Nucleocytoplasmic shuttling of the Cdc42p exchange factor Cdc24p

Cdc24p, the GDP/GTP exchange factor for the regulator of actin cytoskeleton Cdc42p, localizes to sites of polarized growth. Here we show that Cdc24p shuttles in and out of the yeast nucleus during vegetative growth. Far1p is necessary and sufficient for nuclear accumulation of Cdc24p, suggesting that its nuclear import occurs via an association with Far1p. Nuclear export is triggered either by entry into the cell cycle or by mating pheromone. As Far1p is degraded upon entry into the cell cycle, cell cycle-dependent export of Cdc24p occurs in the absence of Far1p, whereas during mating similar export kinetics indicate that a Cdc24p-Far1p complex is exported. Our results suggest that the nucleus serves as a store of preformed Cdc24p-Far1p complex which is required for chemotropism.     

Aleuriospores of Paracoccidioides brasiliensis

In soil extract agar and in Bennett medium abundant aleuriospores ofParacoccidioides brasiliensis have been observed. The possibility that these spores could be the infecting elements for man is discussed.

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Resumen Se han observado abundantes aleuriosporas deParacoccidioides brasiliensis en agar extracto de tierra y en medio de Bennett. Se discute la posibilidad de que éstas esporas puedan ser los elementos que infectan al hombre.

     

Scaffold-mediated symmetry breaking by Cdc42p

Cell polarization generally occurs along a single well-defined axis that is frequently determined by environmental cues such as chemoattractant gradients or cell-cell contacts, but polarization can also occur spontaneously in the apparent absence of such cues, through a process called symmetry breaking. In Saccharomyces cerevisiae, cells are born with positional landmarks that mark the poles of the cell and guide subsequent polarization and bud emergence to those sites, but cells lacking such landmarks polarize towards a random cortical site and proliferate normally. The landmarks employ a Ras-family GTPase, Rsr1p, to communicate with the conserved Rho-family GTPase Cdc42p, which is itself polarized and essential for cytoskeletal polarization. We found that yeast Cdc42p was effectively polarized to a single random cortical site even in the combined absence of landmarks, microtubules and microfilaments. Among a panel of Cdc42p effectors and interacting proteins, we found that the scaffold protein Bem1p was uniquely required for this symmetry-breaking behaviour. Moreover, polarization was dependent on GTP hydrolysis by Cdc42p, suggesting that assembly of a polarization site involves cycling of Cdc42p between GTP- and GDP-bound forms, rather than functioning as a simple on/off switch.     

Identification of novel, evolutionarily conserved Cdc42p-interacting proteins and of redundant pathways linking Cdc24p and Cdc42p to actin polarization in yeast

In the yeast Saccharomyces cerevisiae, Cdc24p functions at least in part as a guanine-nucleotide-exchange factor for the Rho-family GTPase Cdc42p. A genetic screen designed to identify possible additional targets of Cdc24p instead identified two previously known genes, MSB1 and CLA4, and one novel gene, designated MSB3, all of which appear to function in the Cdc24p-Cdc42p pathway. Nonetheless, genetic evidence suggests that Cdc24p may have a function that is distinct from its Cdc42p guanine-nucleotide-exchange factor activity; in particular, overexpression of CDC42 in combination with MSB1 or a truncated CLA4 in cells depleted for Cdc24p allowed polarization of the actin cytoskeleton and polarized cell growth, but not successful cell proliferation. MSB3 has a close homologue (designated MSB4) and two more distant homologues (MDR1 and YPL249C) in S. cerevisiae and also has homologues in Schizosaccharomyces pombe, Drosophila (pollux), and humans (the oncogene tre17). Deletion of either MSB3 or MSB4 alone did not produce any obvious phenotype, and the msb3 msb4 double mutant was viable. However, the double mutant grew slowly and had a partial disorganization of the actin cytoskeleton, but not of the septins, in a fraction of cells that were larger and rounder than normal. Like Cdc42p, both Msb3p and Msb4p localized to the presumptive bud site, the bud tip, and the mother-bud neck, and this localization was Cdc42p dependent. Taken together, the data suggest that Msb3p and Msb4p may function redundantly downstream of Cdc42p, specifically in a pathway leading to actin organization. From previous work, the BNI1, GIC1, and GIC2 gene products also appear to be involved in linking Cdc42p to the actin cytoskeleton. Synthetic lethality and multicopy suppression analyses among these genes, MSB, and MSB4, suggest that the linkage is accomplished by two parallel pathways, one involving Msb3p, Msb4p, and Bni1p, and the other involving Gic1p and Gic2p. The former pathway appears to be more important in diploids and at low temperatures, whereas the latter pathway appears to be more important in haploids and at high temperatures.     

