RESUMO
Malassezia are the dominant commensal yeast species of the human skin microbiota and are associated with inflammatory skin diseases, such as atopic eczema (AE). The Mala s 1 allergen of Malassezia sympodialis is a ß-propeller protein, inducing both IgE and T-cell reactivity in AE patients. We demonstrate by immuno-electron microscopy that Mala s 1 is mainly located in the M. sympodialis yeast cell wall. An anti-Mala s 1 antibody did not inhibit M. sympodialis growth suggesting Mala s 1 may not be an antifungal target. In silico analysis of the predicted Mala s 1 protein sequence identified a motif indicative of a KELCH protein, a subgroup of ß-propeller proteins. To test the hypothesis that antibodies against Mala s 1 cross-react with human skin (KELCH) proteins we examined the binding of the anti-Mala s 1 antibody to human skin explants and visualized binding in the epidermal skin layer. Putative human targets recognized by the anti-Mala s 1 antibody were identified by immunoblotting and proteomics. We propose that Mala s 1 is a KELCH-like ß-propeller protein with similarity to human skin proteins. Mala s 1 recognition may trigger cross-reactive responses that contribute to skin diseases associated with M. sympodialis.
Assuntos
Dermatite Atópica , Malassezia , Humanos , Alérgenos , Dermatite Atópica/microbiologia , Sequência de AminoácidosRESUMO
Monoclonal antibody (mAb)-based immunotherapies targeting systemic and deep-seated fungal infections are still in their early stages of development, with no licensed antifungal mAbs currently being available for patients at risk. The cell wall glycoproteins of Candida albicans are of particular interest as potential targets for therapeutic antibody generation due to their extracellular location and key involvement in fungal pathogenesis. Here, we describe the generation of recombinant human antibodies specifically targeting two key cell wall proteins (CWPs) in C. albicans: Utr2 and Pga31. These antibodies were isolated from a phage display antibody library using peptide antigens representing the surface-exposed regions of CWPs expressed at elevated levels during in vivo infection. Reformatted human-mouse chimeric mAbs preferentially recognized C. albicans hyphal forms compared to yeast cells, and increased binding was observed when the cells were grown in the presence of the antifungal agent caspofungin. In J774.1 macrophage interaction assays, mAb pretreatment resulted in the faster engulfment of C. albicans cells, suggesting a role of the CWP antibodies as opsonizing agents during phagocyte recruitment. Finally, in a series of clinically predictive mouse models of systemic candidiasis, our lead mAb achieved improved survival (83%) and a several-log reduction of the fungal burden in the kidneys, similar to the levels achieved for the fungicidal drug caspofungin and superior to the therapeutic efficacy of any anti-Candida mAb reported to date.
Assuntos
Anticorpos Monoclonais , Candida albicans , Animais , Anticorpos Antifúngicos , Anticorpos Monoclonais/farmacologia , Antifúngicos/farmacologia , Antígenos de Fungos , Caspofungina , Parede Celular , Epitopos , Humanos , CamundongosRESUMO
An increasing number of outbreaks due to resistant non-albicans Candida species have been reported worldwide. Between 2014 and 2016, Candida isolates causing invasive candidiasis were recovered in a Mexican hospital. Isolates were identified to species level and antifungal susceptibility was determined. In the time period studied, 74 invasive candidiasis cases were identified, with 38% (28/74) caused by Candida parapsilosis, out of which 54% (15/28) were fluconazole resistant. The ERG11 gene was sequenced for 12 recoverable fluconazole-resistant C. parapsilosis isolates and SNPs identified. The 12 isolates had one common silent point mutation in ERG11 (T591C) and seven isolates had an additional (A395T) mutation, which corresponded to Y132F. Four of the isolates carrying this mutation were closely related within the same cluster by microsatellite typing. This is the first report of an invasive candidiasis outbreak in Mexico due to azole-resistant C. parapsilosis associated with the Y132F substitution.
