Serum Antibody Signature Directed against <i>Candida albicans</i> Hsp90 and Enolase Detects Invasive Candidiasis in Non-Neutropenic Patients

Invasive candidiasis (IC) adds significantly to the morbidity and mortality of non-neutropenic patients if not diagnosed and treated early. To uncover serologic biomarkers that alone or in combination could reliably detect IC in this population, IgG antibody–reactivity profiles to the <i>Candida albicans</i> intracellular proteome were examined by serological proteome analysis (SERPA) and data mining procedures in a training set of 24 non-neutropenic patients. Despite the high interindividual molecular heterogeneity, unsupervised clustering analyses revealed that serum 22-IgG antibody–reactivity patterns differentiated IC from non-IC patients. Univariate analyses further highlighted that 15 out of the 22 SERPA-identified IgG antibodies could be useful candidate IC biomarkers. The diagnostic performance of one of these candidates (anti-Hsp90 IgG antibodies) was validated using an ELISA prototype in a test set of 59 non-neutropenic patients. We then formulated an IC discriminator based on the combined immunoproteomic fingerprints of this and another SERPA-detected and previously validated IC biomarker (anti-Eno1 IgG antibodies) in the training set. Its consistency was substantiated using their ELISA prototypes in the test set. Receiver-operating-characteristic curve analyses showed that this two-biomarker signature accurately identified IC in non-neutropenic patients and provided better IC diagnostic accuracy than the individual biomarkers alone. We conclude that this serum IgG antibody signature directed against <i>C. albicans</i> Hsp90 and Eno1, if confirmed prospectively, may be useful for IC diagnosis in non-neutropenic patients

<i>Candida albicans</i> Modifies the Protein Composition and Size Distribution of THP-1 Macrophage-Derived Extracellular Vesicles

The effectiveness of macrophages in the response to systemic candidiasis is crucial to an effective clearance of the pathogen. The secretion of proteins, mRNAs, noncoding RNAs and lipids through extracellular vesicles (EVs) is one of the mechanisms of communication between immune cells. EVs change their cargo to mediate different responses, and may play a role in the response against infections. Thus we have undertaken the first quantitative proteomic analysis on the protein composition of THP-1 macrophage-derived EVs during the interaction with <i>Candida albicans</i>. This study revealed changes in EVs sizes and in protein composition, and allowed the identification and quantification of 717 proteins. Of them, 133 proteins changed their abundance due to the interaction. The differentially abundant proteins were involved in functions relating to immune response, signaling, or cytoskeletal reorganization. THP-1-derived EVs, both from control and from <i>Candida</i>-infected macrophages, had similar effector functions on other THP-1-differenciated macrophages, activating ERK and p38 kinases, and increasing both the secretion of proinflammatory cytokines and the candidacidal activity; while in THP-1 nondifferenciated monocytes, only EVs from infected macrophages increased significantly the TNF-α secretion. Our findings provide new information on the role of macrophage-derived EVs in response to <i>C. albicans</i> infection and in macrophages communication

Serum Antibody Profile during Colonization of the Mouse Gut by <i>Candida albicans</i>: Relevance for Protection during Systemic Infection

<i>Candida albicans</i> is a commensal microorganism in the oral cavity and gastrointestinal and urogenital tracts of most individuals that acts as an opportunistic pathogen when the host immune response is reduced. Here, we established different immunocompetent murine models to analyze the antibody responses to the <i>C. albicans</i> proteome during commensalism, commensalism followed by infection, and infection (C, C+I, and I models, respectively). Serum anti-<i>C. albicans</i> IgG antibody levels were higher in colonized mice than in infected mice. The antibody responses during gut commensalism (up to 55 days of colonization) mainly focused on <i>C. albicans</i> proteins involved in stress response and metabolism and differed in both models of commensalism. Different serum IgG antibody-reactivity profiles were also found over time among the three murine models. <i>C. albicans</i> gut colonization protected mice from an intravenous lethal fungal challenge, emphasizing the benefits of fungal gut colonization. This work highlights the importance of fungal gut colonization for future immune prophylactic therapies

Proteomics Unravels Extracellular Vesicles as Carriers of Classical Cytoplasmic Proteins in <i>Candida albicans</i>

