The Sur7 cytoplasmic C terminus regulates morphogenesis and stress responses in Candida albicans.

MCC/eisosome subdomains of the plasma membrane promote proper cell wall morphogenesis that is critical for the fungal pathogen Candida albicans to grow invasively and resist stressful environments in the host. Sur7 localizes to MCC/eisosomes and is needed for their function, so in this work, the role of this tetraspan membrane protein was studied by mutagenesis. Deletion mutant analysis showed that the N-terminal region containing the four transmembrane domains mediates Sur7 localization to MCC/eisosomes. Mutation of 32 conserved residues in the N-terminal region indicated that extracellular loop 1 is important, although these mutants generally displayed weak phenotypes. Surprisingly, two Cys residues in a conserved motif in extracellular loop 1 were not important. However, deletion of the entire 15 amino acid motif revealed that it was needed for proper membrane trafficking of Sur7. Deletion and substitution mutagenesis showed that the C terminus is important for resisting cell wall stress. This is significant as it indicates Sur7 carries out an important role in the cytoplasm. Altogether, these results indicate that the N-terminal region localizes Sur7 to MCC/eisosomes and that the C-terminal domain promotes responses in the cytoplasm needed for cell wall morphogenesis and stress resistance.

Analyses of transcriptomics and metabolomics reveal pathway of vacuolar Sur7 contributed to biocontrol potential of entomopathogenic Beauveria bassiana.

Beauveria bassiana is a critical entomopathogenic fungus for pest biocontrol, whose efficiency depends on fungal development and stress resistance. Unlike its revealed location in plasma membrane patches in other organisms, B. bassiana Sur7 specifically localized in vacuoles. This vacuolar Sur7 was previously demonstrated to affect stress tolerance, hyphal development and virulence. There, however, remain more mechanistic details to be explored. In this study, transcriptomics and metabolomics were applied to investigate the mechanism of vacuolar Sur7. Analyses of transcriptomics and metabolomics displayed many differentially expressed genes and abundant metabolites in response to Sur7 loss, respectively. Together with genes associated with vacuolar biofunction (including transportation and hydrolysis), the altered metabolites contributed to cell wall construction and stress resistance. Particularly, an N-acetylglucosamine-associated Brg1/Nrg1 pathway was enriched and partially affected by Sur7. Absence of Sur7 changed the expression level of Brg1/Nrg1 pathway-related transcript factors, which interfered with downstream phenotype of sporulation. In addition, Sur7 was involved in the accumulation of sphingoid bases, which may affect sphingolipid-related signaling pathway. Although experimental evidence is further required, our studies provide a preliminary framework for future exploring the regulatory mechanism of Sur7, and give a new version of metabolic agency connecting Sur7 and downstream signaling pathway.

Subcellular localization of Sur7 and its pleiotropic effect on cell wall integrity, multiple stress responses, and virulence of Beauveria bassiana.

Sur7 is one of multiple proteins constituting MCC (membrane compartment of Can1 acting as an arginine/H+ symporter), a crucial membrane domain that can form punctuate eisosome spots on the plasma membrane and execute diverse functions in model yeast but remains poorly understood in filamentous fungi. Here, a Sur7 homolog bearing a typical SUR7 domain and four transmembrane domains was shown to localize in the conidial vesicles and enter vacuoles and appear sporadically on the periphery membrane during hyphal growth in the insect-pathogenic fungus Beauveria bassiana, implicating an involvement of Sur7 in cellular events linked to both plasma membrane and vacuoles. Deletion of sur7 resulted in reduced conidiation capacity and impaired conidial quality, which was featured by slower germination, attenuated virulence, and reduced carbohydrate epitopes (ß-N-acetylglucosamine and sialic acids). Also, the hyphal cell walls of the deletion mutant were severely impaired due to ~ 70% reductions in chitin and neutral carbohydrate contents and a moderate increase in alkali-soluble carbohydrate content. Consequently, the deletion mutant became more sensitive to three cell wall perturbing chemicals (Congo red, calcofluor white, and SDS) and an antifungal drug (caspofungin) and surprisingly showed a hypersensitivity to oxidative stress of H2O2 and an increased sensitivity to osmotic stress of NaCl or sorbitol. Its hypersensitivity to H2O2 was associated with transcriptional repression of critical catalase genes required for H2O2 decomposition. These findings unveil that Sur7 takes part in both MCC/eisosome and vacuolar events and hence acts as a sustainer of conidiation capacity, cell wall integrity, multiple stress tolerance, and virulence in B. bassiana. Key points • Sur7 is a component of the crucial membrane domain MCC in Beauveria bassiana. • Sur7 localizes mainly in the vacuoles and sporadically on the periphery membrane. • Sur7 is required for cell wall integrity and has a pleiotropic effect on B. bassiana.

SUR7 deletion in Candida albicans impacts extracellular vesicle features and delivery of virulence factors.

