RESUMO
Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens.
Assuntos
Candida albicans , Saccharomyces cerevisiae , Animais , Camundongos , Humanos , Virulência , Saccharomyces cerevisiae/genética , Prolina/metabolismo , Candida , MamíferosRESUMO
The peptide toxin candidalysin, secreted by Candida albicans hyphae, promotes stimulation of neutrophil extracellular traps (NETs). However, candidalysin alone triggers a distinct mechanism for NET-like structures (NLS), which are more compact and less fibrous than canonical NETs. Candidalysin activates NADPH oxidase and calcium influx, with both processes contributing to morphological changes in neutrophils resulting in NLS formation. NLS are induced by leucotoxic hypercitrullination, which is governed by calcium-induced protein arginine deaminase 4 activation and initiation of intracellular signalling events in a dose- and time-dependent manner. However, activation of signalling by candidalysin does not suffice to trigger downstream events essential for NET formation, as demonstrated by lack of lamin A/C phosphorylation, an event required for activation of cyclin-dependent kinases that are crucial for NET release. Candidalysin-triggered NLS demonstrate anti-Candida activity, which is resistant to nuclease treatment and dependent on the deprivation of Zn2+ . This study reveals that C. albicans hyphae releasing candidalysin concurrently trigger canonical NETs and NLS, which together form a fibrous sticky network that entangles C. albicans hyphae and efficiently inhibits their growth.
Assuntos
Candida albicans , Armadilhas Extracelulares , Candida albicans/metabolismo , Armadilhas Extracelulares/metabolismo , Cálcio/metabolismo , Proteínas Fúngicas/metabolismoRESUMO
Fungal infections are a continuously increasing problem in modern health care. Understanding the complex biology of the emerging pathogens and unraveling the mechanisms of host defense may form the basis for the development of more efficient diagnostic and therapeutic tools. Neutrophils play a pivotal role in the defense against fungal pathogens. These phagocytic hunters migrate towards invading fungal microorganisms and eradicate them by phagocytosis, oxidative burst and release of neutrophil extracellular traps (NETs). In the last decade, the process of NET formation has received unparalleled attention, with numerous studies revealing the relevance of this neutrophil function for control of various mycoses. Here, we describe NET formation and summarize its role as part of the innate immune defense against fungal pathogens. We highlight factors influencing the formation of these structures and molecular mechanisms employed by fungi to impair the formation of NETs or subvert their antifungal effects.
Assuntos
Armadilhas Extracelulares/imunologia , Micoses/imunologia , Neutrófilos/imunologia , Fagocitose/imunologia , Antifúngicos/uso terapêutico , Armadilhas Extracelulares/microbiologia , Fungos/imunologia , Fungos/patogenicidade , Humanos , Micoses/microbiologia , Neutrófilos/microbiologia , Espécies Reativas de Oxigênio/imunologiaRESUMO
Nutritional immunity describes the host-driven manipulation of essential micronutrients, including iron, zinc and manganese. To withstand nutritional immunity and proliferate within their hosts, pathogenic microbes must express efficient micronutrient uptake and homeostatic systems. Here we have elucidated the pathway of cellular zinc assimilation in the major human fungal pathogen Candida albicans. Bioinformatics analysis identified nine putative zinc transporters: four cytoplasmic-import Zip proteins (Zrt1, Zrt2, Zrt3 and orf19.5428) and five cytoplasmic-export ZnT proteins (orf19.1536/Zrc1, orf19.3874, orf19.3769, orf19.3132 and orf19.52). Only Zrt1 and Zrt2 are predicted to localise to the plasma membrane and here we demonstrate that Zrt2 is essential for C. albicans zinc uptake and growth at acidic pH. In contrast, ZRT1 expression was found to be highly pH-dependent and could support growth of the ZRT2-null strain at pH 7 and above. This regulatory paradigm is analogous to the distantly related pathogenic mould, Aspergillus fumigatus, suggesting that pH-adaptation of zinc transport may be conserved in fungi and we propose that environmental pH has shaped the evolution of zinc import systems in fungi. Deletion of C. albicans ZRT2 reduced kidney fungal burden in wild type, but not in mice lacking the zinc-chelating antimicrobial protein calprotectin. Inhibition of zrt2Δ growth by neutrophil extracellular traps was calprotectin-dependent. This suggests that, within the kidney, C. albicans growth is determined by pathogen-Zrt2 and host-calprotectin. As well as serving as an essential micronutrient, zinc can also be highly toxic and we show that C. albicans deals with this potential threat by rapidly compartmentalising zinc within vesicular stores called zincosomes. In order to understand mechanistically how this process occurs, we created deletion mutants of all five ZnT-type transporters in C. albicans. Here we show that, unlike in Saccharomyces cerevisiae, C. albicans Zrc1 mediates zinc tolerance via zincosomal zinc compartmentalisation. This novel transporter was also essential for virulence and liver colonisation in vivo. In summary, we show that zinc homeostasis in a major human fungal pathogen is a multi-stage process initiated by Zrt1/Zrt2-cellular import, followed by Zrc1-dependent intracellular compartmentalisation.
