Bruton's Tyrosine Kinase (BTK) and Vav1 Contribute to Dectin1-Dependent Phagocytosis of <i>Candida albicans</i> in Macrophages

<div><p>Phagocytosis of the opportunistic fungal pathogen <i>Candida albicans</i> 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 <i>C. albicans</i> 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)P₃ 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 PKCε. BTK- or Vav1-deficient peritoneal macrophages display a defect in both zymosan and <i>C. albicans</i> phagocytosis. Bone marrow-derived macrophages that lack BTK or Vav1 show reduced uptake of <i>C. albicans</i>, comparable to Dectin1-deficient cells. BTK- or Vav1-deficient mice are more susceptible to systemic <i>C. albicans</i> infection than wild type mice. This work identifies an important role for BTK and Vav1 in immune responses against <i>C. albicans</i>.</p></div

BTK, Vav1 and Syk colocalize with F-actin and PI(3,4,5)P₃.

<p>(A): Electron microscopy of <i>C. albicans</i> phagocytosis by RAW-Dectin1 macrophages. Cells were fixed to visualize polymerized actin. (B): Localization of LifeAct-RFP detecting F-actin and C1-PKCδ-GFP that binds to DAG after 30 and 90 minutes of coincubation with <i>Candida</i>-BFP. (C): Colocalization of LifeAct-RFP detecting F-actin and PH-BTK-GFP that binds to PI(3,4,5)P₃ after 30 and 90 minutes of coincubation with <i>Candida</i>-BFP. (D): Localization of BTK-mCherry, mCherry-Vav1 and Syk-mCherry with LifeAct-GFP after 90 minutes of coincubation with <i>Candida</i>-BFP. White arrows indicate areas of co-localization, while red and green arrows indicate areas of speciation. Experiments were performed multiple times, representative micrographs are shown.</p

BTK and Vav1 interact with Dectin-1 during <i>C. albicans</i> phagocytosis by macrophages.

<p>(A): Morphology and β-glucan exposure of <i>C. albicans</i> expressing blue fluorescent protein (<i>Candida</i>-BFP) at indicated time points after incubation in DMEM with 10% IFS. <i>Candida</i>-BFP was stained with fluorescent carbohydrate recognition domain of Dectin-1 (Dectin1-CRD-Alexa647) that binds β-glucan. Arrows indicate increases staining at bud scars. (B): Immunoblotting experiment showing expression of different proteins in RAW-Dectin1 cells during co-incubation with live <i>C. albicans</i> for the indicated time points. Phagocytosis of <i>C. albicans</i> occurs throughout the time course, but the morphology of the ingested particles changes over time. (C): Co-incubation of RAW-Dectin1 with <i>C. albicans</i> followed by co-immunoprecipitation with anti-BTK or anti-Vav1 antibody and immunoblotting with anti-HA to detect Dectin-1. BTK/Dectin-1 and Vav1/Dectin-1 complexes were detected at different time points during the co-incubation. (D): Quantification of BTK/Dectin-1 and Vav1/Dectin-1 complexes showing strongest interactions at the 90- and 180-minute time points, respectively. Means +/− SD of three independent experiments are shown.</p

BTK-mCherry and mCherry-Vav1 colocalize with PI(3,4,5)P₃ but not with DAG.

<p>(A): Colocalization of BTK-mCherry and PH-BTK-GFP that binds to PI(3,4,5)P₃ at 30 and 90 minutes of coincubation with <i>Candida</i>-BFP showing phagocytosis of yeast and hyphae, respectively. (B): Colocalization of mCherry-Vav1 and PH-BTK-GFP at 30 and 90 minutes of coincubation with <i>Candida</i>-BFP. (C): Localization of mCherry-Syk and PH-BTK-GFP at 30 and 90 minutes of coincubation with <i>Candida</i>-BFP. (D): Localization of BTK-mCherry and C1-PKCδ-GFP at 30 and 90 minutes of coincubation with <i>Candida</i>-BFP. (E): Localization of mCherry-Vav1 and C1-PKCδ-GFP at 30 and 90 minutes of coincubation with <i>Candida</i>-BFP. (F): Localization of mCherry-Syk and C1-PKCδ-GFP at 30 and 90 minutes of coincubation with <i>Candida</i>-BFP. White arrows indicate areas of co-localization, while red and green arrows indicate areas of speciation. Experiments were performed at least three times, representative micrographs are shown.</p

Localization of BTK-mCherry and mCherry-Vav1 to the <i>Candida</i>-containing phagocytic cup.