The commensal fungus <i>Candida albicans</i> secretes a considerable number of proteins and, as in different fungal pathogens, extracellular vesicles (EVs) have also been observed. Our report contains the first proteomic analysis of EVs in <i>C. albicans</i> and a comparative proteomic study of the soluble secreted proteins. With this purpose, cell-free culture supernatants from <i>C. albicans</i> were separated into EVs and EV-free supernatant and analyzed by LC–MS/MS. A total of 96 proteins were identified including 75 and 61 proteins in EVs and EV-free supernatant, respectively. Out of these, 40 proteins were found in secretome by proteomic analysis for the first time. The soluble proteins were enriched in cell wall and secreted pathogenesis related proteins. Interestingly, more than 90% of these EV-free supernatant proteins were classical secretory proteins with predicted N-terminal signal peptide, whereas all the leaderless proteins involved in metabolism, including some moonlighting proteins, or in the exocytosis and endocytosis process were exclusively cargo of the EVs. We propose a model of the different mechanisms used by <i>C. albicans</i> secreted proteins to reach the extracellular medium. Furthermore, we tested the potential of the Bgl2 protein, identified in vesicles and EV-free supernatant, to protect against a systemic candidiasis in a murine model

Proteomics Unravels Extracellular Vesicles as Carriers of Classical Cytoplasmic Proteins in <i>Candida albicans</i>

The commensal fungus <i>Candida albicans</i> secretes a considerable number of proteins and, as in different fungal pathogens, extracellular vesicles (EVs) have also been observed. Our report contains the first proteomic analysis of EVs in <i>C. albicans</i> and a comparative proteomic study of the soluble secreted proteins. With this purpose, cell-free culture supernatants from <i>C. albicans</i> were separated into EVs and EV-free supernatant and analyzed by LC–MS/MS. A total of 96 proteins were identified including 75 and 61 proteins in EVs and EV-free supernatant, respectively. Out of these, 40 proteins were found in secretome by proteomic analysis for the first time. The soluble proteins were enriched in cell wall and secreted pathogenesis related proteins. Interestingly, more than 90% of these EV-free supernatant proteins were classical secretory proteins with predicted N-terminal signal peptide, whereas all the leaderless proteins involved in metabolism, including some moonlighting proteins, or in the exocytosis and endocytosis process were exclusively cargo of the EVs. We propose a model of the different mechanisms used by <i>C. albicans</i> secreted proteins to reach the extracellular medium. Furthermore, we tested the potential of the Bgl2 protein, identified in vesicles and EV-free supernatant, to protect against a systemic candidiasis in a murine model

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Downregulation of PP2A^Cdc55 promotes Cdc15 activation.

<p>(A) Cdc15 is dephosphorylated upon Zds1-dependent inactivation of PP2A^Cdc55. Strain Y603 (<i>MATa MET-CDC20 GAL1-Flag₃-ZDS1 CDC14-Pk₉ CDC15-HA₆</i>) was arrested in metaphase by Cdc20 depletion and galactose was added to induce Zds1 expression. Cdc15 phosphorylation and Zds1 expression levels were analyzed by western blot. (B) Zds1-dependent inactivation of PP2A^Cdc55 induces Cdc15 asymmetric localization. Strain Y1014 (<i>MATα MET-CDC20 GAL1-Flag₃-ZDS1 CDC14-Pk₉ CDC15-eGFP BFA1-mCherry</i>) was arrested in metaphase by Cdc20 depletion and Zds1 expression was induced. At least 50 cells were scored for each strain. (C) Cdc55 deletion causes premature Cdc15 asymmetric localization in metaphase. Strains Y984 (<i>MATa MET-CDC20 CDC14-myc₉ CDC15-eGFP BFA1-mCherry</i>) and Y966 (as Y984, but <i>cdc55</i>Δ) were arrested in metaphase by Cdc20 depletion and the percentage of cells with asymmetric Cdc15-eGFP was quantified. At least 50 cells were scored for each strain. (D) Cdc15 premature asymmetric localization in the absence of Cdc55 in a metaphase-to-anaphase transition. The same strains as in (C) were released into a synchronous anaphase by Cdc20 depletion and reintroduction and followed by time-lapse microscopy (n = 14 for WT; n = 10 for <i>cdc55</i>Δ). Scale bar, 2 µm.</p

Premature Bfa1 inactivation does not provoke premature exit from mitosis.