Assuntos
Candida albicans/metabolismo , Candida albicans/patogenicidade , Zinco/metabolismo , Adaptação Fisiológica , Animais , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Calgranulina B/genética , Calgranulina B/metabolismo , Candida albicans/genética , Candidíase Invasiva/metabolismo , Candidíase Invasiva/microbiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Compartimento Celular , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Genes Fúngicos , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Concentração de Íons de Hidrogênio , Complexo Antígeno L1 Leucocitário/genética , Complexo Antígeno L1 Leucocitário/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Virulência/genética , Virulência/fisiologia , Zinco/toxicidadeRESUMO
BACKGROUND: Advanced cancer causes necrosis and releases damage-associated molecular patterns (DAMPs). Mitochondrial DAMPs activate neutrophils, including generation of neutrophil extracellular traps (NETs), which are injurious, thrombogenic, and implicated in metastasis. We hypothesised that extracellular mitochondrial DNA (mtDNA) in ascites from patients with epithelial ovarian cancer (EOC) would correlate with worse outcomes. METHODS: Banked ascites supernatants from patients with newly diagnosed advanced EOC were analysed for mtDNA, neutrophil elastase, and activation of healthy donor neutrophils and platelets. TCGA was mined for expression of SELP and ELANE. RESULTS: The highest quartile of ascites mtDNA correlated with reduced progression-free survival (PFS) and a higher likelihood of disease progression within 12-months following primary surgery (n = 68, log-rank, p = 0.0178). NETs were detected in resected tumours. Ascites supernatants chemoattracted neutrophils, induced NETs, and activated platelets. Ascites exposure rendered neutrophils suppressive, based on abrogation of ex vivo stimulated T cell proliferation. Increased SELP mRNA expression correlated with worse overall survival (n = 302, Cox model, p = 0.02). CONCLUSION: In this single-centre retrospective analysis, ascites mtDNA correlated with worse PFS in advanced EOC. Mitochondrial and other DAMPs in ascites may activate neutrophil and platelet responses that facilitate metastasis and obstruct anti-tumour immunity. These pathways are potential prognostic markers and therapeutic targets.