Assuntos
Antifúngicos/farmacologia , Azóis/farmacologia , Candida parapsilosis/efeitos dos fármacos , Candida parapsilosis/genética , Candidíase Invasiva/epidemiologia , Surtos de Doenças/estatística & dados numéricos , Proteínas Fúngicas/genética , Mutação/efeitos dos fármacos , Adulto , Substituição de Aminoácidos , Farmacorresistência Fúngica , Feminino , Hospitais Gerais/estatística & dados numéricos , Humanos , Masculino , México , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Mutação/genética , Estudos RetrospectivosRESUMO
Fungal cells change shape in response to environmental stimuli, and these morphogenic transitions drive pathogenesis and niche adaptation. For example, dimorphic fungi switch between yeast and hyphae in response to changing temperature. The basidiomycete Cryptococcus neoformans undergoes an unusual morphogenetic transition in the host lung from haploid yeast to large, highly polyploid cells termed Titan cells. Titan cells influence fungal interaction with host cells, including through increased drug resistance, altered cell size, and altered Pathogen Associated Molecular Pattern exposure. Despite the important role these cells play in pathogenesis, understanding the environmental stimuli that drive the morphological transition, and the molecular mechanisms underlying their unique biology, has been hampered by the lack of a reproducible in vitro induction system. Here we demonstrate reproducible in vitro Titan cell induction in response to environmental stimuli consistent with the host lung. In vitro Titan cells exhibit all the properties of in vivo generated Titan cells, the current gold standard, including altered capsule, cell wall, size, high mother cell ploidy, and aneuploid progeny. We identify the bacterial peptidoglycan subunit Muramyl Dipeptide as a serum compound associated with shift in cell size and ploidy, and demonstrate the capacity of bronchial lavage fluid and bacterial co-culture to induce Titanisation. Additionally, we demonstrate the capacity of our assay to identify established (cAMP/PKA) and previously undescribed (USV101) regulators of Titanisation in vitro. Finally, we investigate the Titanisation capacity of clinical isolates and their impact on disease outcome. Together, these findings provide new insight into the environmental stimuli and molecular mechanisms underlying the yeast-to-Titan transition and establish an essential in vitro model for the future characterization of this important morphotype.
Assuntos
Cryptococcus neoformans/citologia , Cryptococcus neoformans/patogenicidade , Animais , Criptococose/microbiologia , Cryptococcus neoformans/genética , AMP Cíclico/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas Fúngicas/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Hifas/citologia , Hifas/crescimento & desenvolvimento , Hifas/patogenicidade , Pulmão/microbiologia , Pneumopatias Fúngicas/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Modelos Biológicos , Morfogênese , Poliploidia , Fatores de Transcrição/metabolismo , VirulênciaRESUMO
Candida auris is an emerging pathogenic yeast of significant clinical concern because of its frequent intrinsic resistance to fluconazole and often other antifungal drugs and the high mortality rates associated with systemic infections. Furthermore, C. auris has a propensity for persistence and transmission in health care environments. The reasons for this efficient transmission are not well understood, and therefore we tested whether enhanced resistance to environmental stresses might contribute to the ability of C. auris to spread in health care environments. We compared C. auris to other pathogenic Candida species with respect to their resistance to individual stresses and combinations of stresses. Stress resistance was examined using in vitro assays on laboratory media and also on hospital linen. In general, the 17 C. auris isolates examined displayed similar degrees of resistance to oxidative, nitrosative, cationic and cell wall stresses as clinical isolates of C. albicans, C. glabrata, C. tropicalis, C. parapsilosis, C. krusei, C. guilliermondii, C. lusitaniae and C. kefyr. All of the C. auris isolates examined were more sensitive to low pH (pH 2, but not pH 4) compared to C. albicans, but were more resistant to high pH (pH 13). C. auris was also sensitive to low pH, when tested on contaminated hospital linen. Most C. auris isolates were relatively thermotolerant, displaying significant growth at 47°C. Furthermore, C. auris was relatively resistant to certain combinations of combinatorial stress (e.g., pH 13 plus 47°C). Significantly, C. auris was sensitive to the stress combinations imposed by hospital laundering protocol (pH > 12 plus heat shock at >80°C), suggesting that current laundering procedures are sufficient to limit the transmission of this fungal pathogen via hospital linen.