<p>(A): Confocal images showing localization of BTK-mCherry, mCherry-Vav1 and mCherry-Syk in RAW-Dectin1 macrophages incubated with <i>Candida</i>-BFP. Images were taken without <i>Candida</i>-BFP or after 30 minutes, 90 minutes and 180 minutes of co-incubation with <i>Candida</i>-BFP to study phagocytosis of yeast, hyphae, and very long hyphae, respectively. White arrows indicate areas of mCherry recruitment to the <i>Candida</i>-containing phagocytic cup, visible during ingestion of <i>C. albicans</i> yeast (30 minutes) and <i>C. albicans</i> hyphae (90 and 180 minutes). (B): XYZ images of C. albicans phagocytosis by the BTK-mCherry, mCherry-Vav1 and mCherry-Syk cell lines. Cuff regions of protein recruitment are circular bands around <i>C. albicans</i> hyphae that are being ingested. (C): Quantitation of BTK-mCherry, mCherry-Vav1 and mCherry-Syk recruitment to the phagocytic cup after 90 minutes of coincubation with <i>Candida</i>-BFP. (D): Model showing localization of BTK and Vav1 to the phagocytic cup but not to mature phagosomes during phagocytosis of <i>C. albicans</i> yeast and hyphae by macrophages. Representative micrographs and means +/− SD of 3 independent experiments are shown. For statistical analysis, all data were analyzed by unpaired t test.</p

Phenotypic analysis of BTK- and Vav1-deficient macrophages and mice.

<p>(A): Peritoneal macrophages or bone marrow-derived macrophages (BMDM) from wild type, <i>dectin1−/−</i>, <i>btk</i>−/− or <i>vav1</i>−/− mice were incubated with zymosan-Alexa647 or live <i>Candida</i>-BFP for 30 minutes or 1 hour at an MOI of 10 and the number of internalized <i>Candida</i>-BFP was determined by microscopy. Graphs represent means and standard deviations of experiments with three different mice. (B): The contribution of BTK and Vav1 to overall immune responses to <i>C. albicans</i> was determined using the model for systemic candidiasis. Tail vein injection of wild type, <i>dectin1</i>−/−, <i>btk</i>−/− or <i>vav1</i>−/− mice were performed with 0.5×10⁴ colony forming units (CFU) of <i>C. albicans</i> and disease was monitored over time. (C): <i>C. albicans</i> CFU in kidneys of indicated mice at final stage of disease, means +/− SD are indicated. (D): GMS staining of kidney histology slides of wild type, <i>dectin1</i>−/−, <i>btk</i>−/− or <i>vav1</i>−/− mice, at final stage of disease showing extensive fungal invasion of tissues. (E): H&E staining of kidney histology slides of wild type, <i>dectin1</i>−/−, <i>btk</i>−/− or <i>vav1</i>−/− mice, at final stage of disease showing extensive immune cell invasion of tissues. Representative images are shown. TNFα (F) and IL-6 (G) levels in supernatant of mouse peritoneal macrophages 12 hours after incubation without or with <i>C. albicans</i>. Graphs represent means and standard deviations of experiments with three different mice. TNFα (H) and IL-6 (I) levels in kidney lysates of mice infected with 5×10⁴ CFU <i>C. albicans</i> at 11 days after infection. Each dot represents one mouse; means and standard deviations are indicated. Values did not differ significantly. For statistical analysis, all data were analyzed by unpaired t test.</p

Localization of phospholipids and PKC family proteins during <i>C. albicans</i> phagocytosis.