Assuntos
Alarminas/genética , Carcinoma Epitelial do Ovário/genética , DNA Mitocondrial/genética , Armadilhas Extracelulares/genética , Idoso , Ascite/genética , Ascite/patologia , Plaquetas/metabolismo , Carcinoma Epitelial do Ovário/patologia , Armadilhas Extracelulares/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Elastase de Leucócito/genética , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Neutrófilos/metabolismo , Neutrófilos/patologia , Intervalo Livre de Progressão , Microambiente Tumoral/genéticaRESUMO
BACKGROUND: Neutrophils are traditionally considered transcriptionally inactive. Compared to other immune cells, little is known about their transcriptional profile during interaction with pathogens. METHODS: We analyzed the meta-transcriptome of the neutrophil-Candida albicans interplay and the transcriptome of C. albicans challenged with neutrophil extracellular traps (NETs) by RNA-Seq, considering yeast and hypha individually in each approach. RESULTS: The neutrophil response to C. albicans yeast and hyphae was dominated by a morphotype-independent core response. However, 11 % of all differentially expressed genes were regulated in a specific manner when neutrophils encountered the hyphal form of C. albicans. While involving genes for transcriptional regulators, receptors, and cytokines, the neutrophil core response lacked typical antimicrobial effectors genes. Genes of the NOD-like receptor pathway, including NLRP3, were enriched. Neutrophil- and NET-provoked responses in C. albicans differed. At the same time, the Candida transcriptome upon neutrophil encounter and upon NET challenge included genes from various metabolic processes and indicate a mutual role of the regulators Tup1p, Efg1p, Hap43p, and Cap1p. Upon challenge with neutrophils and NETs, the overall Candida response was partially morphotype-specific. Yet again, actual oppositional regulation in yeasts and hyphae was only detected for the arginine metabolism in neutrophil-infecting C. albicans. CONCLUSIONS: Taken together, our study provides a comprehensive and quantitative transcript profile of the neutrophil-C. albicans interaction. By considering the two major appearances of both, neutrophils and C. albicans, our study reveals yet undescribed insights into this medically relevant encounter. Hence, our findings will facilitate future research and potentially inspire novel therapy developments.
Assuntos
Candida albicans/genética , Candida albicans/imunologia , Perfilação da Expressão Gênica , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Arginina/metabolismo , Candida albicans/fisiologia , Citocinas/genética , Citoesqueleto/metabolismo , Hifas/genética , Neutrófilos/citologia , Neutrófilos/imunologia , Transdução de Sinais/genética , Estresse Fisiológico/genética , Açúcares/metabolismoRESUMO
Plasmacytoid dendritic cells (pDCs) were initially considered as critical for innate immunity to viruses. However, our group has shown that pDCs bind to and inhibit the growth of Aspergillus fumigatus hyphae and that depletion of pDCs renders mice hypersusceptible to experimental aspergillosis. In this study, we examined pDC receptors contributing to hyphal recognition and downstream events in pDCs stimulated by A. fumigatus hyphae. Our data show that Dectin-2, but not Dectin-1, participates in A. fumigatus hyphal recognition, TNF-α and IFN-α release, and antifungal activity. Moreover, Dectin-2 acts in cooperation with the FcRγ chain to trigger signaling responses. In addition, using confocal and electron microscopy we demonstrated that the interaction between pDCs and A. fumigatus induced the formation of pDC extracellular traps (pETs) containing DNA and citrullinated histone H3. These structures closely resembled those of neutrophil extracellular traps (NETs). The microarray analysis of the pDC transcriptome upon A. fumigatus infection also demonstrated up-regulated expression of genes associated with apoptosis as well as type I interferon-induced genes. Thus, human pDCs directly recognize A. fumigatus hyphae via Dectin-2; this interaction results in cytokine release and antifungal activity. Moreover, hyphal stimulation of pDCs triggers a distinct pattern of pDC gene expression and leads to pET formation.
Assuntos
Aspergilose/imunologia , Células Dendríticas/imunologia , Armadilhas Extracelulares/imunologia , Lectinas Tipo C/imunologia , Aspergilose/genética , Aspergillus fumigatus/imunologia , Células Cultivadas , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/imunologia , Humanos , Hifas/imunologia , Microscopia Confocal , Microscopia Eletrônica de Varredura , Análise de Sequência com Séries de OligonucleotídeosRESUMO
The Gram negative bacterium Vibrio cholerae is the causative agent of the secretory diarrheal disease cholera, which has traditionally been classified as a noninflammatory disease. However, several recent reports suggest that a V. cholerae infection induces an inflammatory response in the gastrointestinal tract indicated by recruitment of innate immune cells and increase of inflammatory cytokines. In this study, we describe a colonization defect of a double extracellular nuclease V. cholerae mutant in immunocompetent mice, which is not evident in neutropenic mice. Intrigued by this observation, we investigated the impact of neutrophils, as a central part of the innate immune system, on the pathogen V. cholerae in more detail. Our results demonstrate that V. cholerae induces formation of neutrophil extracellular traps (NETs) upon contact with neutrophils, while V. cholerae in return induces the two extracellular nucleases upon presence of NETs. We show that the V. cholerae wild type rapidly degrades the DNA component of the NETs by the combined activity of the two extracellular nucleases Dns and Xds. In contrast, NETs exhibit prolonged stability in presence of the double nuclease mutant. Finally, we demonstrate that Dns and Xds mediate evasion of V. cholerae from NETs and lower the susceptibility for extracellular killing in the presence of NETs. This report provides a first comprehensive characterization of the interplay between neutrophils and V. cholerae along with new evidence that the innate immune response impacts the colonization of V. cholerae in vivo. A limitation of this study is an inability for technical and physiological reasons to visualize intact NETs in the intestinal lumen of infected mice, but we can hypothesize that extracellular nuclease production by V. cholerae may enhance survival fitness of the pathogen through NET degradation.