Assuntos
Candida/patogenicidade , Candidíase/transmissão , Meio Ambiente , Hospitais , Estresse Fisiológico , Antifúngicos/farmacologia , Roupas de Cama, Mesa e Banho/microbiologia , Candida/classificação , Candida/efeitos dos fármacos , Candidíase/microbiologia , Farmacorresistência Fúngica , Equipamentos e Provisões Hospitalares/microbiologia , Humanos , Concentração de Íons de Hidrogênio , Testes de Sensibilidade Microbiana , Estresse Nitrosativo , Estresse Oxidativo , TermotolerânciaRESUMO
Candida albicans is able to proliferate in environments that vary dramatically in ambient pH, a trait required for colonising niches such as the stomach, vaginal mucosal and the GI tract. Here we show that growth in acidic environments involves cell wall remodelling which results in enhanced chitin and ß-glucan exposure at the cell wall periphery. Unmasking of the underlying immuno-stimulatory ß-glucan in acidic environments enhanced innate immune recognition of C. albicans by macrophages and neutrophils, and induced a stronger proinflammatory cytokine response, driven through the C-type lectin-like receptor, Dectin-1. This enhanced inflammatory response resulted in significant recruitment of neutrophils in an intraperitoneal model of infection, a hallmark of symptomatic vaginal colonisation. Enhanced chitin exposure resulted from reduced expression of the cell wall chitinase Cht2, via a Bcr1-Rim101 dependent signalling cascade, while increased ß-glucan exposure was regulated via a non-canonical signalling pathway. We propose that this "unmasking" of the cell wall may induce non-protective hyper activation of the immune system during growth in acidic niches, and may attribute to symptomatic vaginal infection.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Parede Celular/imunologia , Animais , Candida albicans/fisiologia , Candidíase/microbiologia , Parede Celular/química , Humanos , Concentração de Íons de Hidrogênio , Imunidade Inata , Macrófagos/imunologia , Macrófagos/microbiologia , CamundongosRESUMO
The Ypd1 phosphorelay protein is a central constituent of fungal two-component signal transduction pathways. Inhibition of Ypd1 in Saccharomyces cerevisiae and Cryptococcus neoformans is lethal due to the sustained activation of the 'p38-related' Hog1 stress-activated protein kinase (SAPK). As two-component signalling proteins are not found in animals, Ypd1 is considered to be a prime antifungal target. However, a major fungal pathogen of humans, Candida albicans, can survive the concomitant sustained activation of Hog1 that occurs in cells lacking YPD1. Here we show that the sustained activation of Hog1 upon Ypd1 loss is mediated through the Ssk1 response regulator. Moreover, we present evidence that C. albicans survives SAPK activation in the short-term, following Ypd1 loss, by triggering the induction of protein tyrosine phosphatase-encoding genes which prevent the accumulation of lethal levels of phosphorylated Hog1. In addition, our studies reveal an unpredicted, reversible, mechanism that acts to substantially reduce the levels of phosphorylated Hog1 in ypd1Δ cells following long-term sustained SAPK activation. Indeed, over time, ypd1Δ cells become phenotypically indistinguishable from wild-type cells. Importantly, we also find that drug-induced down-regulation of YPD1 expression actually enhances the virulence of C. albicans in two distinct animal infection models. Investigating the underlying causes of this increased virulence, revealed that drug-mediated repression of YPD1 expression promotes hyphal growth both within murine kidneys, and following phagocytosis, thus increasing the efficacy by which C. albicans kills macrophages. Taken together, these findings challenge the targeting of Ypd1 proteins as a general antifungal strategy and reveal novel cellular adaptation mechanisms to sustained SAPK activation.
Assuntos
Candida albicans/fisiologia , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Sistema de Sinalização das MAP Quinases , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Animais , Candida albicans/enzimologia , Candida albicans/genética , Candida albicans/patogenicidade , Regulação para Baixo , Feminino , Proteínas Fúngicas/genética , Deleção de Genes , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Proteínas Quinases Ativadas por Mitógeno/genética , Modelos Biológicos , Fenótipo , Fosforilação , Estresse Fisiológico , VirulênciaRESUMO
Most fungal pathogens of humans display robust protective oxidative stress responses that contribute to their pathogenicity. The induction of enzymes that detoxify reactive oxygen species (ROS) is an essential component of these responses. We showed previously that ectopic expression of the heme-containing catalase enzyme in Candida albicans enhances resistance to oxidative stress, combinatorial oxidative plus cationic stress, and phagocytic killing. Clearly ectopic catalase expression confers fitness advantages in the presence of stress, and therefore in this study we tested whether it enhances fitness in the absence of stress. We addressed this using a set of congenic barcoded C. albicans strains that include doxycycline-conditional tetON-CAT1 expressors. We show that high basal catalase levels, rather than CAT1 induction following stress imposition, reduce ROS accumulation and cell death, thereby promoting resistance to acute peroxide or combinatorial stress. This conclusion is reinforced by our analyses of phenotypically diverse clinical isolates and the impact of stochastic variation in catalase expression upon stress resistance in genetically homogeneous C. albicans populations. Accordingly, cat1Δ cells are more sensitive to neutrophil killing. However, we find that catalase inactivation does not attenuate C. albicans virulence in mouse or invertebrate models of systemic candidiasis. Furthermore, our direct comparisons of fitness in vitro using isogenic barcoded CAT1, cat1Δ and tetON-CAT1 strains show that, while ectopic catalase expression confers a fitness advantage during peroxide stress, it confers a fitness defect in the absence of stress. This fitness defect is suppressed by iron supplementation. Also high basal catalase levels induce key iron assimilatory functions (CFL5, FET3, FRP1, FTR1). We conclude that while high basal catalase levels enhance peroxide stress resistance, they place pressure on iron homeostasis through an elevated cellular demand for iron, thereby reducing the fitness of C. albicans in iron-limiting tissues within the host.
