RESUMO
BACKGROUND: Echinocandin resistance represents a great concern, as these drugs are recommended as first-line therapy for invasive candidiasis. Echinocandin resistance is conferred by mutations in FKS genes. Nevertheless, pathways are crucial for enabling tolerance, evolution, and maintenance of resistance. Therefore, understanding the biological processes and proteins involved in the response to caspofungin may provide clues indicating new therapeutic targets. OBJECTIVES: We determined the resistance mechanism and assessed the proteome response to caspofungin exposure. We then evaluated the phenotypic impact of calcineurin inhibition by FK506 and cephalosporine A (CsA) on caspofungin-resistant Candida glabrata isolates. METHODS: Twenty-five genes associated with caspofungin resistance were analysed by NGS, followed by studies of the quantitative proteomic response to caspofungin exposure. Then, susceptibility testing of caspofungin in presence of FK506 and CsA was performed. The effects of calcineurin inhibitor/caspofungin combinations on heat stress (40°C), oxidative stress (0.2 and 0.4 mM menadione) and on biofilm formation (polyurethane catheter) were analysed. Finally, a Galleria mellonella model using blastospores (1â×â109 cfu/mL) was developed to evaluate the impact of the combinations on larval survival. RESULTS: F659-del was found in the FKS2 gene of resistant strains. Proteomics data showed some up-regulated proteins are involved in cell-wall biosynthesis, response to stress and pathogenesis, some of them being members of calmodulin-calcineurin pathway. Therefore, the impact of calmodulin inhibition was explored. Calmodulin inhibition restored caspofungin susceptibility, decreased capacity to respond to stress conditions, and reduced biofilm formation and in vivo pathogenicity. CONCLUSIONS: Our findings confirm that calmodulin-calcineurin-Crz1 could provide a relevant target in life-threatening invasive candidiasis.
Assuntos
Candidíase Invasiva , Equinocandinas , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Inibidores de Calcineurina/farmacologia , Inibidores de Calcineurina/uso terapêutico , Candida glabrata , Candidíase Invasiva/tratamento farmacológico , Caspofungina/farmacologia , Caspofungina/uso terapêutico , Farmacorresistência Fúngica/genética , Equinocandinas/farmacologia , Equinocandinas/uso terapêutico , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Testes de Sensibilidade Microbiana , ProteômicaRESUMO
Macrophages are involved in the primary human response to Candida albicans. After pathogen recognition, signaling pathways are activated, leading to the production of cytokines, chemokines, and antimicrobial peptides. ATP binding proteins are crucial for this regulation. Here, a quantitative proteomic and phosphoproteomic approach was carried out for the study of human macrophage ATP-binding proteins after interaction with C. albicans. From a total of 547 nonredundant quantified proteins, 137 were ATP binding proteins and 59 were detected as differentially abundant. From the differentially abundant ATP-binding proteins, 6 were kinases (MAP2K2, SYK, STK3, MAP3K2, NDKA, and SRPK1), most of them involved in signaling pathways. Furthermore, 85 phosphopeptides were quantified. Macrophage proteomic alterations including an increase of protein synthesis with a consistent decrease in proteolysis were observed. Besides, macrophages showed changes in proteins of endosomal trafficking together with mitochondrial proteins, including some involved in the response to oxidative stress. Regarding cell death mechanisms, an increase of antiapoptotic over pro-apoptotic signals is suggested. Furthermore, a high pro-inflammatory response was detected, together with no upregulation of key mi-RNAs involved in the negative feedback of this response. These findings illustrate a strategy to deepen the knowledge of the complex interactions between the host and the clinically important pathogen C. albicans.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Candida albicans/crescimento & desenvolvimento , Proteínas de Transporte/genética , Interações Hospedeiro-Patógeno , Proteínas Mitocondriais/genética , Fosfoproteínas/genética , Trifosfato de Adenosina/imunologia , Trifosfato de Adenosina/metabolismo , Proteínas Reguladoras de Apoptose/classificação , Proteínas Reguladoras de Apoptose/imunologia , Candida albicans/patogenicidade , Proteínas de Transporte/classificação , Proteínas de Transporte/imunologia , Morte Celular/genética , Morte Celular/imunologia , Retroalimentação Fisiológica , Humanos , Marcação por Isótopo , Proteínas Mitocondriais/classificação , Proteínas Mitocondriais/imunologia , Fagocitose/imunologia , Fosfopeptídeos/química , Fosfopeptídeos/isolamento & purificação , Fosfoproteínas/classificação , Fosfoproteínas/imunologia , Biossíntese de Proteínas , Mapeamento de Interação de Proteínas , Proteômica/métodos , Transdução de Sinais , Células THP-1RESUMO