Assuntos
Proteínas de Bactérias , Cólera , Desoxirribonucleases , Viabilidade Microbiana , Neutrófilos , Vibrio cholerae , Animais , Feminino , Humanos , Masculino , Camundongos , Proteínas de Bactérias/genética , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Cólera/enzimologia , Cólera/genética , Cólera/imunologia , Cólera/patologia , Desoxirribonucleases/genética , Desoxirribonucleases/imunologia , Desoxirribonucleases/metabolismo , Imunidade Inata/genética , Camundongos Knockout , Neutrófilos/imunologia , Neutrófilos/metabolismo , Neutrófilos/patologia , Vibrio cholerae/enzimologia , Vibrio cholerae/genética , Vibrio cholerae/imunologiaRESUMO
Chronic granulomatous disease, an inherited disorder of the NADPH oxidase in which phagocytes are defective in the generation of superoxide anion and downstream reactive oxidant species, is characterized by severe bacterial and fungal infections and excessive inflammation. Although NADPH oxidase isoforms exist in several lineages, reactive oxidant generation is greatest in neutrophils, where NADPH oxidase has been deemed vital for pathogen killing. In contrast, the function and importance of NADPH oxidase in macrophages are less clear. Therefore, we evaluated susceptibility to pulmonary aspergillosis in globally NADPH oxidase-deficient mice versus transgenic mice with monocyte/macrophage-targeted NADPH oxidase activity. We found that the lethal inoculum was >100-fold greater in transgenic versus globally NADPH oxidase-deficient mice. Consistent with these in vivo results, NADPH oxidase in mouse alveolar macrophages limited germination of phagocytosed Aspergillus fumigatus spores. Finally, globally NADPH oxidase-deficient mice developed exuberant neutrophilic lung inflammation and proinflammatory cytokine responses to zymosan, a fungal cell wall-derived product composed principally of particulate ß-glucans, whereas inflammation in transgenic and wild-type mice was mild and transient. Taken together, our studies identify a central role for monocyte/macrophage NADPH oxidase in controlling fungal infection and in limiting acute lung inflammation.
Assuntos
Aspergillus fumigatus/imunologia , Macrófagos Alveolares/enzimologia , Macrófagos Alveolares/imunologia , Monócitos/enzimologia , Monócitos/imunologia , NADPH Oxidases/fisiologia , Doença Aguda , Animais , Aspergilose/enzimologia , Aspergilose/imunologia , Aspergilose/patologia , Predisposição Genética para Doença , Inflamação/enzimologia , Inflamação/microbiologia , Inflamação/prevenção & controle , Pulmão/enzimologia , Pulmão/imunologia , Pulmão/microbiologia , Macrófagos Alveolares/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Monócitos/microbiologia , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Zimosan/farmacologiaRESUMO
NADPH oxidase is a crucial enzyme in antimicrobial host defense and in regulating inflammation. Chronic granulomatous disease (CGD) is an inherited disorder of NADPH oxidase in which phagocytes are defective in generation of reactive oxidant intermediates. Aspergillus species are ubiquitous, filamentous fungi, which can cause invasive aspergillosis, a major cause of morbidity and mortality in CGD, reflecting the critical role for NADPH oxidase in antifungal host defense. Activation of NADPH oxidase in neutrophils can be coupled to the release of proteins and chromatin that comingle in neutrophil extracellular traps (NETs), which can augment extracellular antimicrobial host defense. NETosis can be driven by NADPH oxidase-dependent and -independent pathways. We therefore undertook an analysis of whether NADPH oxidase was required for NETosis in Aspergillus fumigatus pneumonia. Oropharyngeal instillation of live Aspergillus hyphae induced neutrophilic pneumonitis in both wild-type and NADPH oxidase-deficient (p47(phox-/-)) mice which had resolved in wild-type mice by day 5 but progressed in p47(phox-/-) mice. NETs, identified by immunostaining, were observed in lungs of wild-type mice but were absent in p47(phox-/-) mice. Using bona fide NETs and nuclear chromatin decondensation as an early NETosis marker, we found that NETosis required a functional NADPH oxidase in vivo and ex vivo. In addition, NADPH oxidase increased the proportion of apoptotic neutrophils. Together, our results show that NADPH oxidase is required for pulmonary clearance of Aspergillus hyphae and generation of NETs in vivo. We speculate that dual modulation of NETosis and apoptosis by NADPH oxidase enhances antifungal host defense and promotes resolution of inflammation upon infection clearance.
