Identification of genes preferentially expressed in the pathogenic yeast phase of Paracoccidioides brasiliensis, using suppression subtraction hybridization and differential macroarray analysis

Abstract

Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in Latin America. Pathogenicity appears to be intimately related to the dimorphic transition from the hyphal to the yeast form, which is induced by a shift from environmental temperature to the temperature of the mammalian host. Little information is available on the P. brasiliensis genes that are necessary during the pathogenic phase. We have therefore undertaken Suppression Subtraction Hybridization (SSH) and macroarray analyses with the aim of identifying genes that are preferentially expressed in the yeast phase. Genes identified by both procedures as being more highly expressed in the yeast phase are involved in basic metabolism, signal transduction, growth and morphogenesis, and sulfur metabolism. In order to test whether the observed changes in gene expression reflect the differences between the growth conditions used to obtain the two morphological forms rather than differences intrinsic to the cell types, we performed real-time RT-PCR experiments using RNAs derived from both yeast cells and mycelia that had been cultured at 37 degrees C and 26 degrees C in either complete medium (YPD or Sabouraud) or minimal medium. Twenty genes, including AGS1 (alpha-1,3-glucan synthase) and TSA1 (thiol-specific antioxidant), were shown to be more highly expressed in the yeast cells than in the hyphae. Although their levels of expression could be different in rich and minimal media, there was a general tendency for these genes to be more highly expressed in the yeast cells.

Interactions of Paracoccidioides brasiliensis with host cells: recent advances

“…However, little is known on which pathway is activated to survive and to escape from the machinery of the host cells. Marques and co-workers [80] have identified four genes, RHO1, SEP1, FLB1, and PCK1, which encode proteins involved in cell signaling and polarity establishment in P. brasiliensis. Interestingly, RHO1 was expressed at 10-to 15-fold higher levels in minimal medium than in complete medium.…”

Transcriptome Analysis of Paracoccidioides brasiliensis Cells Undergoing Mycelium-to-Yeast Transition

“…In a pre-liminary attempt to isolate phase-specific transcripts, Venāncio et al (51) have employed the technique of differential display, which led to the identification of many differentially transcribed cDNA fragments specific for each phase. With the aid of genomics-based approaches, such as electronic subtraction, suppressive subtraction hybridization, real-time reverse transcription-PCR (RT-PCR), and low-density array hybridizations, further information regarding phase-specific gene expression has been obtained from both mycelium and yeast cells (12,13,15,35). Nonetheless, very few genetic determinants identified in this fungus are known to be directly involved in either phase transition and/or pathogenicity, although the experiments mentioned above identified the up-regulation of genes that may play a role in the structural or metabolic changes that take place during morphogenesis or may be necessary for colonization and survival in the host (44).…”

Insights into the pathobiology of Paracoccidioides brasiliensis from transcriptome analysis—advances and perspectives

“…The observations of Paris et al [26] that yeast cells of P. brasiliensis were unable to grow in the presence of inorganic sulphur, were confirmed by the array experiments in which the genes coding sulphur metabolism enzymes such as atp sulphurylase, aps kinase, paps redutase and choline sulphatase showed to be up-regulated in yeast cells of this pathogen, indicating the auxotrophic status for cysteine of the pathogenic phase of this fungus [23,24,27].…”

“…Among the modulated genes from the yeast phase are verprolin (vrp), chitin deacetylase (cda), and a-1, 3-glucan synthase (ags). The a-1,3-glucan synthase also reported in [23] to be positively regulated in the pathogenic form of P. brasiliensis can be easily correlated with the biology of this fungus. The a-1,3-glucan is the main component of the yeast cell wall, and it is closed related to the virulence due to its ability to mask the host recognition mechanism of the pathogen, as also reported in H. capsulatum [25] promoting the fungal escape from the host defenses which contribute to the fungal pathogenesis.…”

“…Marques et al [23] and Andrade et al [24] using the array methodology evaluated the differentially expressed genes in mycelium and yeast cells, which in many cases are important keys to understand the pathobiology of P. brasiliensis. Andrade et al [24] reported 66 transcriptional modulated genes in mycelium or yeast categorized into two classes, the first group, cell organization includes genes involved in maintenance of cell wall, membrane and cytoskeleton, as the mycelium up-regulated gene hex which encodes a hexagonal peroxisome protein controlling cell integrity, and bgl coding 1,3-b-glucosidase involved with cell wall modification during the dimorphism.…”