Currently, 14% of the human proteome is made up of proteins whose existence is not confirmed by mass spectrometry. We performed a proteomic profiling of human mesenchymal stem cells derived from adipose tissue or umbilical cord (PRIDE accession number: PXD009893) and identified peptides derived from 13 of such missing proteins. Remarkably, we found compelling evidence of the expression of hyaluronan synthase 1 (NX_Q92839-1) and confirmed its identification by the fragmentation of four heavy-labeled peptides that coeluted with their endogenous light counterparts. Our data also suggest that mesenchymal stem cells constitute a promising source for the detection of missing proteins.
Assuntos
Tecido Adiposo/citologia , Hialuronan Sintases/isolamento & purificação , Células-Tronco Mesenquimais/química , Cordão Umbilical/citologia , Humanos , Peptídeos/análise , Proteoma/análiseRESUMO
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 Candida albicans. 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 Candida-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 C. albicans infection and in macrophages communication.
Assuntos
Candida albicans/patogenicidade , Vesículas Extracelulares/imunologia , Interações Hospedeiro-Patógeno/imunologia , Macrófagos/imunologia , Proteoma/imunologia , Candida albicans/crescimento & desenvolvimento , Diferenciação Celular , Linhagem Celular , Biologia Computacional , Citocinas/genética , Citocinas/imunologia , Citoesqueleto/imunologia , Citoesqueleto/microbiologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , Vesículas Extracelulares/química , Vesículas Extracelulares/microbiologia , Regulação da Expressão Gênica/imunologia , Ontologia Genética , Humanos , Macrófagos/microbiologia , Anotação de Sequência Molecular , Proteoma/genética , Transdução de Sinais , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
Candida albicans 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 C. albicans proteome during commensalism, commensalism followed by infection, and infection (C, C+I, and I models, respectively). Serum anti-C. albicans 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 C. albicans 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. C. albicans 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.
Assuntos
Anticorpos Antifúngicos/sangue , Candida albicans/imunologia , Candidíase/imunologia , Proteínas Fúngicas/imunologia , Interações Hospedeiro-Patógeno , Imunoglobulina G/sangue , Animais , Candida albicans/crescimento & desenvolvimento , Candidíase/microbiologia , Candidíase/mortalidade , Feminino , Proteínas Fúngicas/genética , Microbioma Gastrointestinal/imunologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Análise de Sobrevida , Simbiose/imunologiaRESUMO
Macrophages may induce fungal apoptosis to fight against C. albicans, as previously hypothesized by our group. To confirm this hypothesis, we analyzed proteins from C. albicans cells after 3 h of interaction with macrophages using two quantitative proteomic approaches. A total of 51 and 97 proteins were identified as differentially expressed by DIGE and iTRAQ, respectively. The proteins identified and quantified were different, with only seven in common, but classified in the same functional categories. The analyses of their functions indicated that an increase in the metabolism of amino acids and purine nucleotides were taking place, while the glycolysis and translation levels dropped after 3 h of interaction. Also, the response to oxidative stress and protein translation were reduced. In addition, seven substrates of metacaspase (Mca1) were identified (Cdc48, Fba1, Gpm1, Pmm1, Rct1, Ssb1, and Tal1) as decreased in abundance, plus 12 proteins previously described as related to apoptosis. Besides, the monitoring of apoptotic markers along 24 h of interaction (caspase-like activity, TUNEL assay, and the measurement of ROS and cell examination by transmission electron microscopy) revealed that apoptotic processes took place for 30% of the fungal cells, thus supporting the proteomic results and the hypothesis of macrophages killing C. albicans by apoptosis.