Assuntos
Regulação Enzimológica da Expressão Gênica/imunologia , NADPH Oxidases/metabolismo , Neutrófilos/fisiologia , Aspergilose Pulmonar/enzimologia , Animais , Aspergillus fumigatus/fisiologia , Hifas/fisiologia , Inflamação , Camundongos , Camundongos Knockout , Alvéolos Pulmonares/citologiaRESUMO
Candida species are the cause of 60% of all mycoses in immunosuppressed individuals, leading to â¼150,000 deaths annually due to systemic infections, whereas the current antifungal therapies either have toxic side effects or are insufficiently efficient. We performed a screening of two compound libraries, the Enzo and the Institute for Molecular Medicine Finland (FIMM) oncology collection library, for anti-Candida activity based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) guidelines. From a total of 844 drugs, 26 agents showed activity against Candida albicans. Of those, 12 were standard antifungal drugs (SADs) and 7 were off-target drugs previously reported to be active against Candida spp. The remaining 7 off-target drugs, amonafide, tosedostat, megestrol acetate, melengestrol acetate, stanozolol, trifluperidol, and haloperidol, were identified with this screen. The anti-Candida activities of the new agents were investigated by three individual assays using optical density, ATP levels, and microscopy. The antifungal activities of these drugs were comparable to those of the SADs found in the screen. The aminopeptidase inhibitor tosedostat, which is currently in a clinical trial phase for anticancer therapy, displayed a broad antifungal activity against different Candida spp., including Candida glabrata. Thus, this screen reveals agents that were previously unknown to be anti-Candida agents, which allows for the design of novel therapies against invasive candidiasis.
Assuntos
Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Reposicionamento de Medicamentos , Glicina/análogos & derivados , Ácidos Hidroxâmicos/farmacologia , Bibliotecas de Moléculas Pequenas/farmacologia , Antifúngicos/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Candida/crescimento & desenvolvimento , Ensaios Clínicos como Assunto , Descoberta de Drogas , Farmacorresistência Fúngica , Glicina/química , Glicina/farmacologia , Ensaios de Triagem em Larga Escala , Humanos , Ácidos Hidroxâmicos/química , Testes de Sensibilidade Microbiana , Bibliotecas de Moléculas Pequenas/químicaRESUMO
Klebsiella (K.) pneumoniae is a common cause of pneumonia-derived sepsis. Myeloid related protein 8 (MRP8, S100A8) and MRP14 (S100A9) are the most abundant cytoplasmic proteins in neutrophils. They can form MRP8/14 heterodimers that are released upon cell stress stimuli. MRP8/14 reportedly exerts antimicrobial activity, but in acute fulminant sepsis models MRP8/14 has been found to contribute to organ damage and death. We here determined the role of MRP8/14 in K. pneumoniae sepsis originating from the lungs, using an established model characterized by gradual growth of bacteria with subsequent dissemination. Infection resulted in gradually increasing MRP8/14 levels in lungs and plasma. Mrp14 deficient (mrp14(-/-)) mice, unable to form MRP8/14 heterodimers, showed enhanced bacterial dissemination accompanied by increased organ damage and a reduced survival. Mrp14(-/-) macrophages were reduced in their capacity to phagocytose Klebsiella. In addition, recombinant MRP8/14 heterodimers, but not MRP8 or MRP14 alone, prevented growth of Klebsiella in vitro through chelation of divalent cations. Neutrophil extracellular traps (NETs) prepared from wildtype but not from mrp14(-/-) neutrophils inhibited Klebsiella growth; in accordance, the capacity of human NETs to kill Klebsiella was strongly impaired by an anti-MRP14 antibody or the addition of zinc. These results identify MRP8/14 as key player in protective innate immunity during Klebsiella pneumonia.
