RESUMO
An important virulence factor for the fungal pathogen Candida glabrata is the ability to adhere to the host cells, which is mediated by the expression of adhesins. Epa1 is responsible for â¼95% of the in vitro adherence to epithelial cells and is the founding member of the Epa family of adhesins. The majority of EPA genes are localized close to different telomeres, which causes transcriptional repression due to subtelomeric silencing. In C. glabrata there are three Sir proteins (Sir2, Sir3 and Sir4) that are essential for subtelomeric silencing. Among a collection of 79 clinical isolates, some display a hyperadherent phenotype to epithelial cells compared to our standard laboratory strain, BG14. These isolates also express several subtelomeric EPA genes simultaneously. We cloned the SIR2, SIR3 and SIR4 genes from the hyperadherent isolates and from the BG14 and the sequenced strain CBS138 in a replicative vector to complement null mutants in each of these genes in the BG14 background. All the SIR2 and SIR4 alleles tested from selected hyper-adherent isolates were functional and efficient to silence a URA3 reporter gene inserted in a subtelomeric region. The SIR3 alleles from these isolates were also functional, except the allele from isolate MC2 (sir3-MC2), which was not functional to silence the reporter and did not complement the hyperadherent phenotype of the BG14 sir3Δ. Consistently, sir3-MC2 allele is recessive to the SIR3 allele from BG14. Sir3 and Sir4 alleles from the hyperadherent isolates contain several polymorphisms and two of them are present in all the hyperadherent isolates analyzed. Instead, the Sir3 and Sir4 alleles from the BG14 and another non-adherent isolate do not display these polymorphisms and are identical to each other. The particular combination of polymorphisms in sir3-MC2 and in SIR4-MC2 could explain in part the hyperadherent phenotype displayed by this isolate.
Assuntos
Candida glabrata/genética , Candidíase/genética , Proteínas Fúngicas/genética , Lectinas/genética , Candida glabrata/patogenicidade , Candidíase/microbiologia , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica , Inativação Gênica , Complexo de Inativação Induzido por RNA/genética , Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/classificação , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Telômero/genéticaRESUMO
Autoimmune-polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) is a primary immunodeficiency caused by mutations in the autoimmune regulator gene (AIRE). Patients with AIRE mutations are susceptible to Candida albicans infection and present with autoimmune disorders. We previously demonstrated that cytoplasmic AIRE regulates the Syk-dependent Dectin-1 pathway. In this study, we further evaluated direct contact with fungal elements, synapse formation, and the response of macrophage-like THP-1 cells to C. albicans hyphae to determine the role of AIRE upon Dectin receptors function and signaling. We examined the fungal synapse (FS) formation in wild-type and AIRE-knockdown THP-1 cells differentiated to macrophages, as well as monocyte-derived macrophages from APECED patients. We evaluated Dectin-2 receptor signaling, phagocytosis, and cytokine secretion upon hyphal stimulation. AIRE co-localized with Dectin-2 and Syk at the FS upon hyphal stimulation of macrophage-like THP-1 cells. AIRE-knockdown macrophage-like THP-1 cells exhibited less Dectin-1 and Dectin-2 receptors accumulation, decreased signaling pathway activity at the FS, lower C. albicans phagocytosis, and less lysosome formation. Furthermore, IL-1ß, IL-6, or TNF-α secretion by AIRE-knockdown macrophage-like THP-1 cells and AIRE-deficient patient macrophages was decreased compared to control cells. Our results suggest that AIRE modulates the FS formation and hyphal recognition and help to orchestrate an effective immune response against C. albicans.
