Transcriptome analysis of Paracoccidioides brasiliensis cells undergoing mycelium-to-yeast transition

Abstract

Paracoccidioides brasiliensis is a thermodimorphic fungus associated with paracoccidioidomycosis (PCM), a systemic mycosis prevalent in South America. In humans, infection starts by inhalation of fungal propagules, which reach the pulmonary epithelium and transform into the yeast parasitic form. Thus, the mycelium-to-yeast transition is of particular interest because conversion to yeast is essential for infection. We have used a P. brasiliensis biochip carrying sequences of 4,692 genes from this fungus to monitor gene expression at several time points of the mycelium-to-yeast morphological shift (from 5 to 120 h). The results revealed a total of 2,583 genes that displayed statistically significant modulation in at least one experimental time point. Among the identified gene homologues, some encoded enzymes involved in amino acid catabolism, signal transduction, protein synthesis, cell wall metabolism, genome structure, oxidative stress response, growth control, and development. The expression pattern of 20 genes was independently verified by real-time reverse transcription-PCR, revealing a high degree of correlation between the data obtained with the two methodologies. One gene, encoding 4-hydroxyl-phenyl pyruvate dioxygenase (4-HPPD), was highly overexpressed during the mycelium-to-yeast differentiation, and the use of NTBC [2-(2-nitro-4-trifluoromethylbenzoyl)-cyclohexane-1,3-dione], a specific inhibitor of 4-HPPD activity, as well as that of NTBC derivatives, was able to inhibit growth and differentiation of the pathogenic yeast phase of the fungus in vitro. These data set the stage for further studies involving NTBC and its derivatives as new chemotherapeutic agents against PCM and confirm the potential of array-based approaches to identify new targets for the development of alternative treatments against pathogenic microorganisms.

FIG. 1.

Temperature-induced morphological switch in P. brasiliensis. Mycelia growing in the early exponential phase were induced to undergo morphological transformation by changing the temperature of incubation from 26 to 37°C. At the different time points, the morphological units were arbitrarily classified as hyphae, differentiating hyphae (characterized by the development of chlamydospore-like cells produced by intercalary or lateral swellings in the fertile hyphae), transforming yeast (characterized by the production of multiple buds by the chlamydospore), or yeast. Relative quantities of each morphotype were obtained after individually scoring a total of at least 300 morphological units for each time point.

FIG. 2.

Hierarchical clustering showing the pattern of expression of P. brasiliensis genes encoding ribosomal proteins during the mycelium-to-yeast transition. The color code displays the log₂(Cy5/Cy3) ratio for each time point and has Cy3 as the reference value (hyphae at time point 0).

FIG. 3.

Hierarchical clustering showing the pattern of expression of P. brasiliensis genes encoding proteins involved in chitin synthesis and degradation (A), heat shock proteins (B), and histones (C) during the mycelium-to-yeast transition. The color code displays the log₂(Cy5/Cy3) ratio for each time point and has Cy3 as the reference value (hyphae at time point 0).

FIG. 4.

Clusters of gene expression generated by the K-means algorithm. The 2,583 genes that showed modulation in expression during the mycelium-to-yeast transition were evaluated by a figure-of-merit algorithm. The obtained results supported their subdivision into 56 clusters, which was achieved with the aid of a K-means algorithm. Groups of genes with similar modulations of gene expression during the mycelium-to-yeast transition are located in each cluster. The figure shows, in the y axis, the variation in the log₂(Cy5/Cy3) ratios (from −7 to 7), along with the different time points of the mycelium-to-yeast transition (x axis), taking as a reference their respective expression levels at time zero (hyphae). Clusters 13 and 55, containing genes that displayed the most intense and consistent up-regulation profiles, are indicated by bold lines and shown in more detail in Fig. 5.

FIG. 5.

Hierarchical clustering showing the pattern of expression of P. brasiliensis genes contained in clusters 13 (A) and 55 (B), which showed the highest levels of up-regulation during the mycelium-to-yeast transition. Cluster numbering is considered from the upper left corner of Fig. 4. The color code displays the log₂(Cy5/Cy3) ratio for each time point and has Cy3 as the reference value (hyphae at time point 0).

FIG. 6.

NTBC inhibits P. brasiliensis yeast growth and mycelium-to-yeast transition. P. brasiliensis mycelium was grown in the absence (a) or presence (b to f) of 5 to 100 μg/ml NTBC for 10 days at 37°C.

FIG. 7.

NTBC derivatives can inhibit P. brasiliensis yeast growth. P. brasiliensis viable yeast cells were inoculated in solid PGY medium. Blank 6-mm-diameter paper disks were impregnated with 5.0, 10.0, 25.0 μg/ml (from right to left on the first rows on the plates), 50.0, 100.0, 200.0 μg/ml (second rows, from right to left on the plates), or 0 μg/ml (third row) of NTBC (A) or NTBC derivatives (B to D and left plate in E) and placed on top of the previously inoculated agar plates after drying. Plates were incubated at 37°C, and the diameters of the inhibition zones were evaluated after 7 days. Each disk diffusion assay was performed twice. As shown on the right plate in E, compound 8 was also tested at lower concentrations of 0.25, 0.5, 0.75 μg/ml (first row, from right to left on the plate), 5.0, 7.5, 10.0 μg/ml (second row, from right to left on the plate), or 0 μg/ml (third row).