Assuntos
Apoptose/imunologia , Candida albicans/citologia , Macrófagos/química , Animais , Biomarcadores/análise , Regulação Fúngica da Expressão Gênica/imunologia , Interações Hospedeiro-Patógeno/imunologia , Macrófagos/imunologia , Macrófagos/microbiologia , Camundongos , Proteômica/métodosRESUMO
Exit from mitosis in budding yeast is triggered by activation of the key mitotic phosphatase Cdc14. At anaphase onset, the protease separase and Zds1 promote the downregulation of PP2A(Cdc55) phosphatase, which facilitates Cdk1-dependent phosphorylation of Net1 and provides the first wave of Cdc14 activity. Once Cdk1 activity starts to decline, the mitotic exit network (MEN) is activated to achieve full Cdc14 activation. Here we describe how the PP2A(Cdc55) phosphatase could act as a functional link between FEAR and MEN due to its action on Bfa1 and Mob1. We demonstrate that PP2A(Cdc55) regulates MEN activation by facilitating Cdc5- and Cdk1-dependent phosphorylation of Bfa1 and Mob1, respectively. Downregulation of PP2A(Cdc55) initiates MEN activity up to Cdc15 by Bfa1 inactivation. Surprisingly, the premature Bfa1 inactivation observed does not entail premature MEN activation, since an additional Cdk1-Clb2 inhibitory signal acting towards Dbf2-Mob1 activity restrains MEN activity until anaphase. In conclusion, we propose a clear picture of how PP2A(Cdc55) functions affect the regulation of various MEN components, contributing to mitotic exit.
Assuntos
Anáfase/genética , Proteínas de Ciclo Celular/genética , Mitose/genética , Proteína Fosfatase 2/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Proteína Quinase CDC2/genética , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas do Citoesqueleto/antagonistas & inibidores , Regulação Fúngica da Expressão Gênica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteína Fosfatase 2/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , RNA Interferente Pequeno , Proteínas de Saccharomyces cerevisiae/antagonistas & inibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Separase/genéticaRESUMO
Candida albicans secretes numerous proteins related to cell wall remodeling, adhesion, nutrient acquisition and host interactions. Also, extracellular vesicles containing cytoplasmic proteins are secreted into the medium. The C. albicans ecm33/ecm33 mutant (RML2U) presents an altered cell wall and is avirulent. The proteomic analysis of proteins secreted by RML2U cells identified a total of 170 proteins: 114 and 154 of which correspond to the vesicle-free secretome and extracellular vesicles, respectively. Notably, 98 proteins were common to both samples, and the groups most represented were metabolic and cell wall-related proteins. The results of this study showed that RML2U had an altered pattern of proteins secreted by the classical secretion pathway as well as the formation of extracellular vesicles, including their size, quantity, and protein composition. Specifically, the secretion of aspartic protease 2 (Sap2) was compromised but not its intracellular expression, with bovine serum albumin (BSA) degradation by RML2U being altered when BSA was used as the sole nitrogen source. Furthermore, as recent research links the expression of Sap2 to the TOR (Target Of Rapamycin) signaling pathway, the sensitivity of RML2U to rapamycin (the inhibitor of TOR kinase) was tested and found to be enhanced, connecting Ecm33 with this pathway.