Assuntos
Candida albicans/imunologia , Hifas/imunologia , Macrófagos/imunologia , Fatores de Transcrição/imunologia , Candida albicans/fisiologia , Candidíase/genética , Candidíase/imunologia , Candidíase/microbiologia , Citocinas/imunologia , Citocinas/metabolismo , Células HEK293 , Humanos , Hifas/fisiologia , Lectinas Tipo C/genética , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Macrófagos/microbiologia , Mutação , Fagocitose/genética , Fagocitose/imunologia , Poliendocrinopatias Autoimunes/genética , Poliendocrinopatias Autoimunes/imunologia , Poliendocrinopatias Autoimunes/microbiologia , Interferência de RNA , Células THP-1 , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteína AIRERESUMO
PURPOSE: Mendelian susceptibility to mycobacterial disease is a rare clinical condition characterized by a predisposition to infectious diseases caused by poorly virulent mycobacteria. Other infections such as salmonellosis and candidiasis are also reported. The purpose of this article is to describe a young boy affected with various infectious diseases caused by Mycobacterium tuberculosis complex, Salmonella sp, Klebsiella pneumonie, Citrobacter sp., and Candida sp, complicated with severe enteropathy and transient hypogammaglobulinemia. METHODS: We reviewed medical records and performed flow cytometry staining for lymphocyte populations, lymphocyte proliferation in response to PHA, and intracellular IFN-γ production in T cell PHA blasts in the patient and a healthy control. Sanger sequencing was used to confirm the genetic variants in the patient and relatives. RESULTS: Genetic analysis revealed a bi-allelic mutation in IL12RB1 (C291Y) resulting in complete IL-12Rß1 deficiency. Functional analysis demonstrated the lack of intracellular production of IFN-γ in CD3+ T lymphocytes from the patient in response to rhIL-12p70. CONCLUSIONS: To our knowledge, this is the third patient with MSMD due to IL-12Rß1 deficiency complicated with enteropathy and hypogammaglobulinemia and the first case of this disease to be described in Colombia.
Assuntos
Agamaglobulinemia/genética , Candidíase/genética , Enterite/genética , Infecções por Bactérias Gram-Negativas/genética , Receptores de Interleucina-12/deficiência , Receptores de Interleucina-12/genética , Agamaglobulinemia/tratamento farmacológico , Vacina BCG , Candidíase/tratamento farmacológico , Farmacorresistência Bacteriana , Enterite/tratamento farmacológico , Predisposição Genética para Doença , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Humanos , Lactente , Mutação , Mycobacterium tuberculosisRESUMO
The echinocandin susceptibilities of 122 Candida glabrata complex strains (including 5 Candida nivariensis and 3 Candida bracarensis strains) were evaluated by microdilution and compared with the results from a molecular tool able to detect FKS mutations. No echinocandin resistance was detected. The PCR results coincide with the MIC data in 99.25% of the cases (1 C. glabrata strain was misidentified as resistant) but were 20 h faster. C. nivariensis FKS genes were sequenced and showed differences with C. glabrataFKS genes.
Assuntos
Antifúngicos/farmacologia , Candida glabrata/efeitos dos fármacos , Equinocandinas/farmacologia , Candida , Candida glabrata/genética , Candidíase/genética , Farmacorresistência Fúngica/genética , Testes de Sensibilidade Microbiana , Mutação/genética , Reação em Cadeia da PolimeraseRESUMO
ArtinM is a D-mannose-binding lectin extracted from the seeds of Artocarpus heterophyllus that interacts with TLR2 N-glycans and activates antigen-presenting cells (APCs), as manifested by IL-12 production. In vivo ArtinM administration induces Th1 immunity and confers protection against infection with several intracellular pathogens. In the murine model of Candida albicans infection, it was verified that, in addition to Th1, ArtinM induces Th17 immunity manifested by high IL-17 levels in the treated animals. Herein, we investigated the mechanisms accounting for the ArtinM-induced IL-17 production. We found that ArtinM stimulates the IL-17 production by spleen cells in BALB/c or C57BL/6 mice, a response that was significantly reduced in the absence of IL-23, MyD88, or IL-1R. Furthermore, we showed that ArtinM directly induced the IL-23 mRNA expression and the IL-1 production by macrophages. Consistently, in cell suspensions depleted of macrophages, the IL-17 production stimulated by ArtinM was reduced by 53% and the exogenous IL-23 acted synergistically with ArtinM in promoting IL-17 production by spleen cell suspensions. We verified that the absence of IL-23, IL-1R, or MyD88 inhibited, but did not block, the IL-17 production by ArtinM-stimulated spleen cells. Therefore, we investigated whether ArtinM exerts a direct effect on CD4+ T cells in promoting IL-17 production. Indeed, spleen cell suspensions depleted of CD4+ T cells responded to ArtinM with very low levels of IL-17 release. Likewise, isolated CD4+ T cells under ArtinM stimulus augmented the expression of TGF-ß mRNA and released high levels of IL-17. Considering the observed synergism between IL-23 and ArtinM, we used cells from IL-23 KO mice to assess the direct effect of lectin on CD4+ T cells. We verified that ArtinM increased the IL-17 production significantly, a response that was inhibited when the CD4+ T cells were pre-incubated with anti-CD3 antibody. In conclusion, ArtinM stimulates the production of IL-17 by CD4+ T cells in two major ways: (I) through the induction of IL-23 and IL-1 by APCs and (II) through the direct interaction with CD3 on the CD4+ T cells. This study contributes to elucidation of mechanisms accounting for the property of ArtinM in inducing Th17 immunity and opens new perspectives in designing strategies for modulating immunity by using carbohydrate recognition agents.