The pathobiology of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis causes one of the most prevalent systemic mycoses in Latin America--paracoccidioidomycosis. It is a dimorphic fungus that undergoes a complex transformation in vivo, with mycelia in the environment producing conidia, which probably act as infectious propagules upon inhalation into the lungs, where they transform to the pathogenic yeast form. This transition is readily induced in vitro by temperature changes, resulting in modulation of the composition of the cell wall. Notably, the polymer linkages change from beta-glucan to alpha-glucan, possibly to avoid beta-glucan triggering the inflammatory response. Mammalian oestrogens inhibit this transition, giving rise to a higher incidence of disease in males. Furthermore, the susceptibility of individuals to paracoccidioidomycosis has a genetic basis, which results in a depressed cellular immune response in susceptible patients; resistance is conferred by cytokine-stimulated granuloma formation and nitric oxide production. The latency period and persistence of the disease and the apparent lack of efficacy of humoral immunity are consistent with P. brasiliensis existing as a facultative intracellular pathogen.     

Evidence for positive selection in putative virulence factors within the Paracoccidioides brasiliensis species complex

Paracoccidioides brasiliensis is a dimorphic fungus that is the causative agent of paracoccidioidomycosis, the most important prevalent systemic mycosis in Latin America. Recently, the existence of three genetically isolated groups in P. brasiliensis was demonstrated, enabling comparative studies of molecular evolution among P. brasiliensis lineages. Thirty-two gene sequences coding for putative virulence factors were analyzed to determine whether they were under positive selection. Our maximum likelihood-based approach yielded evidence for selection in 12 genes that are involved in different cellular processes. An in-depth analysis of four of these genes showed them to be either antigenic or involved in pathogenesis. Here, we present evidence indicating that several replacement mutations in gp43 are under positive balancing selection. The other three genes (fks, cdc42 and p27) show very little variation among the P. brasiliensis lineages and appear to be under positive directional selection. Our results are consistent with the more general observations that selective constraints are variable across the genome, and that even in the genes under positive selection, only a few sites are altered. We present our results within an evolutionary framework that may be applicable for studying adaptation and pathogenesis in P. brasiliensis and other pathogenic fungi.     

Melanin as a virulence factor of Paracoccidioides brasiliensis and other dimorphic pathogenic fungi: a minireview

Melanin pigments are substances produced by a broad variety of pathogenic microorganisms, including bacteria, fungi, and helminths. Microbes predominantly produce melanin pigment via tyrosinases, laccases, catecholases, and the polyketide synthase pathway. In fungi, melanin is deposited in the cell wall and cytoplasm, and melanin particles (“ghosts”) can be isolated from these fungi that have the same size and shape of the original cells. Melanin has been reported in several human pathogenic dimorphic fungi including Paracoccidioides brasiliensis, Sporothrix schenckii, Histoplasma capsulatum, Blastomyces dermatitidis, and Coccidioides posadasii. Melanization appears to contribute to virulence by reducing the susceptibility of melanized fungi to host defense mechanisms and antifungal drugs.     

Functions and functional domains of the GTPase Cdc42p

Cdc42p, a Rho family GTPase of the Ras superfamily, is a key regulator of cell polarity and morphogenesis in eukaryotes. Using 37 site-directed cdc42 mutants, we explored the functions and interactions of Cdc42p in the budding yeast Saccharomyces cerevisiae. Cytological and genetic analyses of these cdc42 mutants revealed novel and diverse phenotypes, showing that Cdc42p possesses at least two distinct essential functions and acts as a nodal point of cell polarity regulation in vivo. In addition, mapping the functional data for each cdc42 mutation onto a structural model of the protein revealed as functionally important a surface of Cdc42p that is distinct from the canonical protein-interacting domains (switch I, switch II, and the C terminus) identified previously in members of the Ras superfamily. This region overlaps with a region (alpha5-helix) recently predicted by structural models to be a specificity determinant for Cdc42p-protein interactions.     

PTEN-mediated apical segregation of phosphoinositides controls epithelial morphogenesis through Cdc42

Formation of the apical surface and lumen is a fundamental, yet poorly understood, step in epithelial organ development. We show that PTEN localizes to the apical plasma membrane during epithelial morphogenesis to mediate the enrichment of PtdIns(4,5)P2 at this domain during cyst development in three-dimensional culture. Ectopic PtdIns(4,5)P2 at the basolateral surface causes apical proteins to relocalize to the basolateral surface. Annexin 2 (Anx2) binds PtdIns(4,5)P2 and is recruited to the apical surface. Anx2 binds Cdc42, recruiting it to the apical surface. Cdc42 recruits aPKC to the apical surface. Loss of function of PTEN, Anx2, Cdc42, or aPKC prevents normal development of the apical surface and lumen. We conclude that the mechanism of PTEN, PtdIns(4,5)P2, Anx2, Cdc42, and aPKC controls apical plasma membrane and lumen formation.     

