63 research outputs found
Hbr1 is a negative regulator of hyphae formation under embedded conditions.
<p><i>C</i>. <i>albicans</i> log-phase yeast cells were embedded in molten YPS agar and incubated at 24°C. Photomicrographs were taken through the agar matrix. <b>(A).</b> Wild type strain BWP17 incubated for 4 days illustrating three modes of filamentation or yeast formation from spindle-shaped colonies. Note that these filamented colony types were only infrequently encountered. Panel 1, Pseudohyphal filaments; Panel 2, a single hyphal sprout; Panel 3, Yeast lateral outcropping from a spindle-shaped colony. Bar = 200 μm. <b>(B).</b> A comparison of colony types according to agar depth. Strains BWP17 (WT) and JKC19 (<i>cph1/cph1</i>) were incubated for 4 days; CAMPR8 (<i>HBR1/hbr1</i>) and HLC52 (<i>efg1/efg1</i>) for 2 days. The frequency that each strain formed a colony with at least 1 hypha is indicated (n >125). Strains CAMPR8 and HLC52 formed filaments at all agar depths. Surface colonies from all strains were smooth and lacked filaments. Arrow, spindle-shaped colony; arrow with line, spindle with yeast outgrowth; y, yeast colony. Bar = 2 mm. <b>(C).</b> Strain CAMPR8 cultured as above. Panel 1, simple hyphae with yeast (arrow), 2 day culture; Panel 2, hyphal sprout (arrow), 6 day culture; Bar = 200 μm; Panel 3, lateral yeast formation, Bar = 100 μm; Panel 4, perpendicular lateral branches (L) and yeast0020colonies originating at branch junctions (Y), Bar = 50 μm.</p
Hbr1 Activates and Represses Hyphal Growth in <i>Candida albicans</i> and Regulates Fungal Morphogenesis under Embedded Conditions
<div><p>Transitions between yeast and hyphae are essential for <i>Candida albicans</i> pathogenesis. The genetic programs that regulate its hyphal development can be distinguished by embedded versus aerobic surface agar invasion. Hbr1, a regulator of white-opaque switching, is also a positive and negative regulator of hyphal invasion. During embedded growth at 24°C, an <i>HBR1/hbr1</i> strain formed constitutively filamentous colonies throughout the matrix, resembling <i>EFG1</i> null colonies, and a subset of long unbranched hyphal aggregates enclosed in a spindle-shaped capsule. Inhibition of adenylate cyclase with farnesol perturbed the filamentation of <i>HBR1/hbr1</i> cells producing cytokinesis-defective hyphae whereas farnesol treated <i>EFG1</i> null cells produced abundant opaque-like cells. Point mutations in the Hbr1 ATP-binding domain caused distinct filamentation phenotypes including uniform radial hyphae, hyphal sprouts, and massive yeast cell production. Conversely, aerobic surface colonies of the <i>HBR1</i> heterozygote on Spider and GlcNAc media lacked filamentation that could be rescued by growth under low (5%) O<sub>2</sub>. Consistent with these morphogenesis defects, the <i>HBR1</i> heterozygote exhibited attenuated virulence in a mouse candidemia model. These data define Hbr1 as an ATP-dependent positive and negative regulator of hyphal development that is sensitive to hypoxia.</p></div
<i>C</i>. <i>albicans</i> strains used in this study.
<p>*Prototrophic strain. The plasmid pCIP20 was obtained from A. Brown [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126919#pone.0126919.ref065" target="_blank">65</a>].</p><p><i>C</i>. <i>albicans</i> strains used in this study.</p
Attenuated virulence of an <i>HBR1</i> heterozygote in a mouse model of disseminated infection.
<p>Strains DAY185 (<i>HBR1/HBR1</i>) and R8PRO (<i>HBR1/hbr1</i> ARG+ URA+ HIS+ prototroph) were introduced into female Balb/c mice <i>via</i> the tail vein at the indicated doses (n = 12, each).</p
Hypoxia can rescue the agar invasion defect of the <i>HBR1</i> heterozygous strain.
