Lois M. Douglas

Sterol-Rich Plasma Membrane Domains in Fungi

Concepts regarding the eukaryotic plasma membrane have been evolving in light of growing evidence that it is segregated into distinct lateral domains known as lipid rafts. These sterol- and sphingolipid-rich raft domains are thought to play important roles in dynamic processes, including protein

Septin Function in Yeast Model Systems and Pathogenic Fungi

The septins were first discovered in the budding yeast Saccharomyces cerevisiae and were named for their role in cytokinesis and septum formation (69). Septins are now known to be highly conserved in fungi and animals, although absent in plants and many protozoans (e.g., Plasmodium fasciculatum and Dictyostelium discoideum). The septin proteins are characterized by presence of a distinct type of GTPase domain and by their ability to form filaments. The S. cerevisiae septin proteins form a series of 10-nm filaments that assemble into a ring on the inner surface of the plasma membrane at the bud neck. Septin rings are thought to function as a scaffold to recruit proteins to the bud neck and to act as a boundary domain to restrict diffusion during budding and cytokinesis (30, 39, 66, 68). However, septins are now implicated in a broad range of dynamic membrane events. In S. cerevisiae, septins have been found to also play a role in conjugation and sporulation. Moreover, analyses of septin function in other organisms, including fungi that undergo different patterns of growth and differentiation, are revealing new aspects of septin function. Therefore, we will provide an overview of the current understanding of the relatively well-studied roles of septins during S. cerevisiae budding and then use this as a context to review studies of septin function during other developmental pathways in S. cerevisiae and other fungi. The other fungi will include the model fission yeast Schizosaccharomyces pombe and two opportunistic fungal pathogens: the multimorphic Candida albicans and the filamentous fungus Aspergillus nidulans.

Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

Recognition of yeast by murine macrophages requires mannan but not glucan.

ABSTRACT The first barrier against infection by Candida albicans involves fungal recognition and destruction by phagocytic cells of the innate immune system. It is well established that interactions between different phagocyte receptors and components of the fungal cell wall trigger phagocytosis and subsequent immune responses, but the fungal ligands mediating the initial stage of recognition have not been identified. Here, we describe a novel assay for fungal recognition and uptake by macrophages which monitors this early recognition step independently of other downstream events of phagocytosis. To analyze infection in live macrophages, we validated the neutrality of a codon-optimized red fluorescent protein (yEmRFP) biomarker in C. albicans; growth, hyphal formation, and virulence in infected mice and macrophages were unaffected by yEmRFP production. This permitted a new approach for studying phagocytosis by carrying out competition assays between red and green fluorescent yeast cells to measure the efficiency of yeast uptake by murine macrophages as a function of dimorphism or cell wall defects. These competition experiments demonstrate that, given a choice, macrophages display strong preferences for phagocytosis based on genus, species, and morphology. Candida glabrata and Saccharomyces cerevisiae are taken up by J774 macrophage cells more rapidly than C. albicans, and C. albicans yeast cells are favored over hyphal cells. Significantly, these preferences are mannan dependent. Mutations that affect mannan, but not those that affect glucan or chitin, reduce the uptake of yeast challenged with wild-type competitors by both J774 and primary murine macrophages. These results suggest that mannose side chains or mannosylated proteins are the ligands recognized by murine macrophages prior to fungal uptake.

The Sur7 Protein Regulates Plasma Membrane Organization and Prevents Intracellular Cell Wall Growth in Candida albicans

The Candida albicans plasma membrane plays important roles in cell growth and as a target for antifungal drugs. Analysis of Ca-Sur7 showed that this four transmembrane domain protein localized to stable punctate patches, similar to the plasma membrane subdomains known as eisosomes or MCC that were discovered in S. cerevisiae. The localization of Ca-Sur7 depended on sphingolipid synthesis. In contrast to S. cerevisiae, a C. albicans sur7Delta mutant displayed defects in endocytosis and morphogenesis. Septins and actin were mislocalized, and cell wall synthesis was very abnormal, including long projections of cell wall into the cytoplasm. Several phenotypes of the sur7Delta mutant are similar to the effects of inhibiting beta-glucan synthase, suggesting that the abnormal cell wall synthesis is related to activation of chitin synthase activity seen under stress conditions. These results expand the roles of eisosomes by demonstrating that Sur7 is needed for proper plasma membrane organization and cell wall synthesis. A conserved Cys motif in the first extracellular loop of fungal Sur7 proteins is similar to a characteristic motif of the claudin proteins that form tight junctions in animal cells, suggesting a common role for these tetraspanning membrane proteins in forming specialized plasma membrane domains.