Extracellular vesicles (EVs) from human fungal pathogens have been implicated in fungal virulence, yet little is known about their role in the host-pathogen interaction. Progress has been hampered by the lack of a specific marker for fungal EVs that can be used to monitor EV isolation and tracking in biological systems. Here we report the effect of a SUR7 gene knockout on the production, properties, and role of EVs in the virulence of Candida albicans. Sur7 is a component of the membrane compartment of Can1 (MCC) complex and is enriched in the EVs from C. albicans and other fungal species. MCC is a plasma membrane complex which together with the eisosome, a cytoplasmic protein complex, is a key regulator in plasma membrane organisation and plasma membrane associated processes. The SUR7 knockout strain produces smaller EVs than the wild-type (WT) with different protein and carbohydrate cargos. Furthermore, proteins with known roles in Candida pathogenesis were present in WT EVs and absent or diminished in the sur7Δ EVs. We demonstrate that the reduced virulence of the sur7Δ cells can be partially restored with EVs from a WT strain. These findings demonstrate the importance of Sur7-like proteins in the biogenesis of EVs in fungi and enhance our understanding of the role of fungal EVs in human pathogenesis.

Transcriptomic analysis of Sur7-mediated response of Beauveria bassiana to different nutritional conditions.

Integrity of the cell wall is requisite for fungal growth and function. Sur7 governs cell wall composition, and affects conidial sporulation and germination in Beauveria bassiana, a filamentous entomopathogenic fungus. The role of Sur7 in fungal growth on various nutrients remains unclear. We have previously reported that Sur7 deletion results in the attenuation of B. bassiana growth on supplemented Sabouraud dextrose agar (SDAY) and minimal Czapek-Dox agar (CDA) compared to wild type (WT). Here, we used transcriptomic analysis to compare WT and Sur7 mutant (ΔSur7) responses to CDA and SDAY. Growth on CDA, compared with that on SDAY, affected the expression of more genes in the WT than in the mutant. Differentially expressed genes were enriched for transportation process terms in the ΔSur7 mutant and metabolic process terms in the WT. Different processes were repressed in the ΔSur7 (metabolic process) and WT (ribosome synthesis) cells. Despite the shared enrichment of nitrogen metabolism genes, differentially expressed genes were enriched in distinct saccharide-energy metabolism terms in each strain. We conclude that Sur7 ensures the growth of B. bassiana in a minimal medium by influencing the expression of genes involved in the consumption of sucrose via specific energy metabolism pathways.

Plasma Membrane MCC/Eisosome Domains Promote Stress Resistance in Fungi.

There is growing appreciation that the plasma membrane orchestrates a diverse array of functions by segregating different activities into specialized domains that vary in size, stability, and composition. Studies with the budding yeast Saccharomyces cerevisiae have identified a novel type of plasma membrane domain known as the MCC (membrane compartment of Can1)/eisosomes that correspond to stable furrows in the plasma membrane. MCC/eisosomes maintain proteins at the cell surface, such as nutrient transporters like the Can1 arginine symporter, by protecting them from endocytosis and degradation. Recent studies from several fungal species are now revealing new functional roles for MCC/eisosomes that enable cells to respond to a wide range of stressors, including changes in membrane tension, nutrition, cell wall integrity, oxidation, and copper toxicity. The different MCC/eisosome functions are often intertwined through the roles of these domains in lipid homeostasis, which is important for proper plasma membrane architecture and cell signaling. Therefore, this review will emphasize the emerging models that explain how MCC/eisosomes act as hubs to coordinate cellular responses to stress. The importance of MCC/eisosomes is underscored by their roles in virulence for fungal pathogens of plants, animals, and humans, which also highlights the potential of these domains to act as novel therapeutic targets.

The Sur7 protein resides in punctate membrane subdomains and mediates spatial regulation of cell wall synthesis in Candida albicans.

The eukaryotic plasma membrane is organized into distinct domains that contribute to its function. One new type of plasma membrane domain was identified by studies on the Sur7 protein, which was discovered in the yeast S. cerevisiae to localize into stable punctate patches known as MCC or eisosomes. Sur7 shares similarities with Claudin proteins that form tight junction domains in animal cells, suggesting common roles for these tetraspanning membrane proteins. Recent analysis of C. albicans revealed broad new roles for Sur7; a sur7Delta mutant mislocalized septins and actin and was defective in morphogenesis. Strikingly, cell wall synthesis was very abnormal, including long projections of chitin-rich cell wall into the cytoplasm. Some phenotypes of the sur7Delta mutant are similar to the effects of inhibiting cell wall beta-glucan synthesis. This suggests that the abnormal cell wall structures are related to the increased chitin synthesis commonly seen under cell wall stress conditions, which could be mediated in part by the altered septin localization. Altogether, these results identify new roles for Sur7 and MCC/eisosomes in plasma membrane organization and coordination of the different aspects of cell wall synthesis.