Assuntos
Candida albicans/enzimologia , Candidíase/microbiologia , Catalase/metabolismo , Proteínas Fúngicas/metabolismo , Ferro/metabolismo , Animais , Candida albicans/genética , Candida albicans/metabolismo , Catalase/genética , Feminino , Proteínas Fúngicas/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Estresse OxidativoRESUMO
Filamentous growth is a hallmark of C. albicans pathogenicity compared to less-virulent ascomycetes. A multitude of transcription factors regulate filamentous growth in response to specific environmental cues. Our work, however, suggests the evolutionary history of C. albicans that resulted in its filamentous growth plasticity may be tied to a change in the general transcription machinery rather than transcription factors and their specific targets. A key genomic difference between C. albicans and its less-virulent relatives, including its closest relative C. dubliniensis, is the unique expansion of the TLO (TeLOmere-associated) gene family in C. albicans. Individual Tlo proteins are fungal-specific subunits of Mediator, a large multi-subunit eukaryotic transcriptional co-activator complex. This amplification results in a large pool of 'free,' non-Mediator associated, Tlo protein present in C. albicans, but not in C. dubliniensis or other ascomycetes with attenuated virulence. We show that engineering a large 'free' pool of the C. dubliniensis Tlo2 (CdTlo2) protein in C. dubliniensis, through overexpression, results in a number of filamentation phenotypes typically associated only with C. albicans. The amplitude of these phenotypes is proportional to the amount of overexpressed CdTlo2 protein. Overexpression of other C. dubliniensis and C. albicans Tlo proteins do result in these phenotypes. Tlo proteins and their orthologs contain a Mediator interaction domain, and a potent transcriptional activation domain. Nuclear localization of the CdTlo2 activation domain, facilitated naturally by the Tlo Mediator binding domain or artificially through an appended nuclear localization signal, is sufficient for the CdTlo2 overexpression phenotypes. A C. albicans med3 null mutant causes multiple defects including the inability to localize Tlo proteins to the nucleus and reduced virulence in a murine systemic infection model. Our data supports a model in which the activation domain of 'free' Tlo protein competes with DNA bound transcription factors for targets that regulate key aspects of C. albicans cell physiology.
Assuntos
Candida albicans/genética , Candidíase/genética , Proteínas de Ligação a Telômeros/genética , Telômero/genética , Animais , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Candidíase/microbiologia , Candidíase/patologia , Fungos/genética , Fungos/crescimento & desenvolvimento , Fungos/patogenicidade , Regulação Fúngica da Expressão Gênica , Genômica , Humanos , Hifas/genética , Hifas/crescimento & desenvolvimento , Hifas/patogenicidade , Camundongos , Fenótipo , Proteínas de Ligação a Telômeros/biossínteseRESUMO
Efficient carbon assimilation is critical for microbial growth and pathogenesis. The environmental yeast Saccharomyces cerevisiae is "Crabtree positive", displaying a rapid metabolic switch from the assimilation of alternative carbon sources to sugars. Following exposure to sugars, this switch is mediated by the transcriptional repression of genes (carbon catabolite repression) and the turnover (catabolite inactivation) of enzymes involved in the assimilation of alternative carbon sources. The pathogenic yeast Candida albicans is Crabtree negative. It has retained carbon catabolite repression mechanisms, but has undergone posttranscriptional rewiring such that gluconeogenic and glyoxylate cycle enzymes are not subject to ubiquitin-mediated catabolite inactivation. Consequently, when glucose becomes available, C. albicans can continue to assimilate alternative carbon sources alongside the glucose. We show that this metabolic flexibility promotes host colonization and virulence. The glyoxylate cycle enzyme isocitrate lyase (CaIcl1) was rendered sensitive to ubiquitin-mediated catabolite inactivation in C. albicans by addition of a ubiquitination site. This mutation, which inhibits lactate assimilation in the presence of glucose, reduces the ability of C. albicans cells to withstand macrophage killing, colonize the gastrointestinal tract and cause systemic infections in mice. Interestingly, most S. cerevisiae clinical isolates we examined (67%) have acquired the ability to assimilate lactate in the presence of glucose (i.e. they have become Crabtree negative). These S. cerevisiae strains are more resistant to macrophage killing than Crabtree positive clinical isolates. Moreover, Crabtree negative S. cerevisiae mutants that lack Gid8, a key component of the Glucose-Induced Degradation complex, are more resistant to macrophage killing and display increased virulence in immunocompromised mice. Thus, while Crabtree positivity might impart a fitness advantage for yeasts in environmental niches, the more flexible carbon assimilation strategies offered by Crabtree negativity enhance the ability of yeasts to colonize and infect the mammalian host.