Assuntos
Ácido Aspártico Endopeptidases/genética , Candida albicans/química , Parede Celular/química , Proteínas Fúngicas/isolamento & purificação , Regulação Fúngica da Expressão Gênica , Proteínas de Membrana/genética , Animais , Antifúngicos/farmacologia , Ácido Aspártico Endopeptidases/metabolismo , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/metabolismo , Bovinos , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Vesículas Extracelulares/química , Vesículas Extracelulares/efeitos dos fármacos , Vesículas Extracelulares/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Proteínas de Membrana/metabolismo , Mutação , Proteólise , Proteômica/métodos , Soroalbumina Bovina/química , Sirolimo/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismoRESUMO
The commensal fungus Candida albicans 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 C. albicans and a comparative proteomic study of the soluble secreted proteins. With this purpose, cell-free culture supernatants from C. albicans 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 C. albicans 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.
Assuntos
Candida albicans/metabolismo , Vesículas Extracelulares/metabolismo , Proteínas Fúngicas/metabolismo , Proteoma/metabolismo , Animais , Candida albicans/imunologia , Candidíase/imunologia , Candidíase/microbiologia , Candidíase/prevenção & controle , Citoplasma/metabolismo , Feminino , Proteínas Fúngicas/imunologia , Vacinas Fúngicas/imunologia , Camundongos Endogâmicos BALB C , Proteoma/imunologia , Proteômica , Espectrometria de Massas em Tandem , VacinaçãoRESUMO
Approximately 18% of all human genes purported to encode proteins have not been directly evidenced at the protein level, according to the validation criteria established by neXtProt, and are considered to be "missing" proteins. One of the goals of the Chromosome-Centric Human Proteome Project (C-HPP) is to identify as many of these missing proteins as possible in human samples using mass spectrometry-based methods. To further this goal, a consortium of C-HPP teams (chromosomes 5, 10, 16, and 19) has joined forces to devise new strategies to identify missing proteins by use of a cell-free in vitro transcription/translation system (IVTT). The proposed strategy employs LC-MS/MS data-dependent acquisition (DDA) and targeted selective reaction monitoring (SRM) methods to scrutinize low-complexity samples derived from IVTT. The optimized assays are then applied to identify missing proteins in human cells and tissues. We describe the approach and show proof-of-concept results for development of LC-SRM assays for identification of 18 missing proteins. We believe that the IVTT system, when coupled with downstream mass spectrometric identification, can be applied to identify proteins that have eluded more traditional methods of detection.
Assuntos
Biossíntese de Proteínas , Proteoma , Transcrição Gênica , Cromatografia Líquida , Técnicas In Vitro , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Saccharomyces cerevisiae multicellular communities are sustained by a scaffolding extracellular matrix, which provides spatial organization, and nutrient and water availability, and ensures group survival. According to this tissue-like biology, the yeast extracellular matrix (yECM) is analogous to the higher Eukaryotes counterpart for its polysaccharide and proteinaceous nature. Few works focused on yeast biofilms, identifying the flocculin Flo11 and several members of the HSP70 in the extracellular space. Molecular composition of the yECM, is therefore mostly unknown. The homologue of yeast Gup1 protein in high Eukaryotes (HHATL) acts as a regulator of Hedgehog signal secretion, therefore interfering in morphogenesis and cell-cell communication through the ECM, which mediates but is also regulated by this signalling pathway. In yeast, the deletion of GUP1 was associated with a vast number of diverse phenotypes including the cellular differentiation that accompanies biofilm formation. METHODS: S. cerevisiae W303-1A wt strain and gup1∆ mutant were used as previously described to generate biofilm-like mats in YPDa from which the yECM proteome was extracted. The proteome from extracellular medium from batch liquid growing cultures was used as control for yECM-only secreted proteins. Proteins were separated by SDS-PAGE and 2DE. Identification was performed by HPLC, LC-MS/MS and MALDI-TOF/TOF. The protein expression comparison between the two strains was done by DIGE, and analysed by DeCyder Extended Data Analysis that included Principal Component Analysis and Hierarchical Cluster Analysis. RESULTS: The proteome of S. cerevisiae yECM from biofilm-like mats was purified and analysed by Nano LC-MS/MS, 2D Difference Gel Electrophoresis (DIGE), and MALDI-TOF/TOF. Two strains were compared, wild type and the mutant defective in GUP1. As controls for the identification of the yECM-only proteins, the proteome from liquid batch cultures was also identified. Proteins were grouped into distinct functional classes, mostly Metabolism, Protein Fate/Remodelling