Septins are a family of GTP-binding proteins considered to be cytoskeletal elements because they self-assemble into filaments and other higher-order structures in vivo. In budding yeast, septins establish a diffusion barrier at the bud neck between a mother and daughter cell, promote membrane curvature there, and serve as a scaffold to recruit other proteins to the site of cytokinesis. However, the mechanism by which any septin engages a partner protein has been unclear. The two most related and recently evolved subunits appear to be Cdc11 and Shs1, and the basic building blocks for assembling septin structures are hetero-octameric rods (Cdc11-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Cdc11 and Shs1-Cdc12-Cdc3-Cdc10-Cdc10-Cdc3-Cdc12-Shs1). Loss of Cdc11 is not normally tolerated, whereas cells lacking Shs1 do not appear grossly abnormal. We established several different sensitized genetic backgrounds wherein Shs1 is indispensable, which allowed us to carry out the first comprehensive and detailed genetic analysis of Shs1 in vivo. Our analysis revealed several novel insights, including: (i) the sole portion of Shs1 essential for its function is a predicted coiled-coil-forming segment in its C-terminal extension (CTE); (ii) the CTE of Cdc11 shares this function; (iii) this role for the CTEs of Cdc11 and Shs1 is quite distinct from that of the CTEs of Cdc3 and Cdc12; and (iv) heterotypic Cdc11 and Shs1 junctions likely occur in vivo. KEYWORDS yeast; cytoskeleton; complexes; filaments; mutants S EPTINS are GTP-binding proteins conserved across Eukarya (except higher plants) (Pan et al. 2007;Nishihama et al. 2011). This protein family was first identified because the corresponding loci were among the temperature-sensitive (ts) cell division cycle (cdc) mutations isolated in Saccharomyces cerevisiae (Hartwell 1978). At the restrictive temperature, cdc3, cdc10, cdc11, and cdc12 mutants continued to bud, replicate, and segregate their chromosomes, yet failed to execute cell division, resulting in the formation of chains of multinucleate cells (Hartwell 1971). Shifting a ts allele of any of these four homologous gene products to restrictive temperature prevented formation of what appeared to be rings of highly ordered membrane-associated filaments at the bud neck (Byers and Goetsch 1976). Antibody decoration (Haarer and Pringle 1987;Ford and Pringle 1991;Kim et al. 1991) and, later, use of fusions to green fluorescent protein (GFP) (Cid et al. 1998) demonstrated that these four proteins colocalized with and were likely constituents of these filaments. Because loss of the function of these proteins prevents cytokinesis and cell septation, and they seemed to be integral components of the filamentous structures erected at the bud neck, they were dubbed septins (Sanders and Field 1994;Pringle 2008). Indeed, subsequent purification of these proteins from yeast (Frazier et al. 1998), and their production as recombinant proteins in bacteria Versele and Thorner 2004;Farkasovsky et al. 2005), demonstrated that Cdc3, Cdc10, Cdc11...
Candida albicans is a fungal species that is part of the normal human microbiota and also an opportunistic pathogen capable of causing mucosal and systemic infections. C. albicans cells proliferate in a planktonic (suspension) state, but they also form biofilms, organized and tightly packed communities of cells attached to a solid surface. Biofilms colonize many niches of the human body and persist on implanted medical devices, where they are a major source of new C. albicans infections. Here, we used an unbiased and global substrate-profiling approach to discover proteolytic activities produced specifically by C. albicans biofilms, compared to planktonic cells, with the goal of identifying potential biofilm-specific diagnostic markers and targets for therapeutic intervention. This activity-based profiling approach, coupled with proteomics, identified Sap5 (Candidapepsin-5) and Sap6 (Candidapepsin-6) as major biofilm-specific proteases secreted by C. albicans. Fluorogenic peptide substrates with selectivity for Sap5 or Sap6 confirmed that their activities are highly upregulated in C. albicans biofilms; we also show that these activities are upregulated in other Candida clade pathogens. Deletion of the SAP5 and SAP6 genes in C. albicans compromised biofilm development in vitro in standard biofilm assays and in vivo in a rat central venous catheter biofilm model. This work establishes secreted proteolysis as a promising enzymatic marker and potential therapeutic target for Candida biofilm formation.
Mucins are large gel-forming polymers inside the mucus barrier that inhibit the yeast to hyphal transition of Candida albicans , a key virulence trait of this important human fungal pathogen. However, the molecular motifs in mucins that inhibit filamentation remain unclear, despite their potential for therapeutic interventions. Here, we determined that mucins display an abundance of virulence-attenuating molecules in the form of mucin O -glycans. We isolated and catalogued >100 mucin O -glycans from three major mucosal surfaces and established that they suppress filamentation and related phenotypes relevant to infection, including surface adhesion, biofilm formation, and cross-kingdom competition between C. albicans and the bacterium Pseudomonas aeruginosa . Using synthetic O -glycans we identified three structures (Core 1, Core 1+fucose, and Core 2+galactose) that are sufficient to inhibit filamentation with potency comparable to the complex O -glycan pool. Overall, this work identifies mucin O -glycans as host molecules with untapped therapeutic potential to manage fungal pathogens.
Murine cytomegalovirus infection was studied in cell and organ cultures. Regardless of the virulence characteristics of input virus, all cultures produced attenuated murine cytomegalovirus. Virulent murine cytomegalovirus has been recovered only from salivary glands of infected mice. In vivo the maintenance of murine cytomegalovirus in a virulent state is apparently due to factors other than virus replication in epithelial cells.
Candida albicans , a species of fungi, can thrive in diverse niches of its mammalian hosts; it is a normal resident of the GI tract and mucosal surfaces but it can also enter the bloodstream and colonize internal organs causing serious disease. The ability of C . albicans to thrive in these different host environments has been attributed, at least in part, to its ability to assume different morphological forms. In this work, we examine one such morphological change known as white-opaque switching. White cells are the default state of C . albicans , and most animal studies have been carried out exclusively with white cells. Here, we compared the proliferation of white and opaque cells in two murine models of infection and also monitored, using specially constructed strains, switching between the two states in the host. We found that white cells outcompeted opaque cells in many niches; however, we show for the first time that in some organs (specifically, the heart and spleen), opaque cells competed favorably with white cells and, when injected on their own, could colonize these organs. In environments where the introduced white cells outcompeted the introduced opaque cells, we observed high rates of opaque-to-white switching. We did not observe white-to-opaque switching in any of the niches we examined.
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