Assuntos
Candida albicans/metabolismo , Candida albicans/patogenicidade , Candidíase/metabolismo , Macrófagos/microbiologia , Saccharomyces cerevisiae/metabolismo , Virulência/fisiologia , Animais , Western Blotting , Metabolismo dos Carboidratos , Linhagem Celular , Feminino , Camundongos , Camundongos Endogâmicos BALB C , UbiquitinaçãoRESUMO
Chitin is an essential structural polysaccharide of fungal pathogens and parasites, but its role in human immune responses remains largely unknown. It is the second most abundant polysaccharide in nature after cellulose and its derivatives today are widely used for medical and industrial purposes. We analysed the immunological properties of purified chitin particles derived from the opportunistic human fungal pathogen Candida albicans, which led to the selective secretion of the anti-inflammatory cytokine IL-10. We identified NOD2, TLR9 and the mannose receptor as essential fungal chitin-recognition receptors for the induction of this response. Chitin reduced LPS-induced inflammation in vivo and may therefore contribute to the resolution of the immune response once the pathogen has been defeated. Fungal chitin also induced eosinophilia in vivo, underpinning its ability to induce asthma. Polymorphisms in the identified chitin receptors, NOD2 and TLR9, predispose individuals to inflammatory conditions and dysregulated expression of chitinases and chitinase-like binding proteins, whose activity is essential to generate IL-10-inducing fungal chitin particles in vitro, have also been linked to inflammatory conditions and asthma. Chitin recognition is therefore critical for immune homeostasis and is likely to have a significant role in infectious and allergic disease.
Assuntos
Candida albicans/química , Quitina/imunologia , Interleucina-10/imunologia , Proteína Adaptadora de Sinalização NOD2/imunologia , Receptor Toll-Like 9/imunologia , Animais , Asma/genética , Asma/imunologia , Asma/patologia , Candida albicans/imunologia , Quitina/química , Feminino , Humanos , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Interleucina-10/genética , Masculino , Camundongos , Proteína Adaptadora de Sinalização NOD2/genética , Receptor Toll-Like 9/genéticaRESUMO
The ability of Candida albicans to cause disease is associated with its capacity to undergo morphological transition between yeast and filamentous forms, but the role of morphology in colonization and dissemination from the gastrointestinal (GI) tract remains poorly defined. To explore this, we made use of wild-type and morphological mutants of C. albicans in an established model of GI tract colonization, induced following antibiotic treatment of mice. Our data reveal that GI tract colonization favours the yeast form of C. albicans, that there is constitutive low level systemic dissemination in colonized mice that occurs irrespective of fungal morphology, and that colonization is not controlled by Th17 immunity in otherwise immunocompetent animals. These data provide new insights into the mechanisms of pathogenesis and commensalism of C. albicans, and have implications for our understanding of human disease.
