Phenotypic characterisation of Candida albicans mutants with deletions of arachidonic acid responsive genes

[摘要] English: Candida albicans is a permanent resident of the healthy microbiome of humans. It co-inhabits the host micro-environment with trillions of species which make up the complete microbiota. Host parameters such as nutrient availability, CO2, and temperature as well as pH fluctuations are some of the key contributory elements to C. albicans phenotypic attributes and virulence. In this study, our analysis was based on arachidonic acid (AA), a polyunsaturated fatty acid, as an inducer of a genome wide transcriptional response. Arachidonic acid is a precursor of prostaglandins (PGs) such as PGE2 which can act on prostanoid receptors and potentially alter important biological functions. Since AA is available in the host, it is of great importance to understand the response of pathogenic yeasts to AA and how this interaction contributes to clinical infections.We analyzed genes up-regulated during AA exposure of C. albicans biofilms. Three mutants, two disrupted for membrane-bound proteins (ABC pheromone exporter transport, Hst6p, and amino acid sensor, Ssy1p) and ORF19.4612 (uncharacterized) were analyzed using phenotypic microarrays (PMs). Additional roles of Hst6p were uncovered in this study, one of which is linked to its up-regulation in the presence of AA. The PM data suggests that Hst6p is required for stress protection and conventional phenotypic screens confirmed this. ΔΔhst6 was found to be sensitive to osmotic, oxidative, cell wall and heavy metal stress. Because AA can induce sensitivity to stress, our phenotypic data suggest that HST6 was up-regulated during AA exposure, to respond to stress. In addition to HST6, other ABC transport genes, also important in stress, were up-regulated. Other groups have recently discovered the role of these proteins in stress in higher organisms such as humans, suggesting that the ABC-stress response system is widely conserved. Another function of Hst6p was suppression of hyphal formation in conditions replete with nitrogen sources. ΔΔhst6 was filamentous under these conditions. In addition, ΔΔhst6 formed highly wrinkled colonies with filamentous borders, whose cells were filamentous, under nitrosative stress. Therefore, our data supports the fact that Hst6p is expressed in all the cell types in C. albicans and has a more generic role as opposed to pheromone export alone.Analysis of phenotypes also revealed additional functions of the amino acid sensor, Ssy1p. According to literature, Ssy1p activates cleavage of Stp1p and Stp2p in response to lower pH and poor nutrient availability. In turn, these proteins activate amino acid permeases, Mep1p and Mep2p, required for pH neutralization and yeast-hyphal transition respectively. We found that under stressful conditions, Ssy1p results in differential stress response. For instance, while the wild type strain cells were inflated, cells of ΔΔssy1 grew normally on high ammonium concentrations and in the presence of nitrosative stress. In addition, ΔΔssy1 was resistant to osmotic and cell wall stress. Furthermore, ΔΔssy1 was sensitive to heat stress at 37 °C and 42 °C. Since AA conversion to PGE2 was conducted at 37 °C, and this fatty acid results in predisposition to stress which may affect membrane fluidity, we argue that SSY1 was up-regulated in response to heat stress.Although the microarray data failed to reveal genes directly involved in PGE2 production, the analysis of the white-gray-opaque-like (WGOL) switching, suggest that up-regulation of ORF19.4612 was necessary for rough pink (RP) cell formation and conversion of AA into PGE2. At 37 °C in RPMI-1640 medium, RP cells of the wild type strain produce significantly more PGE2 from AA. Δorf19.4612 is defective in RP cell formation after 3 days on conditions similar to those of RPMI-1640 medium. Since ORF19.4612 was expressed after 48 h at 37 °C, is therefore not only implicated in WGOL, but also in PGE2 production.

[发布机构] University of the Free State