<p><i>C</i>. <i>albicans</i> strains BWP17 (left column) and CAMPR8 (right column) were cultured for 4 days on the surface of the indicated media under aerobic or hypoxic (5% O<sub>2</sub>) conditions at 30°C. Colonies were washed from the agar surfaces before photography through the agar.</p
Effects of farnesol on embedded growth of an <i>efg1/efg1</i> strain.
<p>Photomicrographs taken <i>in situ</i> of HLC52 cells embedded in YPS agar containing 100 M farnesol after 2 days at 24<sup>°</sup>C. <b>(A).</b> Three focal planes illustrating three colony morphologies. Arrow, non-filamentous spindle-shaped colonies in the lower level of the matrix; Arrowhead, upper level colony with compact pseudohyphal filaments that do not produce yeast; Boxed region, a colony expressing arboreal-shaped branching hyphae that develop midlevel in the matrix. Bar = 2 mm. <b>(B).</b> Higher magnification of area such as boxed in (A). Masses of elongated opaque or opaque-like cells (brackets) produced through invasive pseudohyphal migration. Bar = 150 μm. <b>(C).</b> Higher magnification of a leading edge of the pseudohyphal expansion from (B). Bar = 10 μm. <b>(D).</b> An opaque-like cell aggregation illustrating the final maturation step of opaque-like cells from the pseudohyphae. Calcofluor white stain. Bar = 5 μm.</p
Colony phenotypes under various growth conditions.
<p>(†) Per cent filamented colonies;</p><p>(±) Cell separation / cytokinesis defects</p><p>Colony phenotypes under various growth conditions.</p
Mutations in the Hbr1 P-loop define distinct embedded phenotypes.
<p><b>(A).</b> Locations of key predicted Hbr1 structural domains as indicated in the text. Accession numbers: Hbr1 (AF466197_1), AD-004 (NP_057367), Fap7 (NP_010115). Positions of Hbr1 mutated amino acids, (*). <b>(B).</b> Filamentation in the agar matrix can be modulated through HBR1 P-loop mutations. <i>C</i>. <i>albicans</i> cells were cultured within YPS agar at 24°C as described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0126919#pone.0126919.g001" target="_blank">Fig 1</a>. Photographs were taken through the agar matrix at two depths. Left, G19S mutation and hyphal ‘sprout’ development from yeast colonies at all levels in the matrix except from the lower level spindle-shaped colonies. A yeast mass with developing filaments is indicated by the arrow; Middle, G21S mutation and a uniform production of simple hyphae lacking yeast from spindle-shaped colonies furthest from the agar surface; Right, strain CAMPR8 restored to wild type (Strain R8WT). Arrows indicate sites of yeast production. <b>(C).</b> Manipulation of Hbr1 by increasing copy number using strain R8MET grown without added methionine (left), mutation of solvent-exposed Lys residue using strain R866R (center) and addition of a carboxyl-terminal protein tag using strain R8CTH (right). Arrows indicate sites of yeast production. Bars = 1.5 mm.</p
Breeding for blast resistance at CIAT
<p><b>A</b> Jurkat cells or <b>B</b> BAEC were incubated with 10 µM CD36 antisense morpholino or 10 µM 5-mis-splice CD36 control morpholino for 48 hrs. Following CD36 knockdown, cells were pretreated with 10 µM Aβ followed by 10 µM DEA/NO. Following treatment, cell were lysed and assayed for cGMP production. n = 3, * denotes P<0.05.</p
Fibrillar and soluble Aβ inhibit cellular myristate uptake but not SIRPα binding to CD47.
<p>[<sup>3</sup>H]-Myristic acid uptake after 5 min into human aortic VSMC (<b>A</b>), HUVEC (<b>B</b>), and microglial cells (<b>C</b>) was determined in the presence of the indicated concentrations of Aβ (soluble or fibrillar) after lysis by liquid scintillation counting. <b>D </b><sup>125</sup>I-SIRPα-Fc binding to Jurkat T-cells was measured in the presence of soluble Aβ (0.1-10 µM) or a CD47-specific function-blocking antibody (B6H12) for 1 hour at 25°C.</p
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