<p>(A): PH-Akt-RFP and C1-PKCδ-GFP biosensors showing localization of PI(3,4,5)P₃/PI(3,4)P₂ and DAG, respectively, without challenge or after 30 or 90 minutes of co-incubation with <i>Candida</i>-BFP. White arrows indicate areas of PI(3,4,5)P₃/PI(3,4)P₂ and DAG co-localization, while red and green arrows indicate areas of speciation. (B): Quantitation of PI(3,4,5)P₃/PI(3,4)P₂- and DAG-positive phagosomes after 30 minutes of coincubation with <i>Candida</i>-BFP. (C): Model showing localization of PI(3,4,5)P₃/PI(3,4)P₂ and DAG during engagement and internalization of <i>C. albicans</i> yeast and hyphae by macrophages. (D): Localization of GFP-tagged PKCα, PKCβ, PKCδ, PKCε and PKCζ after 30 or 90 minutes of <i>Candida</i>-BFP phagocytosis. (E): Quantitation of PKCα-GFP, PKCβ-GFP, PKCδ-GFP, PKCε-GFP and PKCζ-GFP recruitment to the phagocytic cup after 90 minutes of coincubation with <i>Candida</i>-BFP. Representative micrographs and means +/− SD of 3 independent experiments are shown. For statistical analysis, all data were analyzed by unpaired t test.</p

BTK is involved in DAG production at the phagocytic cup.

<p>(A): RAW-Dectin1 macrophages were pre-incubated with the indicated concentrations of BTK inhibitor PCI-32765 followed by coincubation with <i>Candida</i>-BFP (MOI 10) for 1 hour. Graphs represent number of internalized <i>Candida</i>-BFP per macrophage as determined by microscopy. (B): DAG measurements in RAW-Dectin1 macrophages preicubated with the indicated inhibitors and in the presence or absense of <i>C. albicans</i>. Thin layer chromatography was performed to visualize the phosphatidic acid (PA) product of the DAG kinase assay. (C): Quantification of PA signal from three independent DAG kinase experiments. (D): Confocal images of C1-PKCδ-GFP (DAG) and PKCε-GFP distribution in RAW-Dectin1 macrophages pre-incubated with 0.5 µM BTK inhibitor PCI-32765. (E): Quantification of C1-PKCδ-GFP and PKCε-GFP recruitment to phagosomes in absence and presence of 0.5 µM BTK inhibitor. All graphs display means +/− SD of three independent experiments. (F): Schematic showing localization of PI(3,4,5)P₃, BTK, Vav1, DAG and PKC family proteins during engagement and internalization of <i>C. albicans</i> yeast and hyphae by macrophages. (G): Model summarizing this studies findings. BTK, Vav1 and Syk interact with Dectin-1 during phagocytosis of <i>C. albicans</i> (left). Phosphatidylinositol 4,5-biphosphate (PI(4,5)P₂) can be converted to phosphatidylinositol 3,4,5-triphosphate (PI(3,4,5)P₃) by PI3K or to diacylglycerol (DAG) by phospholipase C γ (PLCγ). Specialized PI(3,4,5)P₃- and DAG-rich phagosomal membranes can be distinguished during <i>C. albicans</i> phagocytosis. Bruton's Tyrosine Kinase (BTK) and Vav1 localize to PI(3,4,5)P₃-rich membrane regions and colocalize with F-actin. Vav1 might play an active role in actin rearrangements at the phagocytic cup through activation of small GTPases Rac1, Cdc42 and/or Rho1. BTK is involved in the production of DAG at the phagocytic cup, possibly through the activation of PLCγ. Protein Kinase C (PKC) family proteins localize to DAG-rich membranes. For statistical analysis, all data were analyzed by unpaired t test.</p

Tuning Hsf1 levels drives distinct fungal morphogenetic programs with depletion impairing Hsp90 function and overexpression expanding the target space

