Genomic and Phenotypic Variation in Morphogenetic Networks of Two Candida albicans Isolates Subtends Their Different Pathogenic Potential

Abstract

The transition from commensalism to pathogenicity of Candida albicans reflects both the host inability to mount specific immune responses and the microorganism's dimorphic switch efficiency. In this study, we used whole genome sequencing and microarray analysis to investigate the genomic determinants of the phenotypic changes observed in two C. albicans clinical isolates (YL1 and YQ2). In vitro experiments employing epithelial, microglial, and peripheral blood mononuclear cells were thus used to evaluate C. albicans isolates interaction with first line host defenses, measuring adhesion, susceptibility to phagocytosis, and induction of secretory responses. Moreover, a murine model of peritoneal infection was used to compare the in vivo pathogenic potential of the two isolates. Genome sequence and gene expression analysis of C. albicans YL1 and YQ2 showed significant changes in cellular pathways involved in environmental stress response, adhesion, filamentous growth, invasiveness, and dimorphic transition. This was in accordance with the observed marked phenotypic differences in biofilm production, dimorphic switch efficiency, cell adhesion, invasion, and survival to phagocyte-mediated host defenses. The mutations in key regulators of the hyphal growth pathway in the more virulent strain corresponded to an overall greater number of budding yeast cells released. Compared to YQ2, YL1 consistently showed enhanced pathogenic potential, since in vitro, it was less susceptible to ingestion by phagocytic cells and more efficient in invading epithelial cells, while in vivo YL1 was more effective than YQ2 in recruiting inflammatory cells, eliciting IL-1β response and eluding phagocytic cells. Overall, these results indicate an unexpected isolate-specific variation in pathways important for host invasion and colonization, showing how the genetic background of C. albicans may greatly affect its behavior both in vitro and in vivo. Based on this approach, we propose that the co-occurrence of changes in sequence and expression in genes and pathways driving dimorphic transition and pathogenicity reflects a selective balance between traits favoring dissemination of the pathogen and traits involved in host defense evasion. This study highlights the importance of investigating strain-level, rather than species level, differences, when determining fungal-host interactions and defining commensal or pathogen behavior.

Keywords: Candida albicans; biofilm; fungal isolates; genomic; host adaptation; pathogenic traits; phagocytes.

Figure 1

Gene ontologies enrichment analysis of the most polymorphic genes. Gene Ontology Term Finder tool on Candida Genome Database were used for functional enrichment analysis, using default parameters. Valid results were considered with a p-value <0.05.

Figure 2

Kinetic analysis of biofilm morphology. To examine cell morphology, Candida albicans cells were seeded for 3 an 24 h at 37°C under 5% CO2. After fixation with 2.5% glutaraldehyde, cells were exposed for 1 h at room temperature with 1% osmium tetroxide. The samples were critical point dried before being gold coated and examined with a scanning electron microscope.

Figure 3

YL1 and YQ2 isolates showed different sugar cell wall composition. Sugars’ composition has been assessed on YL1 and YQ2 cell culture in RPMI in presence or absence of 10% fetal bovine serum and 5% CO₂. Mean of three independent replicates is shown. *p < 0.05, YL1 glucose content vs YQ2 glucose content in the same cultural condition. **p < 0.01, YL1 mannose content vs YQ2 mannose content in the same culture conditions.

Figure 4

YL1 and YQ2 isolates showed different transcriptional regulation during biofilm formation. Heatmap visualization supported by hierarchical clustering (obtained using distance metric selection by Pearson correlation and average linkage clustering methods) of the gene differentially expressed in at least two condition. The complete list of differentially expressed genes is present as Table S8 in Supplementary Material.

Figure 5

YL1 and YQ2 isolates differed in their ability to adhere to epithelial cells. Caco-2 cells were seeded in Lab-Tek II chamber slides, grown at 37°C to confluence (24 h) and then infected with a suspension of Candida in complete RPMI for 1.5 or 3 h at 37°C under 5% CO₂. Uvitex 2B fluorescence of bound C. albicans was visualized by epifluorescence microscopy. At least 200 epithelial cells from each sample were examined and the percentage of Caco-2 cells with adherent yeasts was defined as the ratio of the number of Caco-2 cells with one or more C. albicans to the total number of Caco-2 cells examined. Data are represented as mean ± SD (N = 3). *p < 0.05.

Figure 6

Peripheral blood mononucleated cells (PBMCs)’ cytokine response to YL1 and YQ2 infection. PBMC were exposed to Candida YL1 and YQ2 isolates (E:T = 1:10) for 5 days. Then, supernatants were harvested and assessed for innate (A) and adaptive (B) cytokine levels, as detailed above. Data are represented as mean ± SD. *p < 0.05, YL1 vs YQ2; **p < 0.01, YL1 vs YQ2. US indicates unstimulated cells.

Figure 7

YL1 and YQ2 isolates differently resisted to microglial cell-mediated antifungal activity. (A) Susceptibility of the YL1 and YQ2 isolates to phagocytosis was assessed by epifluorescence microscopy using BV2 microglial cells. Oregon green 488 prelabelled yeast cells were exposed to BV2 cells (E:T = 1:5) for 1.5 and 3 h. At each end point, Uvitex 2B was added for 15 min; the cultures were then washed, fixed, and analyzed by epifluorescence microscopy. The percent of phagocytic cells was calculated as detailed in Section “Materials and Methods.” Data are shown as mean ± SD (N = 3). (B) Susceptibility of YL1 and YQ2 isolates to antifungal activity by microglia. Yeast cells were exposed to BV2 cells at E:T = 10:1. After 3, 6, and 20 h, the percent of antifungal activity was determined as detailed in Section “Materials and methods.” Data are shown as mean ± SD (N = 43). *p < 0.05, YL1 vs YQ2. (C) Acidification of phagolysosomes containing YL1 and YQ2 cells. Oregon green 488 prelabelled yeast cells were exposed to BV2 cells (E:T = 1:5); then, LysoTraker dye was added. After counterstaining with Uvitex 2B, samples were fixed and then visualized by epifluorescence microscopy. Representative images of acidification of phagolysosomes by means of epifluorescence are shown. (D) Percent of acidic phagolysosomes, containing YL1 or YQ2 cells. The values were calculated by evaluating the number of red-stained vacuoles among 200 yeast-containing vacuoles. Data are shown as mean ± SD (N = 3). *p < 0.05, YL1 vs YQ2.