Assuntos
Calgranulina B/imunologia , Infecções por Klebsiella/imunologia , Klebsiella pneumoniae/imunologia , Neutrófilos/imunologia , Pneumonia Bacteriana/imunologia , Sepse/imunologia , Transportadores de Cassetes de Ligação de ATP/imunologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Calgranulina B/genética , Calgranulina B/metabolismo , Linhagem Celular , Humanos , Infecções por Klebsiella/microbiologia , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/microbiologia , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fagocitose , Pneumonia Bacteriana/microbiologia , Sepse/microbiologiaRESUMO
Candida albicans belongs to our commensal mucosal flora and in immune-competent individuals in the absence of epithelial damage, this fungus is well tolerated and controlled by our immune defense. However, C. albicans is an opportunistic microorganism that can cause different forms of infections, ranging from superficial to life-threatening systemic infections. C. albicans is polymorphic and switches between different phenotypes (e.g. from yeast form to hyphal form). C. albicans hyphae are invasive and can grow into tissues to eventually reach circulation. During fungal infections, neutrophils in particular play a critical role for the defense, but how neutrophils are directed toward the invasive forms of fungi is less well understood. We set out to investigate possible neutrophil chemoattractants released by C. albicans into culture supernatants. We found that cell-free culture supernatants from the hyphal form of C. albicans induced both neutrophil chemotaxis and concomitant intracellular calcium transients. Size separation and hydrophobic sorting of supernatants indicated small hydrophilic factors as responsible for the activity. Further analysis showed that the culture supernatants contained high levels of short-chain fatty acids with higher levels from hyphae as compared to yeast. Short-chain fatty acids are known neutrophil chemoattractants acting via the neutrophil free fatty acid receptor 2. In line with this, the calcium signaling in neutrophils induced by hyphae culture supernatants was blocked by a free fatty acid receptor 2 antagonist and potently increased in the presence of a positive allosteric modulator. Our data imply that short-chain fatty acids may act as a recruitment signal whereby neutrophils can detect C. albicans hyphae.
Assuntos
Candida albicans , Neutrófilos , Humanos , Ácidos Graxos não Esterificados/análise , Hifas/química , Hifas/genética , Quimiotaxia , Ácidos Graxos Voláteis/análise , Fatores QuimiotáticosRESUMO
The enteropathogen Yersinia pseudotuberculosis can survive in the harsh environment of lymphoid compartments that abounds in immune cells. This capacity is dependent on the plasmid-encoded Yersinia outer proteins (Yops) that are delivered into the host cell via a mechanism involving the Yersinia type III secretion system. We show that the virulence protein YopK has a role in the mechanism by which Y. pseudotuberculosis avoids the polymorphonuclear leukocyte or neutrophil (PMN) defense. A yopK mutant, which is attenuated in the mouse infection model, where it fails to cause systemic infection, was found to colonize Peyer's patches and mesenteric lymph nodes more rapidly than the wild-type strain. Further, in mice lacking PMNs, the yopK mutant caused full disease with systemic spread and typical symptoms. Analyses of effects on PMNs revealed that both the wild-type strain and the yopK mutant inhibited internalization and reactive oxygen species production, as well as neutrophil extracellular trap formation by PMNs. However, the wild-type strain effectively avoided induction of PMN death, whereas the mutant caused a necrosis-like PMN death. Taken together, our results indicate that YopK is required for the ability of Yersinia to resist the PMN defense, which is critical for the virulence of the pathogen. We suggest a mechanism whereby YopK functions to prevent unintended Yop delivery and thereby PMN disruption, resulting in necrosis-like cell death, which would enhance the inflammatory response favoring the host.