<div><p>The capacity to respond to temperature fluctuations is critical for microorganisms to survive within mammalian hosts, and temperature modulates virulence traits of diverse pathogens. One key temperature-dependent virulence trait of the fungal pathogen <i>Candida albicans</i> is its ability to transition from yeast to filamentous growth, which is induced by environmental cues at host physiological temperature. A key regulator of temperature-dependent morphogenesis is the molecular chaperone Hsp90, which has complex functional relationships with the transcription factor Hsf1. Although Hsf1 controls global transcriptional remodeling in response to heat shock, its impact on morphogenesis remains unknown. Here, we establish an intriguing paradigm whereby overexpression or depletion of <i>C</i>. <i>albicans HSF1</i> induces morphogenesis in the absence of external cues. <i>HSF1</i> depletion compromises Hsp90 function, thereby driving filamentation. <i>HSF1</i> overexpression does not impact Hsp90 function, but rather induces a dose-dependent expansion of Hsf1 direct targets that drives overexpression of positive regulators of filamentation, including Brg1 and Ume6, thereby bypassing the requirement for elevated temperature during morphogenesis. This work provides new insight into Hsf1-mediated environmentally contingent transcriptional control, implicates Hsf1 in regulation of a key virulence trait, and highlights fascinating biology whereby either overexpression or depletion of a single cellular regulator induces a profound developmental transition.</p></div

Hsf1 binds to an expanded set of taget genes upon overexpression, inducing the expression of positive regulators of filamentation.

<p>a) ChIP-seq was performed to determine the targets of Hsf1 in strains expressing basal (<i>HSF1-TAP/HSF1</i>) and overexpressed (<i>tetO-HSF1-TAP/tetO-HSF1</i>) levels of Hsf1, which identified an expansion of Hsf1 targets upon overexpression. Genes previously identified as Hsf1 targets and genes involved in <i>C</i>. <i>albicans</i> filamenation are indicated. b) Hsf1 signals at the summit of Hsf1 ChIP-seq peaks in wild-type (basal conditions, top) and <i>HSF1</i> overexpression (bottom) strains. Weak Hsf1 ChIP-seq signal can be seen at the overexpression-specific target sites under basal conditions, suggesting that Hsf1 also binds these sites under basal conditions albeit at a lower level. Legend shows Hsf1 ChIP-seq signal across a 2 kb window spanning Hsf1 binding summits identified by MACS analysis. c) Both basal and <i>HSF1</i> overexpression-specific targets contain the conserved heat shock element (HSE) binding sites. The percentage of ChIP-seq peaks with the HSE motifs TTCnnGAA, TTCn⁷TTC, TTCnnGAAnnTTC, or variations of the motifs termed triple cis motifs (GAA n^0-3 GAA n^0-3 GAA), triple trans motifs (GAA n^0-3 TTC n^0-3 GAA) or triple trans/cis motifs (TTC n^0-3 GAA n^0-3 GAA, GAA n^0-3 GAA n^0-3 TTC, TTC n^0-3 TTC n^0-3 GAA, GAA n^0-3 TTC n^0-3 TTC) are shown for basal and overexpression-specific targets. d) MA plot showing difference in gene expression between wild-type and <i>HSF1</i> overexpression strains. Genes more than 1.5-fold up-regulated in the <i>HSF1</i> overexpression strain as compared to the wild-type strain are shown in red, whereas genes more than 1.5-fold down-regulated are shown in blue. Genes of interest based on their roles in filamentation are indicated. <i>HSF1</i> is highlighted in purple. e) A Euler diagram depicting the overlap between differentially regulated genes upon <i>HSF1</i> overexpression determined by RNA-seq and Hsf1 bound targets determined by ChIP-seq. Hsf1 targets includes those promoters bound in either the wild-type or overexpression strain. Selected genes associated with filamentation are indicated in the diagram. f) A heat map showing gene expression changes upon <i>HSF1</i> overexpression for filamentation regulators. Colored dots are included to indicate whether the respective gene is bound by Hsf1 in the wild-type strain (in red) or only when Hsf1 is overexpressed (in orange). The colour bar depicts the change in expression as determined by RNA-seq.</p