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 fzo1 Δ/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, fzo1 Δ/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 fzo1 Δ/Δ cells. Collectively, these findings reinforce the connection between functional mitochondria and azole tolerance, oxidant-mediated stress, and iron homeostasis in C. albicans .

Candida albicans, the dimorphic opportunistic human fungal pathogen, is capable of forming highly drug-resistant biofilms in the human host. Formation of biofilm is a multistep and multiregulatory process involving various adaptive mechanisms. The ability of cells in a biofilm to alter membrane lipid composition is one such adaptation crucial for biofilm development in C. albicans. Lipids modulate mixed species biofilm formation in vivo and inherent antifungal resistance associated with these organized communities. Cells in C. albicans biofilms display phase-dependent changes in phospholipid classes and in levels of lipid raft formation. Systematic studies with genetically modified strains in which the membrane phospholipid composition can be manipulated are limited in C. albicans. In this review, we summarize the knowledge accumulated on the impact that alterations in phospholipids may have on the biofilm forming ability of C. albicans in the human host. This review may provide the requisite impetus to analyze lipids from a therapeutic standpoint in managing C. albicans biofilms.

The unfolded protein response (UPR), crucial for the maintenance of ER homeostasis, is tied to the regulation of multiple cellular processes in pathogenic fungi. Here we show that C. albicans relies on an ER‐resident protein, inositol‐requiring enzyme 1 (Ire1) for sensing ER stress and activating the UPR. Compromised Ire1 function impacts cellular processes that are dependent on functional secretory homeostasis, as inferred from transcriptional profiling. Concordantly, an Ire1 mutant strain exhibits pleiotropic roles in ER stress response, antifungal tolerance, cell wall regulation and virulence‐related traits. Hac1 is the downstream target of C. albicans Ire1 as it initiates the unconventional splicing of the 19 bp intron from HAC1 mRNA during tunicamycin‐induced ER stress. Ire1 also activates the UPR in response to perturbations in cell wall integrity and cell membrane homeostasis in a manner that does not necessitate the splicing of HAC1 mRNA. Furthermore, the Ire1 mutant strain was severely defective in hyphal morphogenesis and biofilm formation as well as in establishing a successful infection in vivo. Together, these findings demonstrate that C. albicans Ire1 functions to regulate traits that are essential for virulence and suggests its importance in responding to multiple stresses, thus integrating various stress signals to maintain ER homeostasis.

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.

Adaptation to ER stress is linked to the pathogenicity of C. albicans. The fungus responds to ER stress primarily by activating the conserved Ire1-Hac1-dependent unfolded protein response (UPR) pathway. Subsequently, when ER homeostasis is re-established, the UPR is attenuated in a timely manner, a facet that is unexplored in C. albicans. Here, we show that C. albicans licenses the HOG (high-osmolarity glycerol) MAPK pathway for abating ER stress as evidenced by activation and translocation of Hog1 to the nucleus during tunicamycin-induced ER stress. We find that, once activated, Hog1 attenuates the activity of Ire1-dependent UPR, thus facilitating adaptation to ER stress. We use the previously established assay, where the disappearance of the UPR-induced spliced HAC1 mRNA correlates with the re-establishment of ER homeostasis, to investigate attenuation of the UPR in C. albicans. hog1Δ/Δ cells retain spliced HAC1 mRNA levels for longer duration reflecting the delay in attenuating Ire1-dependent UPR. Conversely, compromising the expression of Ire1 (ire1 DX mutant strain) results in diminished levels of phosphorylated Hog1, restating the cross-talk between Ire1 and HOG pathways. Phosphorylation signal to Hog1 MAP kinase is relayed through Ssk1 in response to ER stress as inactivation of Ssk1 abrogates Hog1 phosphorylation in C. albicans. Additionally, Hog1 depends on its cytosolic as well as nuclear activity for mediating ER stress-specific responses in the fungus. Our results show that HOG pathway serves as a point of cross-talk with the UPR pathway, thus extending the role of this signaling pathway in promoting adaptation to ER stress in C. albicans. Additionally, this study integrates this MAPK pathway into the little known frame of ER stress adaptation pathways in C. albicans.

Iron homeostasis mechanisms allow the prime commensal-pathogen Candida albicans to cope with the profound shift in iron levels in the mammalian host. The regulators, Sef1 and Sfu1 influence activation and repression of genes required for iron uptake and acquisition by inducing the expression of iron regulon genes in iron-deplete conditions and inactivating them in iron-replete condition. Our study for the first time shows that C. albicans coordinates the activation of the iron regulon with the mitochondrial use of iron for Fe–S cluster biosynthesis, a cellular process that is connected to cellular iron metabolism. We took advantage of a mutant defective in mitochondrial biogenesis (fzo1Δ/Δ) to assess the aforesaid link as this mutant exhibited sustained expression of the Sef1 iron regulon, signifying an iron-starved state in the mutant. Our analysis demonstrates that mitochondrion is pivotal for regulation of Fe–S cluster synthesis such that the disruption of this cellular process in fzo1Δ/Δ cells lead to excessive mitochondrial iron accumulation and reduced activity of the Fe–S cluster-containing enzyme aconitase. Sef1 responds to defective Fe–S cluster synthesis by regulated changes in its subcellular localization; it was retained in the nucleus resulting in the induced expression of the iron regulon. We predict that the mitochondrial Fe–S assembly generates a molecule that is critical for ensuring iron-responsive transcriptional activation of the Sef1 regulon. All told, our data marks Fe–S biogenesis as a mechanism that meshes cellular iron procurement with mitochondrial iron metabolism resulting in regulating the Sef1 regulon in C. albicans.