Assuntos
Candida albicans/citologia , Candida albicans/fisiologia , Candidíase/imunologia , Candidíase/microbiologia , Trato Gastrointestinal/imunologia , Trato Gastrointestinal/microbiologia , Células Th17/imunologia , Animais , Candida albicans/crescimento & desenvolvimento , Candida albicans/imunologia , Modelos Animais de Doenças , CamundongosRESUMO
Candida albicans remains the fungus most frequently associated with nosocomial bloodstream infection. In disseminated candidiasis, the role of Foxp3(+) regulatory T (Treg) cells remains largely unexplored. Our aims were to characterize Foxp3(+) Treg-cell activation in a murine intravenous challenge model of disseminated C. albicans infection, and determine the contribution to disease. Flow cytometric analyses demonstrated that C. albicans infection drove in vivo expansion of a splenic CD4(+) Foxp3(+) population that correlated positively with fungal burden. Depletion from Foxp3(hCD2) reporter mice in vivo confirmed that Foxp3(+) cells exacerbated fungal burden and inflammatory renal disease. The CD4(+) Foxp3(+) population expanded further after in vitro stimulation with C. albicans antigens (Ags), and included at least three cell types. These arose from proliferation of the natural Treg-cell subset, together with conversion of Foxp3(-) cells to the induced Treg-cell form, and to a cell type sharing effector Th17-cell characteristics, expressing ROR-γt, and secreting IL-17A. The expanded Foxp3(+) T cells inhibited Th1 and Th2 responses, but enhanced Th17-cell responses to C. albicans Ags in vitro, and in vivo depletion confirmed their ability to enhance the Th17-cell response. These data lead to a model for disseminated candidiasis whereby expansion of Foxp3(+) T cells promotes Th17-cell responses that drive pathology.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Proliferação de Células , Fatores de Transcrição Forkhead/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologia , Animais , Candida albicans/fisiologia , Candidíase/microbiologia , Células Cultivadas , Citocinas/imunologia , Citocinas/metabolismo , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Subunidade alfa de Receptor de Interleucina-2/imunologia , Subunidade alfa de Receptor de Interleucina-2/metabolismo , Nefropatias/imunologia , Nefropatias/virologia , Camundongos , Camundongos Endogâmicos C57BL , Baço/imunologia , Baço/virologia , Linfócitos T Reguladores/metabolismo , Linfócitos T Reguladores/microbiologia , Células Th17/metabolismo , Células Th17/microbiologiaRESUMO
Nutritional immunity--the withholding of nutrients by the host--has long been recognised as an important factor that shapes bacterial-host interactions. However, the dynamics of nutrient availability within local host niches during fungal infection are poorly defined. We have combined laser ablation-inductively coupled plasma mass spectrometry (LA-ICP MS), MALDI imaging and immunohistochemistry with microtranscriptomics to examine iron homeostasis in the host and pathogen in the murine model of systemic candidiasis. Dramatic changes in the renal iron landscape occur during disease progression. The infection perturbs global iron homeostasis in the host leading to iron accumulation in the renal medulla. Paradoxically, this is accompanied by nutritional immunity in the renal cortex as iron exclusion zones emerge locally around fungal lesions. These exclusion zones correlate with immune infiltrates and haem oxygenase 1-expressing host cells. This local nutritional immunity decreases iron availability, leading to a switch in iron acquisition mechanisms within mature fungal lesions, as revealed by laser capture microdissection and qRT-PCR analyses. Therefore, a complex interplay of systemic and local events influences iron homeostasis and pathogen-host dynamics during disease progression.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Ferro/imunologia , Animais , Candida albicans/metabolismo , Candidíase/metabolismo , Candidíase/patologia , Modelos Animais de Doenças , Feminino , Ferro/metabolismo , Rim/imunologia , Rim/metabolismo , Rim/patologia , Camundongos , Camundongos Endogâmicos BALB CRESUMO
The fungal cell wall is the first point of interaction between an invading fungal pathogen and the host immune system. The outer layer of the cell wall is comprised of GPI anchored proteins, which are post-translationally modified by both N- and O-linked glycans. These glycans are important pathogen associated molecular patterns (PAMPs) recognised by the innate immune system. Glycan synthesis is mediated by a series of glycosyl transferases, located in the endoplasmic reticulum and Golgi apparatus. Mnn2 is responsible for the addition of the initial α1,2-mannose residue onto the α1,6-mannose backbone, forming the N-mannan outer chain branches. In Candida albicans, the MNN2 gene family is comprised of six members (MNN2, MNN21, MNN22, MNN23, MNN24 and MNN26). Using a series of single, double, triple, quintuple and sextuple mutants, we show, for the first time, that addition of α1,2-mannose is required for stabilisation of the α1,6-mannose backbone and hence regulates mannan fibril length. Sequential deletion of members of the MNN2 gene family resulted in the synthesis of lower molecular weight, less complex and more uniform N-glycans, with the sextuple mutant displaying only un-substituted α1,6-mannose. TEM images confirmed that the sextuple mutant was completely devoid of the outer mannan fibril layer, while deletion of two MNN2 orthologues resulted in short mannan fibrils. These changes in cell wall architecture correlated with decreased proinflammatory cytokine induction from monocytes and a decrease in fungal virulence in two animal models. Therefore, α1,2-mannose of N-mannan is important for both immune recognition and virulence of C. albicans.