Assuntos
Artocarpus/química , Complexo CD3/imunologia , Interleucina-17/imunologia , Interleucina-1/imunologia , Interleucina-23/imunologia , Lectina de Ligação a Manose , Lectinas de Plantas , Células Th17/imunologia , Animais , Complexo CD3/genética , Candida albicans/imunologia , Candidíase/tratamento farmacológico , Candidíase/genética , Candidíase/imunologia , Interleucina-1/genética , Interleucina-17/genética , Interleucina-23/genética , Lectina de Ligação a Manose/química , Lectina de Ligação a Manose/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/genética , Fator 88 de Diferenciação Mieloide/imunologia , Lectinas de Plantas/química , Lectinas de Plantas/farmacologia , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/imunologia , Células Th1/imunologiaRESUMO
Amphotericin B (AMB) is an antifungal drug that binds to ergosterol and forms pores at the cell membrane, causing the loss of ions. In addition, AMB induces the accumulation of reactive oxygen species (ROS), and although these molecules have multiple deleterious effects on fungal cells, their specific role in the action mechanism of AMB remains unknown. In this work, we studied the role of ROS in the action mechanism of AMB. We determined the intracellular induction of ROS in 44 isolates of different pathogenic yeast species (Candida albicans, Candida parapsilosis, Candida glabrata, Candida tropicalis, Candida krusei, Cryptococcus neoformans, and Cryptococcus gattii). We also characterized the production of ROS in AMB-resistant isolates. We found that AMB induces the formation of ROS in all the species tested. The inhibition of the mitochondrial respiratory chain by rotenone blocked the induction of ROS by AMB and provided protection from the killing action of the antifungal. Moreover, this phenomenon was absent in strains that displayed resistance to AMB. These strains showed an alteration in the respiration rate and mitochondrial membrane potential and also had higher catalase activity than that of the AMB-susceptible strains. Consistently, AMB failed to induce protein carbonylation in the resistant strains. Our data demonstrate that the production of ROS by AMB is a universal and important action mechanism that is correlated with the fungicidal effect and might explain the low rate of resistance to the molecule. Finally, these data provide an opportunity to design new strategies to improve the efficacy of this antifungal.
Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Candida/efeitos dos fármacos , Cryptococcus/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Candida/genética , Candidíase/tratamento farmacológico , Candidíase/genética , Candidíase/microbiologia , Catalase/metabolismo , Membrana Celular/efeitos dos fármacos , Criptococose/tratamento farmacológico , Criptococose/microbiologia , Farmacorresistência Fúngica , Transporte de Elétrons/efeitos dos fármacos , Ergosterol/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estresse Oxidativo/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Rotenona/farmacologia , Desacopladores/farmacologiaRESUMO
BACKGROUND: Patients with X-linked hyper-IgM syndrome (X-HIGM) due to CD40 ligand (CD40L) mutations are susceptible to fungal pathogens; however, the underlying susceptibility mechanisms remain poorly understood. OBJECTIVE: To determine whether monocyte-derived dendritic cells (DCs) from patients with X-HIGM exhibit normal responses to fungal pathogens. METHODS: DCs from patients and controls were evaluated for the expression of costimulatory (CD80 and CD86) and MHC class II molecules and for their ability to produce IL-12 and IL-10 in response to Candida albicans and Paracoccidioides brasiliensis. We also evaluated the ability of C albicans- and P brasiliensis-pulsed mature DCs to induce autologous T-cell proliferation, generation of T helper (T(H)) 17 cells, and production of IFN-γ, TGF-ß, IL-4, IL-5, and IL-17. RESULTS: Immature DCs from patients with X-HIGM showed reduced expression of CD80, CD86, and HLA-DR, which could be reversed by exogenous trimeric soluble CD40L. Most important, mature DCs from patients with X-HIGM differentiated by coculturing DCs with fungi secreted minimal amounts of IL-12 but substantial amounts of IL-10 compared with mature DCs from normal individuals. Coculture of mature DCs from X-HIGM patients with autologous T cells led to low IFN-γ production, whereas IL-4 and IL-5 production was increased. T-cell proliferation and IL-17 secretion were normal. Finally, in vitro incubation with soluble CD40L reversed the decreased IL-12 production and the skewed T(H)2 pattern response. CONCLUSION: Absence of CD40L during monocyte/DC differentiation leads to functional DC abnormalities, which may contribute to the susceptibility to fungal infections in patients with X-HIGM.
Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Células Dendríticas/metabolismo , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/imunologia , Paracoccidioides/imunologia , Paracoccidioidomicose/imunologia , Adolescente , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Antígeno B7-2/genética , Antígeno B7-2/metabolismo , Ligante de CD40/genética , Ligante de CD40/imunologia , Ligante de CD40/metabolismo , Candida albicans/patogenicidade , Candidíase/complicações , Candidíase/genética , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/genética , Proliferação de Células , Células Cultivadas , Criança , Pré-Escolar , Técnicas de Cocultura , Citocinas/metabolismo , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Células Dendríticas/patologia , Células Dendríticas/virologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Antígenos de Histocompatibilidade Classe II/genética , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/complicações , Síndrome de Imunodeficiência com Hiper-IgM Tipo 1/genética , Ativação Linfocitária/efeitos dos fármacos , Ativação Linfocitária/genética , Masculino , Mutação/genética , Paracoccidioides/patogenicidade , Paracoccidioidomicose/complicações , Paracoccidioidomicose/genética , Células Th17/imunologia , Células Th17/metabolismo , Células Th17/patologiaRESUMO
Candida albicans is the most common opportunistic fungal pathogen and causes local and systemic disease in immunocompromised patients. Alveolar macrophages (AMs) are pivotal for the clearance of C. albicans from the lung. Activated AMs secrete 5-lipoxygenase-derived leukotrienes (LTs), which in turn enhance phagocytosis and microbicidal activity against a diverse array of pathogens. Our aim was to investigate the role of LTB(4) and LTD(4) in AM antimicrobial functions against C. albicans and the signaling pathways involved. Pharmacologic and genetic inhibition of LT biosynthesis as well as receptor antagonism reduced phagocytosis of C. albicans when compared with untreated or WT controls. Conversely, exogenous LTs of both classes augmented base-line C. albicans phagocytosis by AMs. Although LTB(4) enhanced mainly mannose receptor-dependent fungal ingestion, LTD(4) enhanced mainly dectin-1 receptor-mediated phagocytosis. LT enhancement of yeast ingestion was dependent on protein kinase C-δ (PKCδ) and PI3K but not PKCα and MAPK activation. Both LTs reduced activation of cofilin-1, whereas they enhanced total cellular F-actin; however, LTB(4) accomplished this through the activation of LIM kinases (LIMKs) 1 and 2, whereas LTD(4) did so exclusively via LIMK-2. Finally, both exogenous LTB(4) and LTD(4) enhanced AM fungicidal activity in an NADPH oxidase-dependent manner. Our data identify LTB(4) and LTD(4) as key mediators of innate immunity against C. albicans, which act by both distinct and conserved signaling mechanisms to enhance multiple antimicrobial functions of AMs.
