illustrates the powerful use of signature tagging in conjunction with gene overexpression for the identification of genes involved in processes pertaining to C. albicans virulence. A total of 8 samples are included in this study. For fitness profiling of planktonic cells, 2 biological replicates were analyzed (samples Pool_Fitness_planktonic_rep1 and Pool_Fitness_planktonic_rep2). For quantification of strain abundance during biofilm formation, 6 biological replicates were analyzed (Pool_Biofilm_rep1, Pool_Biofilm_rep2, Pool_Biofilm_rep3, Pool_Biofilm_rep4, Pool_Biofilm_rep5, Pool_Biofilm_rep6). Genomic DNA was purified and used as template to PCR-amplify barcodes, which were then used as probes for microarray hybridization. This experiment was done twice independently. For quantification of strain abundance during multi-strain biofilm formation, strain pools were grown in minimal GHAUM medium with or without doxycycline, each inoculum was then diluted to an OD600 of 1 in fresh minimal GHAUM medium with or without doxycycline and left at room temperature for 30 min, to allow further overexpression. Plastic slides (ThermanoxM-bM-^DM-"; Nunc) were immersed in the inoculum for 30 min at room temperature to allow adhesion of cells to the plastic substrate. The plastic slides were then transferred to the glass vessel of a 40-mL incubation chamber. This vessel has two glass tubes inserted to drive the entry of medium and air, while used medium is evacuated through a third tube. The flow of medium is controlled by a recirculation pump (IsmatecM-BM-.) set at 0.6 mL.min-1 and pushed by pressured air supplied at 105 Pa, conditions minimizing planktonic phase growth and promoting biofilm formation. The chambers with the plastic substrate were incubated at 37C and biofilms were grown for 40h followed by genomic DNA extraction, barcode amplification and differential labeling (dox-treated samples with Cy5, untreated samples with Cy3) and hybridization to barcode microarrays.

Project description:Biofilm formation is an important virulence trait of the pathogenic yeast Candida albicans. We have combined gene overexpression, strain barcoding and microarray profiling to screen a library of 531 C. albicans conditional overexpression strains (~10% of the genome) for genes affecting biofilm development in mixed-population experiments. The overexpression of 16 genes increased strain occupancy within a multi-strain biofilm, whereas overexpression of 4 genes decreased it. The set of 16 genes was significantly enriched for those encoding predicted glycosylphosphatidylinositol (GPI)-modified proteins, namely Ihd1/Pga36, Phr2, Pga15, Pga19, Pga22, Pga32, Pga37, Pga42 and Pga59; eight of which have been classified as pathogen-specific. Validation experiments using either individually- or competitively-grown overexpression strains revealed that the contribution of these genes to biofilm formation was variable and stage-specific. Deeper functional analysis of PGA59 and PGA22 at a single-cell resolution using atomic force microscopy showed that overexpression of either gene increased C. albicans ability to adhere to an abiotic substrate. However, unlike PGA59, PGA22 overexpression led to cell cluster formation that resulted in increased sensitivity to shear forces and decreased ability to form a single-strain biofilm. Within the multi-strain environment provided by the PGA22-non overexpressing cells, PGA22-overexpressing cells were protected from shear forces and fitter for biofilm development. Ultrastructural analysis, genome-wide transcript profiling and phenotypic analyses in a heterologous context suggested that PGA22 affects cell adherence through alteration of cell wall structure and/or function. Taken together, our findings reveal that several novel predicted GPI-modified proteins contribute to the cooperative behaviour between biofilm cells and are important participants during C. albicans biofilm formation. Moreover, they illustrate the power of using signature tagging in conjunction with gene overexpression for the identification of novel genes involved in processes pertaining to C. albicans virulence.

Project description:Biofilms have been implicated in delayed wound healing, although the mechanisms by which biofilms impair wound healing are poorly understood. Many species of bacteria produce exotoxins and exoenzymes that may inhibit healing. In addition, oxygen consumption by biofilms, as well as responding leukocytes, may impede wound healing. In this study, we used oxygen microsensors to measure oxygen transects through in vitro-cultured biofilms, biofilms formed in vivo within scabs from a diabetic (db/db) mouse model, and ex vivo human chronic wound specimens. The results show that oxygen levels within mouse scabs had steep gradients that reached minima ranging from 17-72 mmHg on live mice and 6.4-1.1 mmHg on euthanized mice. The oxygen gradients in the mouse scabs were similar to those observed for clinical isolates cultured in vitro and for human ex vivo specimens. No oxygen gradients were observed for heat-killed mouse scabs, suggesting that active metabolism by the viable bacteria and host cells contributed to the reduced oxygen partial pressure of the scabs. To characterize the metabolic activities of the bacteria in the mouse scabs, we performed transcriptomics analyses of Pseudomonas aeruginosa biofilms associated with the db/db mice wounds using Affymetrix microarrays. The results demonstrated that the bacteria expressed genes for metabolic activities associated with cell growth. Interestingly, the transcriptome results indicated that the bacteria within the wounds also experienced oxygen-limitation stress. Among the bacterial genes that were expressed in vivo were genes associated with the Anr-mediated hypoxia-stress response. Other bacterial stress response genes highly expressed in vivo were genes associated with stationary-phase growth, osmotic stress, and RpoH-mediated heat shock stress. Overall, the results support the hypothesis that bacterial biofilms in chronic wounds promote chronicity by contributing to the maintenance of localized low oxygen tensions. Transcriptional profiling of two independent biological replicates of Pseudomonas aeruginosa biofilms, as grown to 72 hours and used as inocula applied to the murine wounds, was performed. A principle components analysis (PCA) was used to provide an overview of the transcriptome data from the 28-day mouse wound scab, comparing the data to the biofilm inoculum, and to published reports of P. aeruginosa biofilm and planktonic samples. The analysis shows that the transcriptome of the mouse wound scab was distinct from the biofilm inoculum that was applied to the wound, demonstrating a shift in biofilm gene expression following 28 days of infection. We sought to characterize P. aeruginosa activity within biofilms in the mouse wound model by isolating and identifying mRNA from the biofilms used as inocula and from the wound scabs 28 days post infection.

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