Assuntos
Proteínas da Membrana Bacteriana Externa/imunologia , Neutrófilos/imunologia , Infecções por Yersinia pseudotuberculosis/imunologia , Yersinia pseudotuberculosis/imunologia , Animais , Morte Celular/imunologia , Feminino , Humanos , Linfonodos/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Necrose/imunologia , Nódulos Linfáticos Agregados/imunologia , Espécies Reativas de Oxigênio/imunologia , Infecções por Yersinia pseudotuberculosis/sangueRESUMO
BACKGROUND: Aspergillus spp infection is a potentially lethal disease in patients with neutropenia or impaired neutrophil function. We showed previously that Aspergillus hyphae, too large for neutrophil phagocytosis, are inhibited by reactive oxygen species-dependent neutrophil extracellular trap (NET) formation. This process is defective in chronic granulomatous disease (CGD) because of impaired phagocyte nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function. OBJECTIVE: To determine the antifungal agent and mechanism responsible for reconstitution of Aspergillus growth inhibition within NETs after complementation of NADPH oxidase function by gene therapy (GT) for CGD. METHODS: Antifungal activity of free and NET-released calprotectin was assessed by incubation of Aspergillus nidulans with purified calprotectin, induced NETs from human controls, and CGD neutrophils after GT in the presence or absence of Zn(2+) or α-S100A9 antibody, and with induced NETs from wild-type or S100A9(-/-) mouse neutrophils. RESULTS: We identified the host Zn(2+) chelator calprotectin as a neutrophil-associated antifungal agent expressed within NETs, reversibly preventing A nidulans growth at low concentrations, and leading to irreversible fungal starvation at higher concentrations. Specific antibody-blocking and Zn(2+) addition abolished calprotectin-mediated inhibition of A nidulans proliferation in vitro. The role of calprotectin in anti-Aspergillus defense was confirmed in calprotectin knockout mice. CONCLUSION: Reconstituted NET formation by GT for human CGD was associated with rapid cure of pre-existing therapy-refractory invasive pulmonary aspergillosis in vivo, underlining the role of functional NADPH oxidase in NET formation and calprotectin release for antifungal activity. These results demonstrate the critical role of calprotectin in human innate immune defense against Aspergillus infection.
Assuntos
Aspergilose/imunologia , Aspergillus nidulans/efeitos dos fármacos , Terapia Genética/métodos , Doença Granulomatosa Crônica/terapia , Complexo Antígeno L1 Leucocitário/metabolismo , Neutrófilos/imunologia , Animais , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Aspergilose/microbiologia , Criança , Doença Granulomatosa Crônica/complicações , Doença Granulomatosa Crônica/imunologia , Humanos , Complexo Antígeno L1 Leucocitário/farmacologia , Masculino , Camundongos , NADPH Oxidases/genética , NADPH Oxidases/metabolismo , Neutrófilos/metabolismo , Neutrófilos/fisiologia , Espécies Reativas de Oxigênio/metabolismoRESUMO
Neutrophils are the first line of defense at the site of an infection. They encounter and kill microbes intracellularly upon phagocytosis or extracellularly by degranulation of antimicrobial proteins and the release of Neutrophil Extracellular Traps (NETs). NETs were shown to ensnare and kill microbes. However, their complete protein composition and the antimicrobial mechanism are not well understood. Using a proteomic approach, we identified 24 NET-associated proteins. Quantitative analysis of these proteins and high resolution electron microscopy showed that NETs consist of modified nucleosomes and a stringent selection of other proteins. In contrast to previous results, we found several NET proteins that are cytoplasmic in unstimulated neutrophils. We demonstrated that of those proteins, the antimicrobial heterodimer calprotectin is released in NETs as the major antifungal component. Absence of calprotectin in NETs resulted in complete loss of antifungal activity in vitro. Analysis of three different Candida albicans in vivo infection models indicated that NET formation is a hitherto unrecognized route of calprotectin release. By comparing wild-type and calprotectin-deficient animals we found that calprotectin is crucial for the clearance of infection. Taken together, the present investigations confirmed the antifungal activity of calprotectin in vitro and, moreover, demonstrated that it contributes to effective host defense against C. albicans in vivo. We showed for the first time that a proportion of calprotectin is bound to NETs in vitro and in vivo.
