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D Cup Mature


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"In 2008, I wasn't a very mature athlete, mentally," Mourao said. "Today - perhaps because I've become a mother twice and had all these other experiences - I'm more focused on myself and my performance and can better channel the energy at what I want to do."


Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P-3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKC epsilon. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK-or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.


N2 - Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P-3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKC epsilon. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK-or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.


AB - Phagocytosis of the opportunistic fungal pathogen Candida albicans by cells of the innate immune system is vital to prevent infection. Dectin-1 is the major phagocytic receptor involved in anti-fungal immunity. We identify two new interacting proteins of Dectin-1 in macrophages, Bruton's Tyrosine Kinase (BTK) and Vav1. BTK and Vav1 are recruited to phagocytic cups containing C. albicans yeasts or hyphae but are absent from mature phagosomes. BTK and Vav1 localize to cuff regions surrounding the hyphae, while Dectin-1 lines the full length of the phagosome. BTK and Vav1 colocalize with the lipid PI(3,4,5)P-3 and F-actin at the phagocytic cup, but not with diacylglycerol (DAG) which marks more mature phagosomal membranes. Using a selective BTK inhibitor, we show that BTK contributes to DAG synthesis at the phagocytic cup and the subsequent recruitment of PKC epsilon. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and C. albicans phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of C. albicans, comparable to Dectin1-deficient cells. BTK-or Vav1-deficient mice are more susceptible to systemic C. albicans infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against C. albicans.


Specificity of the prespore antibody. Phase contrast and fluorescence images of vegetative cells (A), cells from dissociated late culminants (B) and intact culminating (C) and mature fruiting bodies (D) of D. discoideum, which were stained with 1:2000 diluted antibodies, raised in rabbit against a mixture of D. discoideum and P. pallidum spores, and post stained with Fluorescein isothiocyanate (FITC)-conjugated donkey-anti-rabbit IgG. The spore antibodies react to spore walls (white arrows in B), and with vesicles in prespore cells, which presynthesize wall components (pink arrows in B). In multicellular structures stained prespore cells are separated from the unstained prestalk/upper cells (white arrows in C,D) by a well-defined boundary. The unstained rearguard cells (C, pink arrow) will form the lower cup and basal disc of the mature fruiting body (pink arrows in D). Scale bars, A/B: 10 μm; C/D: 100 μm.


Prespore cells in a number of species were previously shown to be specifically detected by antibodies raised against spores of a single species [11, 20]. We raised a universal antispore antibody by inoculating rabbits with a 1:1 mixture of spores from the group-4 species D. discoideum and the group-2 species P. pallidum. After pre-adsorbtion to fixed vegetative cells of both species, the antibody specifically recognised the vesicles in prespore cells, which partially presynthesize the spore wall of mature spores (Figure 1, Additional file 1: Figures A1-A3). In sorogens of D. discoideum, the spore antibody typically yields granular staining of prespore vesicles in the posterior 70% of the structure, leaving the most rearguard 5 to 10% and the anterior 20 to 25% free of granular staining. In these regions there is low homogeneous staining throughout the cells, which conveniently outlines the non-prespore tissue.


Expression patterns ofP.pallidum ecmAandecmBhomologues.P. pallidum cells, transformed with fusion constructs of LacZ and the promoters of the closest P. pallidum ecmA/ecmB homologues PPL_07208 (A-D), PPL_07586 (E-G) and PPL_04427 (H-K), were developed to completed aggregates (A, E, H), tipped mounds/early sorogens (B, F, K) and more mature sorogens (C, D, G, I, J) and then fixed and stained with X-gal. PPL_07208::LacZ structures stained very strongly within 1 hr (A-C) and were also stained more briefly (D). PPL_07586::LacZ stained weakly after 24 h and PPL_04427::LacZ required 15 min for staining to develop. Bar: 100 μm. 041b061a72


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