Assuntos
Candida albicans/imunologia , Candida albicans/patogenicidade , Mananas/imunologia , Manose/metabolismo , Manosiltransferases/metabolismo , Glicoproteínas de Membrana/imunologia , Animais , Candida albicans/enzimologia , Candidíase/imunologia , Parede Celular/química , Parede Celular/imunologia , Feminino , Proteínas Fúngicas/genética , Proteínas Fúngicas/imunologia , Proteínas Fúngicas/metabolismo , Humanos , Mananas/química , Manose/química , Manosiltransferases/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Polissacarídeos/metabolismo , Receptores de Reconhecimento de Padrão/imunologia , Receptores de Reconhecimento de Padrão/metabolismo , Alinhamento de Sequência , Deleção de SequênciaRESUMO
The ß-glucan receptor Dectin-1 is a member of the C-type lectin family and functions as an innate pattern recognition receptor in antifungal immunity. In both mouse and man, Dectin-1 has been found to play an essential role in controlling infections with Candida albicans, a normally commensal fungus in man which can cause superficial mucocutaneous infections as well as life-threatening invasive diseases. Here, using in vivo models of infection, we show that the requirement for Dectin-1 in the control of systemic Candida albicans infections is fungal strain-specific; a phenotype that only becomes apparent during infection and cannot be recapitulated in vitro. Transcript analysis revealed that this differential requirement for Dectin-1 is due to variable adaptation of C. albicans strains in vivo, and that this results in substantial differences in the composition and nature of their cell walls. In particular, we established that differences in the levels of cell-wall chitin influence the role of Dectin-1, and that these effects can be modulated by antifungal drug treatment. Our results therefore provide substantial new insights into the interaction between C. albicans and the immune system and have significant implications for our understanding of susceptibility and treatment of human infections with this pathogen.
Assuntos
Antifúngicos/farmacologia , Candida albicans/imunologia , Parede Celular/efeitos dos fármacos , Lectinas Tipo C/imunologia , Animais , Candida albicans/genética , Caspofungina , Parede Celular/química , Quitina/metabolismo , Equinocandinas/farmacologia , Lectinas Tipo C/genética , Lipopeptídeos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Reconhecimento de Padrão/imunologia , beta-Glucanas/metabolismoRESUMO
The survival of all microbes depends upon their ability to respond to environmental challenges. To establish infection, pathogens such as Candida albicans must mount effective stress responses to counter host defences while adapting to dynamic changes in nutrient status within host niches. Studies of C. albicans stress adaptation have generally been performed on glucose-grown cells, leaving the effects of alternative carbon sources upon stress resistance largely unexplored. We have shown that growth on alternative carbon sources, such as lactate, strongly influence the resistance of C. albicans to antifungal drugs, osmotic and cell wall stresses. Similar trends were observed in clinical isolates and other pathogenic Candida species. The increased stress resistance of C. albicans was not dependent on key stress (Hog1) and cell integrity (Mkc1) signalling pathways. Instead, increased stress resistance was promoted by major changes in the architecture and biophysical properties of the cell wall. Glucose- and lactate-grown cells displayed significant differences in cell wall mass, ultrastructure, elasticity and adhesion. Changes in carbon source also altered the virulence of C. albicans in models of systemic candidiasis and vaginitis, confirming the importance of alternative carbon sources within host niches during C. albicans infections.