Assuntos
Actinas/metabolismo , Candida albicans/metabolismo , Candidíase/metabolismo , Cofilina 1/metabolismo , Imunidade Inata/fisiologia , Leucotrieno B4/metabolismo , Macrófagos Alveolares/metabolismo , Actinas/genética , Actinas/imunologia , Animais , Candida albicans/imunologia , Candidíase/genética , Candidíase/imunologia , Cofilina 1/genética , Cofilina 1/imunologia , Ativação Enzimática/genética , Ativação Enzimática/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Lectinas Tipo C , Leucotrieno B4/genética , Leucotrieno B4/imunologia , Quinases Lim/genética , Quinases Lim/imunologia , Quinases Lim/metabolismo , Macrófagos Alveolares/imunologia , Proteínas de Membrana/genética , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/imunologia , Proteínas do Tecido Nervoso/metabolismo , Fagocitose/genética , Fagocitose/imunologia , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/imunologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C-delta , Ratos , Ratos WistarRESUMO
Con el objeto de conocer las especies causantes de candidosis humanas en pacientes HIV positivos o con otras inmunodeficiencias secundarias y la incidencia de especies con capacidad de resistencia a antifúngicos, se estudiaron 76 aislamientos de Candida procedentes de 61 casos de candidosis superficiales y profundas de niños y adultos. Obtenidas desde piel, anexos, mucosas, abscesos, catéteres y secreciones diversas, entre otras. La identificación de las especies fue realizada por estudios de características morfológicas, cromogénicas y bioquímicas (CHROMagar , Candifast, API 20 y API 32). Los resultados revelan predominio de especies noalbicans (52.7 por ciento), obteniéndose las siguientes frecuencias de aislamientos: C.albicans (47,3 por ciento), C. parapsilosis: 15,8 por ciento, C. glabrata: 13,2 por ciento, C. krusei: 11,8 por ciento, C. tropicalis: 10,6 por ciento y C. dubliniensis: 1,3 por ciento. Algunas de ellas pueden presentar resistencia primaria o secundaria a algunos antifúngicos de uso habitual, por lo cual es necesario incluir estudios de sensibilidad a estos, para una mejor conducta terapéutica.
In order to find out species causing human candidosis in positive HIV patients or in individuals suffering from other secondary immunodeficiencies and the incidence of species bearing a resistance ability to antifungal agents, 76 Candida isolations obtained from 61 cases of superficial and deep candidosis in children and adults were studied. Samples were collected from skin, annexa, mucosities, abscesses, catheters and diverse secretions, among others. The identification of species was carried out through studies on morphological, chromogenic and biochemical characteristics (CHROMagar, Candifast, API 20 and API 32). Results reveal a predominance of non-albican species (52,7 percent), and the following frequencies of isolation: C.albicans (47.3 percent), C. parapsilosis: 15.8 percent, C.glabrata: 13.2 percent, C. krusei: 11.8 percent, C. tropicalis: 10.6 percent and C. dubliniensis: 1.3 percent. Some of them may exhibit some primary or secondary resistance to certain antifungal agents of common use, this is why it is necessary to include studies on sensitivity of them so as to attain a better therapeutical behaviour.
Assuntos
Humanos , Masculino , Adulto , Feminino , Criança , Infecções Oportunistas Relacionadas com a AIDS , Antifúngicos/antagonistas & inibidores , Antifúngicos/uso terapêutico , Candidíase/classificação , Candidíase/etiologia , Candidíase/genética , Candidíase/microbiologia , Candidíase/prevenção & controle , Candidíase/terapia , Candidíase/transmissão , Argentina , Doenças do Sistema Imunitário/complicaçõesRESUMO
Paracoccidioides brasiliensis, a thermodimorphic fungus, is the causative agent of the prevalent systemic mycosis in Latin America, paracoccidioidomycosis. We present here a survey of expressed genes in the yeast pathogenic phase of P. brasiliensis. We obtained 13,490 expressed sequence tags from both 5' and 3' ends. Clustering analysis yielded the partial sequences of 4,692 expressed genes that were functionally classified by similarity to known genes. We have identified several Candida albicans virulence and pathogenicity homologues in P. brasiliensis. Furthermore, we have analyzed the expression of some of these genes during the dimorphic yeast-mycelium-yeast transition by real-time quantitative reverse transcription-PCR. Clustering analysis of the mycelium-yeast transition revealed three groups: (i) RBT, hydrophobin, and isocitrate lyase; (ii) malate dehydrogenase, contigs Pb1067 and Pb1145, GPI, and alternative oxidase; and (iii) ubiquitin, delta-9-desaturase, HSP70, HSP82, and HSP104. The first two groups displayed high mRNA expression in the mycelial phase, whereas the third group showed higher mRNA expression in the yeast phase. Our results suggest the possible conservation of pathogenicity and virulence mechanisms among fungi, expand considerably gene identification in P. brasiliensis, and provide a broader basis for further progress in understanding its biological peculiarities.