Assuntos
Candida albicans/imunologia , Complexo Antígeno L1 Leucocitário/imunologia , Neutrófilos/imunologia , Abscesso Abdominal/imunologia , Abscesso Abdominal/microbiologia , Análise de Variância , Animais , Antifúngicos/química , Antifúngicos/metabolismo , Células Cultivadas , Estruturas Celulares/química , Estruturas Celulares/imunologia , Estruturas Celulares/ultraestrutura , Histonas/química , Histonas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Imunidade Inata , Imuno-Histoquímica , Complexo Antígeno L1 Leucocitário/química , Complexo Antígeno L1 Leucocitário/metabolismo , Pneumopatias Fúngicas/imunologia , Pneumopatias Fúngicas/microbiologia , Camundongos , Camundongos Knockout , Ativação de NeutrófiloRESUMO
Interaction between microorganisms and their surroundings are generally mediated via the cell wall or cell envelope. An understanding of the overall chemical composition of these surface layers may give clues on how these interactions occur and suggest mechanisms to manipulate them. This knowledge is key, for instance, in research aiming to reduce colonization of medical devices and device-related infections from different types of microorganisms. In this context, X-ray photoelectron spectroscopy (XPS) is a powerful technique as its analysis depth below 10 nm enables studies of the outermost surface structures of microorganism. Of specific interest for the study of biological systems is cryogenic XPS (cryo-XPS). This technique allows studies of intact fast-frozen hydrated samples without the need for pre-treatment procedures that may cause the cell structure to collapse or change due to the loss of water. Previously, cryo-XPS has been applied to study bacterial and algal surfaces with respect to their composition of lipids, polysaccharides and peptide (protein and/or peptidoglycan). This contribution focuses onto two other groups of microorganisms with widely different architecture and modes of life, namely fungi and viruses. It evaluates to what extent existing models for data treatment of XPS spectra can be applied to understand the chemical composition of their very different surface layers. XPS data from model organisms as well as reference substances representing specific building blocks of their surface were collected and are presented. These results aims to guide future analysis of the surface chemical composition of biological systems.
RESUMO
The Phagocytosis of fungal structures by neutrophils is a well-documented function of these immune cells. However, neutrophil phagocytosis of hyphal structures in the urine sediment is not usually observed during routine sample evaluation. This is a case of hyphal phagocytosis by neutrophils in the urine of a kidney allograft recipient patient.
Assuntos
Hifas , Neutrófilos , Humanos , FagocitoseRESUMO
Intra-abdominal infection (peritonitis) is a leading cause of severe disease in surgical intensive care units, as over 70% of patients diagnosed with peritonitis develop septic shock. A critical role of the immune system is to return to homeostasis after combating infection. S100A8/A9 (calprotectin) is an antimicrobial and pro-inflammatory protein complex used as a biomarker for diagnosis of numerous inflammatory disorders. Here we describe the role of S100A8/A9 in inflammatory collateral tissue damage (ICTD). Using a mouse model of disseminated intra-abdominal candidiasis (IAC) in wild-type and S100A8/A9-deficient mice in the presence or absence of S100A9 inhibitor paquinimod, the role of S100A8/A9 during ICTD and fungal clearance were investigated. S100A8/A9-deficient mice developed less ICTD than wild-type mice. Restoration of S100A8/A9 in knockout mice by injection of recombinant protein resulted in increased ICTD and fungal clearance comparable to wild-type levels. Treatment with paquinimod abolished ICTD and S100A9-deficient mice showed increased survival compared to wild-type littermates. The data indicates that S100A8/A9 controls ICTD levels and antimicrobial activity during IAC and that targeting of S100A8/A9 could serve as promising adjunct therapy against this challenging disease.