Cell Cycle Dynamics and Quorum Sensing in Candida albicans Chlamydospores Are Distinct from Budding and Hyphal Growth

ABSTRACT The regulation of morphogenesis in the human fungal pathogen Candida albicans is under investigation to better understand how the switch between budding and hyphal growth is linked to virulence. Therefore, in this study we examined the ability of C. albicans to undergo a distinct type of morphogenesis to form large thick-walled chlamydospores whose role in infection is unclear, but they act as a resting form in other species. During chlamydospore morphogenesis, cells switch to filamentous growth and then develop elongated suspensor cells that give rise to chlamydospores. These filamentous cells were distinct from true hyphae in that they were wider and were not inhibited by the quorum-sensing factor farnesol. Instead, farnesol increased chlamydospore production, indicating that quorum sensing can also have a positive role. Nuclear division did not occur across the necks of chlamydospores, as it does in budding. Interestingly, nuclei divided within the suspensor cells, and then one daughter nucleus subsequently migrated into the chlamydospore. Septins were not detected near mitotic nuclei but were localized at chlamydospore necks. At later stages, septins localized throughout the chlamydospore plasma membrane and appeared to form long filamentous structures. Deletion of the CDC10 or CDC11 septins caused greater curvature of cells growing in a filamentous manner and morphological defects in suspensor cells and chlamydospores. These studies identify aspects of chlamydospore morphogenesis that are distinct from bud and hyphal morphogenesis.

Fungal Membrane Organization: The Eisosome Concept

The fungal plasma membrane is organized into specialized domains that vary in size, stability, and composition. Membrane compartment of Can1(MCC)/eisosome domains that were recently discovered in the budding yeast Saccharomyces cerevisiae are interesting because they represent a novel type of membrane domain that is important for plasma membrane organization, sphingolipid homeostasis, and cell wall morphogenesis. The MCC portion was identified as stable punctate patches that correspond to furrows in the plasma membrane that are about 300 nm long and 50 nm deep. These domains contain integral membrane proteins, including the tetraspan proteins Sur7 and Nce102. The eisosome portion includes proteins peripherally associated with the cytoplasmic side of the MCC, including the Bin/amphiphysin/Rvs-domain proteins Pil1 and Lsp1, which assemble into filaments that curve the membrane to form the furrows. By comparing MCC/eisosome domains in diverse fungi, researchers are identifying common features that further our understanding of their unique biogenesis, structure, and function.

BAR Domain Proteins Rvs161 and Rvs167 Contribute to Candida albicans Endocytosis, Morphogenesis, and Virulence

ABSTRACT The Candida albicans plasma membrane plays critical roles in growth and virulence and as a target for antifungal drugs. Three C. albicans genes that encode Bin-Amphiphysin-Rvs homology domain proteins were mutated to define their roles in plasma membrane function. The deletion of RVS161 and RVS167, but not RVS162, caused strong defects. The rvs161Δ mutant was more defective in endocytosis and morphogenesis than rvs167Δ, but both were strongly defective in polarizing actin patches. Other plasma membrane constituents were still properly localized, including a filipin-stained domain at the hyphal tips. An analysis of growth under different in vitro conditions showed that the rvs161Δ and rvs167Δ mutants grew less invasively in agar and also suggested that they have defects in cell wall synthesis and Rim101 pathway signaling. These mutants were also more resistant to the antimicrobial peptide histatin 5 but showed essentially normal responses to the drugs caspofungin and amphotericin. Surprisingly, the rvs161Δ mutant was more sensitive to fluconazole, whereas the rvs167Δ mutant was more resistant, indicating that these mutations cause overlapping but distinct effects on cells. The rvs161Δ and rvs167Δ mutants both showed greatly reduced virulence in mice. However, the mutants were capable of growing to high levels in kidneys. Histological analyses of infected kidneys revealed that these rvsΔ mutants grew in a large fungal mass that was walled off by leukocytes, rather than forming disseminated microabscesses as seen for the wild type. The diminished virulence is likely due to a combination of the morphogenesis defects that reduce invasive growth and altered cell wall construction that exposes proinflammatory components to the host immune system.

