Distinct roles of the 7-transmembrane receptor protein Rta3 in regulating the asymmetric distribution of phosphatidylcholine across the plasma membrane and biofilm formation in Candida albicans.

Fungal pathogens such as Candida albicans exhibit several survival mechanisms to evade attack by antifungals and colonise host tissues. Rta3, a member of the Rta1-like family of lipid-translocating exporters has a 7-transmembrane domain topology, similar to the G-protein-coupled receptors and is unique to the fungal kingdom. Our findings point towards a role for the plasma membrane localised Rta3 in providing tolerance to miltefosine, an analogue of alkylphosphocholine, by maintaining mitochondrial energetics. Concurrent with miltefosine susceptibility, the rta3Δ/Δ strain displays increased inward translocation (flip) of fluorophore-labelled phosphatidylcholine (PC) across the plasma membrane attributed to enhanced PC-specific flippase activity. We also assign a novel role to Rta3 in the Bcr1-regulated pathway for in vivo biofilm development. Transcriptome analysis reveals that Rta3 regulates expression of Bcr1 target genes involved in cell surface properties, adhesion, and hyphal growth. We show that rta3Δ/Δ mutant is biofilm-defective in a rat venous catheter model of infection and that BCR1 overexpression rescues this defect, indicating that Bcr1 functions downstream of Rta3 to mediate biofilm formation in C. albicans. The identification of this novel Rta3-dependent regulatory network that governs biofilm formation and PC asymmetry across the plasma membrane will provide important insights into C. albicans pathogenesis.

The activity of RTA2, a downstream effector of the calcineurin pathway, is required during tunicamycin-induced ER stress response in Candida albicans.

In this study, we demonstrate a novel function of a downstream effector molecule of the calcineurin pathway, RTA2 (Resistance To Aminocholesterol), in ER stress response. The deletion of RTA2 increases susceptibility to the ER stressor tunicamycin and morpholine-like drug, 7-aminocholesterol. Additionally, the expression of RTA2 is also transcriptionally induced by ergosterol biosynthesis inhibitors and cell-wall-damaging agents. As tunicamycin induces the unfolded protein response pathway (UPR) via the transcription factor, HAC1, we monitored the expression of a subset of HAC1-dependent UPR target genes in rta2Δ/Δ cells. Upon tunicamycin exposure, rta2Δ/Δ cells displayed a significantly reduced expression of UPR genes, in spite of only a moderate decrease in the HAC1 spliced mRNA levels and no change in Hac1 protein levels. Furthermore, hac1Δ/Δrta2Δ/Δ cells display an exacerbated sensitivity to tunicamycin compared to the single mutants. We propose that functional RTA2 is requisite for the regulation of Hac1p-dependent UPR target genes to maximal levels, thereby assisting survival during ER stress. Collectively, this study proposes, for the first time, existence of an interplay between the Hac1p- and calcineurin- controlled networks via a downstream effector molecule of the latter, RTA2, to facilitate survival during ER stress in Candida albicans.

Mitochondria influence CDR1 efflux pump activity, Hog1-mediated oxidative stress pathway, iron homeostasis, and ergosterol levels in Candida albicans.

Mitochondrial dysfunction in Candida albicans is known to be associated with drug susceptibility, cell wall integrity, phospholipid homeostasis, and virulence. In this study, we deleted CaFZO1, a key component required during biogenesis of functional mitochondria. Cells with FZO1 deleted displayed fragmented mitochondria, mitochondrial genome loss, and reduced mitochondrial membrane potential and were rendered sensitive to azoles and peroxide. In order to understand the cellular response to dysfunctional mitochondria, genome-wide expression profiling of fzo1Δ/Δ cells was performed. Our results show that the increased susceptibility to azoles was likely due to reduced efflux activity of CDR efflux pumps, caused by the missorting of Cdr1p into the vacuole. In addition, fzo1Δ/Δ cells showed upregulation of genes involved in iron assimilation, in iron-sufficient conditions, characteristic of iron-starved cells. One of the consequent effects was downregulation of genes of the ergosterol biosynthesis pathway with a commensurate decrease in cellular ergosterol levels. We therefore connect deregulated iron metabolism to ergosterol biosynthesis pathway in response to dysfunctional mitochondria. Impaired activation of the Hog1 pathway in the mutant was the basis for increased susceptibility to peroxide and increase in reactive oxygen species, indicating the importance of functional mitochondria in controlling Hog1-mediated oxidative stress response. Mitochondrial phospholipid levels were also altered as indicated by an increase in phosphatidylserine and phosphatidylethanolamine and decrease in phosphatidylcholine in fzo1Δ/Δ cells. Collectively, these findings reinforce the connection between functional mitochondria and azole tolerance, oxidant-mediated stress, and iron homeostasis in C. albicans.