<p>(A) Cdc15 phosphorylation in the absence of Cdc55. Strains Y2961 (<i>MATa MET-CDC20 CDC14-Pk₉ CDC15-HA₆</i>) and Y528 (as Y2961, but <i>cdc55</i>Δ) were released into a synchronous anaphase by Cdc20 depletion and reintroduction. Cdc15 phosphorylation was analyzed by western blot. The budding index was used to monitor cell cycle progression. Pgk1 served as a loading control. (B) Cdc14 activity in <i>cdc55</i>Δ mutant cells. Strains Y2810 (<i>MATa MET-CDC20 CDC14-</i>Pk₉) and Y2118 (as Y2810, but <i>cdc55</i>Δ) were arrested in metaphase by Cdc20 depletion. Anaphase samples were taken 20 min after release when more than 80% of the cells showed anaphase spindles. Phosphatase activity from immunopurified Cdc14 was measured as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003966#s4" target="_blank">Materials and Methods</a>. Means and standard deviations are shown. (C) The MEN is not prematurely active in the absence of Cdc55. Strains Y547 (<i>MATa MET-CDC20 CDC14-Pk₉ HA₃-CDH1</i>) and Y548 (as Y547, but <i>cdc55</i>Δ) were released into synchronous anaphase by Cdc20 depletion and reintroduction. Cdh1 phosphorylation and Cdc5, Clb2 and Sic1 proteins levels were analyzed by western blot.</p

A model for MEN regulation by PP2A^Cdc55.

<p>In early anaphase, FEAR-induced inactivation of the PP2A^Cdc55 promotes the first wave of Cdc14 release and contributes to the accumulation of the Cdc5-phosphorylated form of Bfa1. In this way, FEAR alleviates the inhibitory signal on Tem1 by promoting the Bfa1 phosphorylation and stimulates Cdc15 activity through the FEAR-induced Cdc14 released. However, the downstream MEN kinase Dbf2–Mob1 is kept inactive by Cdk1–Clb2 phosphorylation, restraining MEN activity. Mob1 phosphorylation is invariable despite the progressive decrease in Cdk1 activity because the counteracting phosphatase PP2A^Cdc55 is also downregulated in early anaphase. In late anaphase, the increase in Cdc14 activity and the decrease in Cdk1–Clb2 activity alleviate the Cdk1 inhibitory signal towards Dbf2–Mob1 and the MEN is fully active.</p

Bfa1 localizes asymmetrically at the dSPB in the absence of PP2A^Cdc55 activity.

<p>(A) Cdc55 deletion causes premature Bfa1 asymmetric localization at the dSPB in metaphase-arrested cells. Strains Y559 (<i>MATa MET-CDC20 CDC14-Pk₉ BFA1-eGFP</i>) and Y560 (as Y559, but <i>cdc55</i>Δ) were arrested in metaphase by Cdc20 depletion. Percentages of equal, high/low and single distribution of Bfa1-eGFP were determined. At least 100 cells were scored for each strain. (B) Bfa1 localization becomes asymmetric upon Zds1-dependent inactivation of PP2A^Cdc55. Strain Y875 (<i>MATα MET-CDC20 GAL1-Flag₃-ZDS1 CDC14-Pk₉ BFA1-mCherry SPC42-YFP</i>) was arrested in metaphase by Cdc20 depletion and galactose was added to induce Zds1 overexpression. Asymmetric Bfa1-mCherry signal was quantified as the SPB relative intensity ratio as described in the <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003966#s4" target="_blank">Materials and Methods</a>. (C) Premature Bfa1 asymmetric localization in the absence of Cdc55 in a metaphase-to-anaphase transition. Strains Y951 (<i>MATa MET-CDC20 CDC28F19 CDC14-myc₉ BFA1-mCherry SPC42-YFP</i>) and Y1017 (as Y951, but <i>cdc55</i>Δ) were arrested in metaphase by Cdc20 depletion and released into synchronous anaphase by Cdc20 reintroduction. Time-lapse microscopy was used to visualize Bfa1-mCherry localization dynamics. Bfa1 SPB ratios were measured during mitosis progression (n = 15 for WT; n = 10 for <i>cdc55</i>Δ). The distance between the two SPBs was used to calculate the spindle length. (D) Premature Bfa1 asymmetric localization in the absence of Cdc55 in a synchronous cell cycle after G1 release. Strains Y876 (<i>MATa CDC28F19 CDC14-myc₉ BFA1-mCherry SPC42-YFP</i>) and Y1006 (as Y876, but <i>cdc55</i>Δ) were arrested at G1 with α-factor and released into a synchronous cell cycle. Time-lapse microscopy was performed as in (C) (n = 26 for WT; n = 25 for <i>cdc55</i>Δ). Scale bar, 2 µm.</p