Sur7 Promotes Plasma Membrane Organization and Is Needed for Resistance to Stressful Conditions and to the Invasive Growth and Virulence of Candida albicans

ABSTRACT The human fungal pathogen Candida albicans causes lethal systemic infections because of its ability to grow and disseminate in a host. The C. albicans plasma membrane is essential for virulence by acting as a protective barrier and through its key roles in interfacing with the environment, secretion of virulence factors, morphogenesis, and cell wall synthesis. Difficulties in studying hydrophobic membranes have limited the understanding of how plasma membrane organization contributes to its function and to the actions of antifungal drugs. Therefore, the role of the recently discovered plasma membrane subdomains termed the membrane compartment containing Can1 (MCC) was analyzed by assessing the virulence of a sur7Δ mutant. Sur7 is an integral membrane protein component of the MCC that is needed for proper localization of actin, morphogenesis, cell wall synthesis, and responding to cell wall stress. MCC domains are stable 300-nm-sized punctate patches that associate with a complex of cytoplasmic proteins known as an eisosome. Analysis of virulence-related properties of a sur7Δ mutant revealed defects in intraphagosomal growth in macrophages that correlate with increased sensitivity to oxidation and copper. The sur7Δ mutant was also strongly defective in pathogenesis in a mouse model of systemic candidiasis. The mutant cells showed a decreased ability to initiate an infection and greatly diminished invasive growth into kidney tissues. These studies on Sur7 demonstrate that the plasma membrane MCC domains are critical for virulence and represent an important new target for the development of novel therapeutic strategies. IMPORTANCE Candida albicans, the most common human fungal pathogen, causes lethal systemic infections by growing and disseminating in a host. The plasma membrane plays key roles in enabling C. albicans to grow in vivo, and it is also the target of the most commonly used antifungal drugs. However, plasma membrane organization is poorly understood because of the experimental difficulties in studying hydrophobic components. Interestingly, recent studies have identified a novel type of plasma membrane subdomain in fungi known as the membrane compartment containing Can1 (MCC). Cells lacking the MCC-localized protein Sur7 display broad defects in cellular organization and response to stress in vitro. Consistent with this, C. albicans cells lacking the SUR7 gene were more susceptible to attack by macrophages than cells with the gene and showed greatly reduced virulence in a mouse model of systemic infection. Thus, Sur7 and other MCC components represent novel targets for antifungal therapy. Candida albicans, the most common human fungal pathogen, causes lethal systemic infections by growing and disseminating in a host. The plasma membrane plays key roles in enabling C. albicans to grow in vivo, and it is also the target of the most commonly used antifungal drugs. However, plasma membrane organization is poorly understood because of the experimental difficulties in studying hydrophobic components. Interestingly, recent studies have identified a novel type of plasma membrane subdomain in fungi known as the membrane compartment containing Can1 (MCC). Cells lacking the MCC-localized protein Sur7 display broad defects in cellular organization and response to stress in vitro. Consistent with this, C. albicans cells lacking the SUR7 gene were more susceptible to attack by macrophages than cells with the gene and showed greatly reduced virulence in a mouse model of systemic infection. Thus, Sur7 and other MCC components represent novel targets for antifungal therapy.

The Candida albicans Sur7 Protein Is Needed for Proper Synthesis of the Fibrillar Component of the Cell Wall That Confers Strength

ABSTRACT The Candida albicans plasma membrane plays important roles in interfacing with the environment, morphogenesis, and cell wall synthesis. The role of the Sur7 protein in cell wall structure and function was analyzed, since previous studies showed that this plasma membrane protein is needed to prevent abnormal intracellular growth of the cell wall. Sur7 localizes to stable patches in the plasma membrane, known as MCC (membrane compartment occupied by Can1), that are associated with eisosome proteins. The sur7Δ mutant cells displayed increased sensitivity to factors that exacerbate cell wall defects, such as detergent (SDS) and the chitin-binding agents calcofluor white and Congo red. The sur7Δ cells were also slightly more sensitive to inhibitors that block the synthesis of cell wall chitin (nikkomycin Z) and β-1,3-glucan (caspofungin). In contrast, Fmp45, a paralog of Sur7 that also localizes to punctate plasma membrane patches, did not have a detectable role in cell wall synthesis. Chemical analysis of cell wall composition demonstrated that sur7Δ cells contain decreased levels of β-glucan, a glucose polymer that confers rigidity on the cell wall. Consistent with this, sur7Δ cells were more sensitive to lysis, which could be partially rescued by increasing the osmolarity of the medium. Interestingly, Sur7 is present in static patches, whereas β-1,3-glucan synthase is mobile in the plasma membrane and is often associated with actin patches. Thus, Sur7 may influence β-glucan synthesis indirectly, perhaps by altering the functions of the cell signaling components that localize to the MCC and eisosome domains.