In this study, we explored the sphingolipid (SL) landscape in Candida auris, which plays pivotal roles in fungal biology and drug susceptibility. The composition of SLs exhibited substantial variations at both the SL class and molecular species levels among clade isolates. Utilizing principal component analysis, we successfully differentiated the five clades based on their SL class composition. While phytoceramide (PCer) was uniformly the most abundant SL class in all the isolates, other classes showed significant variations. These variations were not limited to SL class level only as the proportion of different molecular species containing variable number of carbons in fatty acid chains also differed between the isolates. Also a comparative analysis revealed abundance of ceramides and glucosylceramides in fluconazole susceptible isolates. Furthermore, by comparing drug-resistant and susceptible isolates within clade IV, we uncovered significant intraclade differences in key SL classes such as high PCer and low long chain base (LCB) content in resistant strains, underscoring the impact of SL heterogeneity on drug resistance development in C. auris. These findings shed light on the multifaceted interplay between genomic diversity, SLs, and drug resistance in this emerging fungal pathogen.

Certain species of Mucorales have been identified as causative agents of mucormycosis, a rare yet often lethal fungal infection. Notably, these fungi exhibit intrinsic resistance to common azole drugs, which target lipids. Given the pivotal role of lipids in drug resistance and their contribution to innate resistance to azoles, this study provides a comprehensive overview of key lipid classes, including sphingolipids (SLs), glycerophospholipids (GPLs), and sterols, in Rhizopus delemar 99-880, a well-characterized reference strain among Mucorales. Using shotgun lipidomics as well as liquid- and gas-chromatography-based mass spectrometric analyses, we identified the lipid intermediates and elucidated the biosynthetic pathways of SLs, PGLs, and sterols. The acidic SLs were not found, probably because the acidic branch of the SL biosynthesis pathway terminates at α-hydroxy phytoceramides, as evident by their high abundance. Intermediates in the neutral SL pathway incorporated higher levels of 16:0 fatty acid compared to other pathogenic fungi. A strikingly high phosphatidylethanolamine (PE)/phosphatdylcholine (PC) ratio was observed among GPLs. Ergosterol remains the major sterol, similar to other fungi, and our analysis confirms the existence of alternate ergosterol biosynthesis pathways. The total lipidomic profile of R. delemar 99-880 offers insights into its lipid metabolism and potential implications for studying pathogenesis and drug resistance mechanisms.

Certain species of Mucorales have been identified as causative agents of mucormycosis, a rare yet often lethal fungal infection. Notably, these fungi exhibit intrinsic resistance to common azole drugs, which target lipids. Given the pivotal role of lipids in drug resistance and their contribution to the innate resistance to azoles, this study provides a comprehensive overview of key lipid classes, including sphingolipids (SLs), glycerophospholipids(GPLs), and sterols, in Rhizopus delemar 99-880, a well-characterized reference strain among Mucorales. Through high-throughput analysis involving the identification and quantification of intermediates, we have elucidated the biosynthetic pathways of SLs, PGLs, and sterols. Notably, the acidic SLs were not found probably because the acidic branch of the SL biosynthesis pathway terminates at α-hydroxy phytoceramides as evident by their high abundance. Intermediates in the neutral SL pathway incorporated higher levels of 16:0 fatty acid compared to other pathogenic fungi. A strikingly high phosphatidylethanolamine (PE)/phosphatidylcholine (PC) ratio was observed among GPLs. Ergosterol remains the major sterol, similar to other fungi, and our analysis confirms the existence of alternate ergosterol biosynthesis pathways. The total lipidomic profile of R. delemar 99-880 offers insights into its lipid metabolism and potential implications for studying pathogenesis and drug resistance mechanisms.

The intrinsic resistance of Candidozyma auris (C. auris) to antifungal drugs poses a major therapeutic challenge, with conventional resistance mechanisms providing only partial explanations. Sphingolipids (SLs), known for their interclade heterogeneity, play a crucial role in antifungal resistance. This study examined the SL landscape in two drug-susceptible clade II isolates, C-line and P-line, from distinct geographical origins, which were experimentally evolved to develop stable fluconazole (FLC) resistance. The progenitors displayed distinct SL profiles, P1 had higher PhytoCer and αOHPhytoCer, indicating a more active acidic SL biosynthesis branch, whereas C1 exhibited elevated αOHGlcCer, αOHCer, and LCBs, reflecting a greater role of the neutral biosynthesis branch. The principal component analysis also confirmed distinct segregation of the two progenitors. Upon evolution, P1.1 and C1.1 adaptors showed significant SL alterations. P1.1 exhibited PhytoCer enrichment, while C1.1 showed reduced αOHGlcCer alongside increased PhytoCer, dhCer, and αOHPhytoCer levels. Notably, αOHGlcCer remained unchanged in P1.1, whereas LCBs and αOHPhytoCer decreased compared to P1. Despite these lineage-specific differences between the progenitors, both evolved replicates exhibited increased PhytoCer as a common denominator like what is also observed in clinical FLC-resistant isolates. These findings highlight intraclade SL variability and suggest that specific SLs contribute to FLC resistance in C. auris.

Flippases, composed of catalytic and regulatory subunits, preserve cellular integrity by maintaining the asymmetric distribution of phospholipids across the plasma membrane. Here, we provide the first functional characterization of Lem3, the regulatory subunit of the flippase complex in Candida albicans , and reveal its critical role in membrane homeostasis and pathogenesis. We demonstrate that Lem3 localizes to both the endoplasmic reticulum and plasma membrane, consistent with its role in phospholipid translocation. Phenotypic analysis of a lem3 Δ/Δ mutant showed decreased susceptibility to the alkylphosphocholine analog, miltefosine. Concurrently, the mutant was defective in the inward translocation (flip) of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) across the plasma membrane, accompanied by altered levels of ergosterol and sphingolipids, indicating the requirement of Lem3 for maintaining membrane lipid composition. Notably, LEM3 deletion suppressed the enhanced phospholipid internalization seen in an rta3 Δ/Δ mutant, suggesting that Rta3 negatively regulates Lem3 activity. The lem3 Δ/Δ strain exhibited increased susceptibility to azole antifungals, likely due to increased membrane permeability and impaired function of the major drug efflux pump Cdr1. Strikingly, the absence of LEM3 reversed drug resistance in an azole-resistant clinical isolate, reinforcing its therapeutic relevance. Finally, Lem3 was important for biofilm formation and in vivo virulence, establishing it as a key determinant of C. albicans pathogenicity. Together, these findings position Lem3 and the flippase machinery as promising targets for antifungal therapy. IMPORTANCE: Plasma membrane lipid composition, maintained by lipid translocators, is a key determinant of virulence in pathogenic fungi. Here, we demonstrate that Lem3, the regulatory subunit of the flippase complex, by regulating the asymmetric distribution of phospholipids, impacts the expression of virulence traits and modulates drug resistance in Candida albicans . These findings highlight how diverse cellular processes are interconnected through flippase-mediated lipid homeostasis. With the alarming rise in fungal infections and antifungal resistance, targeting lipid translocators such as Lem3 offers a promising avenue for the development of novel therapeutic targets.