and Cell Rescue and Defence mechanisms, standing out the presence of heat shock chaperones, metalloproteinases, broad signalling cross-talkers and other putative signalling proteins. The data has been deposited to the ProteomeXchange with identifier PXD001133. CONCLUSIONS: yECM, as the mammalian counterpart, emerges as highly proteinaceous. As in higher Eukaryotes ECM, numerous proteins that could allow dynamic remodelling, and signalling events to occur in/and via yECM were identified. Importantly, large sets of enzymes encompassing full antagonistic metabolic pathways, suggest that mats develop into two metabolically distinct populations, suggesting that either extensive moonlighting or actual metabolism occurs extracellularly. The gup1∆ showed abnormally loose ECM texture. Accordingly, the correspondent differences in proteome unveiled acetic and citric acid producing enzymes as putative players in structural integrity maintenance.
Assuntos
Matriz Extracelular/química , Proteoma/análise , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Biofilmes/crescimento & desenvolvimento , Cromatografia Líquida de Alta Pressão , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Deleção de Genes , Proteínas de Membrana Transportadoras/deficiência , Proteômica , Saccharomyces cerevisiae/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
The cell wall integrity (CWI) pathway of the model organism Saccharomyces cerevisiae has been thoroughly studied as a paradigm of the mitogen-activated protein kinase (MAPK) pathway. It consists of a classic MAPK module comprising the Bck1 MAPK kinase kinase, two redundant MAPK kinases (Mkk1 and Mkk2), and the Slt2 MAPK. This module is activated under a variety of stimuli related to cell wall homeostasis by Pkc1, the only member of the protein kinase C family in budding yeast. Quantitative phosphoproteomics based on stable isotope labeling of amino acids in cell culture is a powerful tool for globally studying protein phosphorylation. Here we report an analysis of the yeast phosphoproteome upon overexpression of a PKC1 hyperactive allele that specifically activates CWI MAPK signaling in the absence of external stimuli. We found 82 phosphopeptides originating from 43 proteins that showed enhanced phosphorylation in these conditions. The MAPK S/T-P target motif was significantly overrepresented in these phosphopeptides. Hyperphosphorylated proteins provide putative novel targets of the Pkc1-cell wall integrity pathway involved in diverse functions such as the control of gene expression, protein synthesis, cytoskeleton maintenance, DNA repair, and metabolism. Remarkably, five components of the plasma-membrane-associated protein complex known as eisosomes were found among the up-regulated proteins. We show here that Pkc1-induced phosphorylation of the eisosome core components Pil1 and Lsp1 was not exerted directly by Pkc1, but involved signaling through the Slt2 MAPK module.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosfoproteínas/metabolismo , Proteína Quinase C/metabolismo , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Sítios de Ligação/genética , Western Blotting , Membrana Celular/metabolismo , Parede Celular/metabolismo , Espectrometria de Massas , Microscopia de Fluorescência , Proteínas Quinases Ativadas por Mitógeno/genética , Dados de Sequência Molecular , Mutação , Fosfopeptídeos/metabolismo , Fosfoproteínas/genética , Fosforilação , Proteína Quinase C/genética , Proteoma/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Serina/genética , Serina/metabolismo , Treonina/genética , Treonina/metabolismoRESUMO
In response to different stimuli, macrophages can differentiate into either a pro-inflammatory subtype (M1, classically activated macrophages) or acquire an anti-inflammatory phenotype (M2, alternatively activated macrophages). Candida albicans is the most important opportunistic fungus in nosocomial infections, and it is contended by neutrophils and macrophages during the first steps of the invasive infection. Murine macrophages responses to C. albicans have been widely studied, whereas the responses of human-polarized macrophages remain less characterized. In this study, we have characterized the proteomic differences between human M1- and M2-polarized macrophages, both in basal conditions and in response to C. albicans, by quantitative proteomics (2DE). This proteomic approach allowed us to identify metabolic routes and cytoskeletal rearrangement components that are the most relevant differences between M1 and M2 macrophages. The analysis has revealed fructose-1,6-bisphosphatase 1, a critical enzyme in gluconeogenesis, up-regulated in M1, as a novel protein marker for macrophage polarization. Regarding the response to C. albicans, an M1-to-M2 switch in polarization was observed. This M1-to-M2 switch might contribute to Candida pathogenicity by decreasing the generation of specific immune responses, thus enhancing fungal survival and colonization, or instead, may be part of the host attempt to reduce the inflammation and limit the damage of the infection.