Assuntos
Candida albicans/metabolismo , Candida albicans/patogenicidade , Carbono/metabolismo , Parede Celular/metabolismo , Farmacorresistência Fúngica , Interações Hospedeiro-Patógeno , Metabolismo dos Carboidratos , Parede Celular/ultraestrutura , Contagem de Colônia Microbiana , Meios de Cultura/química , Viabilidade Microbiana , Estresse Fisiológico , VirulênciaRESUMO
The Hog1 stress-activated protein kinase regulates both stress responses and morphogenesis in Candida albicans and is essential for the virulence of this major human pathogen. Stress-induced Hog1 phosphorylation is regulated by the upstream MAPKK, Pbs2, which in turn is regulated by the MAPKKK, Ssk2. Here, we have investigated the role of phosphorylation of Hog1 and Pbs2 in Hog1-mediated processes in C. albicans. Mutation of the consensus regulatory phosphorylation sites of Hog1 (Thr-174/Tyr-176) and Pbs2 (Ser-355/Thr-359), to nonphosphorylatable residues, resulted in strains that phenocopied hog1Δ and pbs2Δ cells. Consistent with this, stress-induced phosphorylation of Hog1 was abolished in cells expressing nonphosphorylatable Pbs2 (Pbs2(AA)). However, mutation of the consensus sites of Pbs2 to phosphomimetic residues (Pbs2(DD)) failed to constitutively activate Hog1. Furthermore, Ssk2-independent stress-induced Hog1 activation was observed in Pbs2(DD) cells. Collectively, these data reveal a previously uncharacterized MAPKKK-independent mechanism of Hog1 activation in response to stress. Although Pbs2(DD) cells did not exhibit high basal levels of Hog1 phosphorylation, overexpression of an N-terminal truncated form of Ssk2 did result in constitutive Hog1 activation, which was further increased upon stress. Significantly, both Pbs2(AA) and Pbs2(DD) cells displayed impaired stress resistance and attenuated virulence in a mouse model of disease, whereas only Pbs2(AA) cells exhibited the morphological defects associated with loss of Hog1 function. This indicates that Hog1 mediates C. albicans virulence by conferring stress resistance rather than regulating morphogenesis.
Assuntos
Proteínas de Bactérias/metabolismo , Candida albicans/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Fúngica da Expressão Gênica , MAP Quinase Quinase Quinases/metabolismo , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Alelos , Animais , Proteínas de Bactérias/genética , Modelos Animais de Doenças , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Mutagênese , Mutação , Osmose , Fosforilação , Proteínas Quinases/genética , Transdução de Sinais , VirulênciaRESUMO
Candida albicans is normally found as a commensal microbe, commonly colonizing the gastrointestinal tract in humans. However, this fungus can also cause mucosal and systemic infections once immune function is compromised. Dectin-1 is an innate pattern recognition receptor essential for the control of fungal infections in both mice and humans; however, its role in the control of C. albicans colonization of the gastrointestinal tract has not been defined. Here, we demonstrate that in mice dectin-1 is essential for the control of gastrointestinal invasion during systemic infection, with dectin-1 deficiency associating with impaired fungal clearance and dysregulated cytokine production. Surprisingly, however, following oral infection, dectin-1 was not required for the control of mucosal colonization of the gastrointestinal tract, in terms of either fungal burdens or cytokine response. Thus, in mice, dectin-1 is essential for controlling systemic infection with C. albicans but appears to be redundant for the control of gastrointestinal colonization.
Assuntos
Candida albicans/crescimento & desenvolvimento , Trato Gastrointestinal/microbiologia , Lectinas Tipo C/metabolismo , Mucosa/microbiologia , Animais , Candida albicans/efeitos dos fármacos , Candida albicans/isolamento & purificação , Candida albicans/patogenicidade , Candidíase/imunologia , Candidíase/microbiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Post-translational modifications of proteins play key roles in eukaryotic growth, differentiation and environmental adaptation. In model systems the ubiquitination of specific proteins contributes to the control of cell cycle progression, stress adaptation and metabolic reprogramming. We have combined molecular, cellular and proteomic approaches to examine the roles of ubiquitination in Candida albicans, because little is known about ubiquitination in this major fungal pathogen of humans. Independent null (ubi4/ubi4) and conditional (MET3p-UBI4/ubi4) mutations were constructed at the C. albicans polyubiquitin-encoding locus. These mutants displayed morphological and cell cycle defects, as well as sensitivity to thermal, oxidative and cell wall stresses. Furthermore, ubi4/ubi4 cells rapidly lost viability under starvation conditions. Consistent with these phenotypes, proteins with roles in stress responses (Gnd1, Pst2, Ssb1), metabolism (Acs2, Eno1, Fba1, Gpd2, Pdx3, Pgk1, Tkl1) and ubiquitination (Ubi4, Ubi3, Pre1, Pre3, Rpt5) were among the ubiquitination targets we identified, further indicating that ubiquitination plays key roles in growth, stress responses and metabolic adaptation in C. albicans. Clearly ubiquitination plays key roles in the regulation of fundamental cellular processes that underpin the pathogenicity of this medically important fungus. This was confirmed by the observation that the virulence of C. albicans ubi4/ubi4 cells is significantly attenuated.