Cytosolic Neutral Proteinases of Paracoccidioides brasiliensis

Cytosolic proteinases were assayed in both morphological phases of Paracoccidioides brasiliensis. Preparations from the mycelial phase were more active in vitro than those from the yeast cells. Optimal proteinase activities for both phases occurred at pH's between 6.0 and 9.0, and at 45°C. Gelatin-SDS-PAGE electrophoresis separated several bands (58–112 kDa) in mycelial preparations; a single band (70 kDa) was seen in yeast preparations. Enzymatic activities were inhibited by antipain, phenyl methyl sulfonyl fluoride (PMSF), and chymostatin, suggestive of serine proteinases. Partial inhibition of the mycelial enzymes by ethylene diamine tetraacetic acid (EDTA), 1,10-phenanthroline, and iodoacetamide, also suggested the presence of cysteine- and metallo-proteinases. The enzymatic activity increased in preparations extracted from yeast cells transforming to mycelia, and decreased in preparations obtained from the reverse process. Received: 29 September 1997 / Accepted: 19 February 1998     

Characteristics of 17Paracoccidioides brasiliensis isolates

We have studied the physiological and morphological features of 17 isolates ofParacoccidioides brasiliensis in order to define their phenotypes. The isolates were cultured at room temperature on potato dextrose agar (PDA, Difco) slants for mycelial growth and in 1% dextrose brain heart infusion agar (BHIA, Difco) at 37°C for the study of yeast forms. Most mycelial and yeast forms grew well between pH 5.6–9.4. In their response to osmotic pressure the isolates were separated in three groups: intolerant, intermediate and tolerant. They also varied in carbohydrate assimilation tests, which indicated important metabolic variation. No clear differences were observed in phenol oxidase tests, KNO₃, starch, casein and arbutin assimilation tests. Only 1 of the isolates, Bt-19, had gelatinase activity. No correlation was observed between the above differences and virulence. Two patterns of growth were observed in the mycelial cultures, glabrous and cottonous, the latter being correlated with increased virulence for ddY mice. Most yeast forms grew as cerebriform colonies, but Pb-HC and Bt-19 colonies had a cobblestone-like surface.     

Opposing roles for actin in Cdc42p polarization

In animal and fungal cells, the monomeric GTPase Cdc42p is a key regulator of cell polarity that itself exhibits a polarized distribution in asymmetric cells. Previous work showed that in budding yeast, Cdc42p polarization is unaffected by depolymerization of the actin cytoskeleton (Ayscough et al., J. Cell Biol. 137, 399-416, 1997). Surprisingly, we now report that unlike complete actin depolymerization, partial actin depolymerization leads to the dispersal of Cdc42p from the polarization site in unbudded cells. We provide evidence that dispersal is due to endocytosis associated with cortical actin patches and that actin cables are required to counteract the dispersal and maintain Cdc42p polarity. Thus, although Cdc42p is initially polarized in an actin-independent manner, maintaining that polarity may involve a reinforcing feedback between Cdc42p and polarized actin cables to counteract the dispersing effects of actin-dependent endocytosis. In addition, we report that once a bud has formed, polarized Cdc42p becomes more resistant to dispersal, revealing an unexpected difference between unbudded and budded cells in the organization of the polarization site.     

Cdc42-dependent actin dynamics controls maturation and secretory activity of dendritic cells

Cell division cycle 42 (Cdc42) is a member of the Rho guanosine triphosphatase family and has pivotal functions in actin organization, cell migration, and proliferation. To further study the molecular mechanisms of dendritic cell (DC) regulation by Cdc42, we used Cdc42-deficient DCs. Cdc42 deficiency renders DCs phenotypically mature as they up-regulate the co-stimulatory molecule CD86 from intracellular storages to the cell surface. Cdc42 knockout DCs also accumulate high amounts of invariant chain–major histocompatibility complex (MHC) class II complexes at the cell surface, which cannot efficiently present peptide antigens (Ag’s) for priming of Ag-specific CD4 T cells. Proteome analyses showed a significant reduction in lysosomal MHC class II–processing proteins, such as cathepsins, which are lost from DCs by enhanced secretion. As these effects on DCs can be mimicked by chemical actin disruption, our results propose that Cdc42 control of actin dynamics keeps DCs in an immature state, and cessation of Cdc42 activity during DC maturation facilitates secretion as well as rapid up-regulation of intracellular molecules to the cell surface.     

Production of proteinase and phospholipase by Paracoccidioides brasiliensis

We have investigated the production of proteinase and phospholipase by 20 different isolates of Paracoccidioides brasiliensis. Isolates were grown in Bacto-peptone, Dextrose, pH 5.5, agar slants, at 27 °C for 30 days, and cultures were transferred onto Petri dishes containing basis medium and bovine serum albumin fraction V and sterile egg yolk as substrates for enzyme production, and incubated at 27 °C. After 30 days net enzyme activity was visualized and quantitavely evaluated, measuring a ratio between colony diameter and diameter of the transparent (proteinase) or white (phospholipase) ring zone surrounding it. Results demonstrated that all isolates had the ability to produce proteinase and phospholipase, even though variability in enzyme production was noted among different isolates of P. brasiliensis. This revised version was published online in June 2006 with corrections to the Cover Date.