Assuntos
Anti-Inflamatórios/metabolismo , Biomarcadores/análise , Candida albicans/metabolismo , Candidíase/metabolismo , Inflamação/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiologia , Western Blotting , Candida albicans/patogenicidade , Candidíase/imunologia , Candidíase/microbiologia , Células Cultivadas , Citocinas/genética , Citocinas/metabolismo , Eletroforese em Gel Bidimensional , Ensaio de Imunoadsorção Enzimática , Humanos , Inflamação/imunologia , Inflamação/microbiologia , Macrófagos/imunologia , Microscopia de Fluorescência , Fagocitose , Proteômica , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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 Candida albicans 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 C. albicans Hsp90 and Eno1, if confirmed prospectively, may be useful for IC diagnosis in non-neutropenic patients.
Assuntos
Anticorpos Antifúngicos/sangue , Candida albicans/patogenicidade , Candidíase Invasiva/diagnóstico , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas de Choque Térmico HSP90/imunologia , Fosfopiruvato Hidratase/imunologia , Adulto , Idoso , Biomarcadores/sangue , Proteínas Sanguíneas/análise , Candida albicans/imunologia , Candidíase Invasiva/microbiologia , Estudos de Casos e Controles , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Proteínas de Choque Térmico HSP90/genética , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Neutropenia/microbiologia , Proteômica/métodos , Reprodutibilidade dos TestesRESUMO
The combination of stable isotope labeling (SIL) with mass spectrometry (MS) allows comparison of the abundance of thousands of proteins in complex mixtures. However, interpretation of the large data sets generated by these techniques remains a challenge because appropriate statistical standards are lacking. Here, we present a generally applicable model that accurately explains the behavior of data obtained using current SIL approaches, including (18)O, iTRAQ, and SILAC labeling, and different MS instruments. The model decomposes the total technical variance into the spectral, peptide, and protein variance components, and its general validity was demonstrated by confronting 48 experimental distributions against 18 different null hypotheses. In addition to its general applicability, the performance of the algorithm was at least similar than that of other existing methods. The model also provides a general framework to integrate quantitative and error information fully, allowing a comparative analysis of the results obtained from different SIL experiments. The model was applied to the global analysis of protein alterations induced by low H2O2 concentrations in yeast, demonstrating the increased statistical power that may be achieved by rigorous data integration. Our results highlight the importance of establishing an adequate and validated statistical framework for the analysis of high-throughput data.
Assuntos
Modelos Estatísticos , Proteoma/análise , Proteômica/métodos , Proteínas de Saccharomyces cerevisiae/análise , Saccharomyces cerevisiae/genética , Mineração de Dados , Expressão Gênica/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Marcação por Isótopo , Anotação de Sequência Molecular , Isótopos de Oxigênio , Proteoma/genética , Proteoma/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The Spanish team of the Human Proteome Project (SpHPP) marked the annotation of Chr16 and data analysis as one of its priorities. Precise annotation of Chromosome 16 proteins according to C-HPP criteria is presented. Moreover, Human Body Map 2.0 RNA-Seq and Encyclopedia of DNA Elements (ENCODE) data sets were used to obtain further information relative to cell/tissue specific chromosome 16 coding gene expression patterns and to infer the presence of missing proteins. Twenty-four shotgun 2D-LC-MS/MS and gel/LC-MS/MS MIAPE compliant experiments, representing 41% coverage of chromosome 16 proteins, were performed. Furthermore, mapping of large-scale multicenter mass spectrometry data sets from CCD18, MCF7, Jurkat, and Ramos cell lines into RNA-Seq data allowed further insights relative to correlation of chromosome 16 transcripts and proteins. Detection and quantification of chromosome 16 proteins in biological matrices by SRM procedures are also primary goals of the SpHPP. Two strategies were undertaken: one focused on known proteins, taking advantage of MS data already available, and the second, aimed at the detection of the missing proteins, is based on the expression of recombinant proteins to gather MS information and optimize SRM methods that will be used in real biological samples. SRM methods for 49 known proteins and for recombinant forms of 24 missing proteins are reported in this study.
Assuntos
Cromossomos Humanos Par 16 , Proteoma , Transcriptoma , Cromatografia Líquida , Humanos , Espectrometria de Massas , Análise de Sequência de RNARESUMO
BACKGROUND: In a multicellular organism, the extracellular matrix (ECM) provides a cell-supporting scaffold and helps maintaining the biophysical integrity of tissues and organs. At the same time it plays crucial roles in cellular communication and signalling, with implications in spatial organisation, motility and differentiation. Similarly, the presence of an ECM-like extracellular polymeric substance is known to support and protect bacterial and fungal multicellular aggregates, such as biofilms or colonies. However, the roles and composition of this microbial ECM are still poorly understood. RESULTS: This work presents a protocol to produce S. cerevisiae and C. albicans ECM in an equally highly reproducible manner. Additionally, methodologies for the extraction and fractionation into protein and glycosidic analytical pure fractions were improved. These were subjected to analytical procedures, respectively SDS-PAGE, 2-DE, MALDI-TOF-MS and LC-MS/MS, and DAE and FPLC. Additional chemical methods were also used to test for uronic acids and sulphation. CONCLUSIONS: The methodologies hereby presented were equally efficiently applied to extract high amounts of ECM material from S. cerevisiae and C. albicans mats, therefore showing their robustness and reproducibility for yECM molecular and structural characterization. yECM from S. cerevisiae and C. albicans displayed a different proteome and glycoside fractions. S. cerevisiae yECM presented two well-defined polysaccharides with different mass/charge, and C. albicans ECM presented a single different one. The chemical methods further suggested the presence of uronic acids, and chemical modification, possibly through sulphate substitution. All taken, the procedures herein described present the first sensible and concise approach to the molecular and chemical characterisation of the yeast ECM, opening the way to the in-depth study of the microbe multicellular aggregates structure and life-style.
Assuntos
Proteínas Fúngicas/análise , Proteínas Fúngicas/isolamento & purificação , Glicômica/métodos , Polissacarídeos/análise , Polissacarídeos/isolamento & purificação , Proteômica/métodos , Candida albicans/química , Técnicas de Química Analítica/métodos , Matriz Extracelular/química , Reprodutibilidade dos Testes , Saccharomyces cerevisiae/químicaRESUMO
Candida albicans Prn1 is a protein with an unknown function similar to mammalian Pirin. It also has orthologues in other pathogenic fungi, but not in Saccharomyces cerevisiae. Prn1 highly increases its abundance in response to H2O2 treatment; thus, to study its involvement in the oxidative stress response, a C. albicans prn1∆ mutant and the corresponding wild-type strain SN250 have been studied. Under H2O2 treatment, Prn1 absence led to a higher level of reactive oxygen species (ROS) and a lower survival rate, with a higher percentage of death by apoptosis, confirming its relevant role in oxidative detoxication. The quantitative differential proteomics studies of both strains in the presence and absence of H2O2 indicated a lower increase in proteins with oxidoreductase activity after the treatment in the prn1∆ strain, as well as an increase in proteasome-activating proteins, corroborated by in vivo measurements of proteasome activity, with respect to the wild type. In addition, remarkable differences in the abundance of some transcription factors were observed between mutant and wild-type strains, e.g., Mnl1 or Nrg1, an Mnl1 antagonist. orf19.4850, a protein orthologue to S. cerevisiae Cub1, has shown its involvement in the response to H2O2 and in proteasome function when Prn1 is highly expressed in the wild type.
RESUMO
Better prognostic predictors for invasive candidiasis (IC) are needed to tailor and individualize therapeutic decision-making and minimize its high morbidity and mortality. We investigated whether molecular profiling of IgG-antibody response to the whole soluble Candida proteome could reveal a prognostic signature that may serve to devise a clinical-outcome prediction model for IC and contribute to known IC prognostic factors. By serological proteome analysis and data-mining procedures, serum 31-IgG antibody-reactivity patterns were examined in 45 IC patients randomly split into training and test sets. Within the training cohort, unsupervised two-way hierarchical clustering and principal-component analyses segregated IC patients into two antibody-reactivity subgroups with distinct prognoses that were unbiased by traditional IC prognostic factors and other patients-related variables. Supervised discriminant analysis with leave-one-out cross-validation identified a five-IgG antibody-reactivity signature as the most simplified and accurate IC clinical-outcome predictor, from which an IC prognosis score (ICPS) was derived. Its robustness was confirmed in the test set. Multivariate logistic-regression and receiver-operating-characteristic curve analyses demonstrated that the ICPS was able to accurately discriminate IC patients at high risk for death from those at low risk and outperformed conventional IC prognostic factors. Further validation of the five-IgG antibody-reactivity signature on a multiplexed immunoassay supported the serological proteome analysis results. The five IgG antibodies incorporated in the ICPS made biologic sense and were associated either with good-prognosis and protective patterns (those to Met6p, Hsp90p, and Pgk1p, putative Candida virulence factors and antiapoptotic mediators) or with poor-prognosis and risk patterns (those to Ssb1p and Gap1p/Tdh3p, potential Candida proapoptotic mediators). We conclude that the ICPS, with additional refinement in future larger prospective cohorts, could be applicable to reliably predict patient clinical-outcome for individualized therapy of IC. Our data further provide insights into molecular mechanisms that may influence clinical outcome in IC and uncover potential targets for vaccine design and immunotherapy against IC.
Assuntos
Anticorpos Antifúngicos/sangue , Anticorpos Antifúngicos/imunologia , Candida/imunologia , Candidíase Invasiva/sangue , Candidíase Invasiva/imunologia , Mapeamento de Peptídeos/métodos , Adulto , Idoso , Candidíase Invasiva/terapia , Análise por Conglomerados , Estudos de Coortes , Feminino , Humanos , Imunoensaio , Imunoglobulina G/imunologia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , Prognóstico , Reprodutibilidade dos Testes , Solubilidade , Resultado do TratamentoRESUMO
In this chapter, detailed procedures for stable isotope labeling with amino acids in cell culture, SILAC labeling of yeast auxotroph, optimization and evaluation of phosphopeptide enrichment, and sample preparation and analysis by high-resolution LC-MS/M, identification of phosphosites, and quantification methods are described.We report methods for the application of double SILAC to yeast using a combination of labeled lysine and labeled arginine.The combination of SILAC-based quantitation with phosphopeptides enrichment by TiO2 in a batch that enables measurement of protein posttranslational modifications is a powerful application to analyze the global phosphoproteome for studies in signaling pathways.