RESUMO
Candida albicans is an opportunistic fungal pathogen that colonizes human gastro-intestinal mucosal tissues. Its effect on the immune response in intestinal epithelial cells and on the intestinal mucosal barrier are not yet fully understood. In this study, we investigated Caco-2 cells, a monolayer model of intestinal epithelial cells, with or without treatment with C. albicans SC5314 (CA) or heat-inactivated CA (CA-inact). RNA sequencing was conducted, and the mRNA and protein levels of NOD-like receptor pyrin domain-containing protein 3 (NLRP3) or NLRP6/ASC/caspase-1 inflammasome signaling pathway components, inflammatory cytokines (interleukin-18 [IL-18] and IL-1ß), anti-microbial peptides (AMPs; ß-defensin-2 [BD-2], BD-3, and LL-37), and tight junction proteins (occludin and zona occludens-1 [ZO-1]) were examined by real-time PCR, western blotting, and/or immunofluorescence microscopy. Lactase dehydrogenase (LDH) activity in the Caco-2 cell supernatant were measured by enzyme kinetics analysis. Our results showed that the NOD-like receptor signaling pathway participates in the CA- and CA-inact-infected Caco-2 cells, as shown by microarray analysis of total mRNA expression. The expression of NLRP3, NLRP6, ASC, BD-2, BD-3, occludin, and ZO-1 were significantly decreased in Caco-2 cells infected with CA and CA-inact compared to that in the untreated control. IL-1ß expression was decreased in the Caco-2 cells in both the CA- and CA-inact-infected groups compared to that in the control. Caspase-1 and IL-18 levels were not markedly affected by CA or CA-inact in Caco-2 cells. Our findings indicate that CA can inhibit the NLRP3 and NLRP6 pathways and dampen human intestinal mucosal barrier activity by decreasing the production of AMPs and tight junction proteins, independent of CA activity.
Assuntos
Candida albicans/metabolismo , Candidíase/metabolismo , Células Epiteliais/metabolismo , Inflamassomos/metabolismo , Mucosa Intestinal/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteínas de Junções Íntimas/metabolismo , Peptídeos Catiônicos Antimicrobianos/genética , Peptídeos Catiônicos Antimicrobianos/metabolismo , Células CACO-2 , Candidíase/enzimologia , Candidíase/genética , Células Epiteliais/enzimologia , Células Epiteliais/microbiologia , Humanos , Inflamassomos/genética , Interleucina-18/genética , Interleucina-18/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Mucosa Intestinal/microbiologia , Peptídeos e Proteínas de Sinalização Intracelular/genética , L-Lactato Desidrogenase/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Ocludina/genética , Ocludina/metabolismo , RNA-Seq , Proteínas de Junções Íntimas/genética , Proteína da Zônula de Oclusão-1/genética , Proteína da Zônula de Oclusão-1/metabolismo , beta-Defensinas/metabolismo , CatelicidinasRESUMO
ß-Glucan-induced trained immunity in myeloid cells leads to long-term protection against secondary infections. Although previous studies have characterized this phenomenon, strategies to boost trained immunity remain undefined. We found that ß-glucan-trained macrophages from mice with a myeloid-specific deletion of the phosphatase SHIP-1 (LysMΔSHIP-1) showed enhanced proinflammatory cytokine production in response to lipopolysaccharide. Following ß-glucan training, SHIP-1-deficient macrophages exhibited increased phosphorylation of Akt and mTOR targets, correlating with augmented glycolytic metabolism. Enhanced training in the absence of SHIP-1 relied on histone methylation and acetylation. Trained LysMΔSHIP-1 mice produced increased amounts of proinflammatory cytokines upon rechallenge in vivo and were better protected against Candida albicans infection compared with control littermates. Pharmacological inhibition of SHIP-1 enhanced trained immunity against Candida infection in mouse macrophages and human peripheral blood mononuclear cells. Our data establish proof of concept for improvement of trained immunity and a strategy to achieve it by targeting SHIP-1.
Assuntos
Candidíase/enzimologia , Candidíase/imunologia , Imunidade , Células Mieloides/enzimologia , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/metabolismo , beta-Glucanas/farmacologia , Animais , Candida albicans/fisiologia , Candidíase/microbiologia , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/microbiologia , Camundongos Endogâmicos C57BL , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatases/antagonistas & inibidoresRESUMO
We previously reported the importance of induced nuclear transglutaminase (TG) 2 activity, which results in hepatic cell death, in ethanol-induced liver injury. Here, we show that co-incubation of either human hepatic cells or mouse primary hepatocytes derived from wild-type but not TG2-/- mice with pathogenic fungi Candida albicans and C. glabrata, but not baker's yeast Saccharomyces cerevisiae, induced cell death in host cells by enhancing cellular, particularly nuclear, TG activity. Further pharmacological and genetic approaches demonstrated that this phenomenon was mediated partly by the production of reactive oxygen species (ROS) such as hydroxyl radicals, as detected by a fluorescent probe and electron spin resonance. A ROS scavenger, N-acetyl cysteine, blocked enhanced TG activity primarily in the nuclei and inhibited cell death. In contrast, deletion of C. glabrata nox-1, which encodes a ROS-generating enzyme, resulted in a strain that failed to induce the same phenomena. A similar induction of hepatic ROS and TG activities was observed in C. albicans-infected mice. An antioxidant corn peptide fraction inhibited these phenomena in hepatic cells. These results address the impact of ROS-generating pathogens in inducing nuclear TG2-related liver injuries, which provides novel therapeutic targets for preventing and curing alcoholic liver disease.
Assuntos
Acetilcisteína/farmacologia , Candida albicans/patogenicidade , Candida glabrata/patogenicidade , Núcleo Celular/enzimologia , Sequestradores de Radicais Livres/farmacologia , Hepatócitos/enzimologia , Peptídeos/farmacologia , Animais , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/genética , Candida glabrata/efeitos dos fármacos , Candida glabrata/enzimologia , Candida glabrata/genética , Candidíase/tratamento farmacológico , Candidíase/enzimologia , Candidíase/genética , Candidíase/microbiologia , Morte Celular/efeitos dos fármacos , Linhagem Celular , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/deficiência , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/imunologia , Deleção de Genes , Regulação da Expressão Gênica , Hepatócitos/efeitos dos fármacos , Hepatócitos/microbiologia , Interações Hospedeiro-Patógeno , Humanos , Radical Hidroxila , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , NADPH Oxidases/deficiência , NADPH Oxidases/genética , Cultura Primária de Células , Proteína 2 Glutamina gama-Glutamiltransferase , Saccharomyces cerevisiae/fisiologia , Transdução de Sinais , Transglutaminases/deficiência , Transglutaminases/genética , Transglutaminases/imunologiaRESUMO
Regulation of protein function by reversible post-translational modification, SUMOylation, is widely conserved in the eukaryotic kingdom. SUMOylation is essential for cell growth, division, and adaptation to stress in most organisms, including fungi. As these are key factors in determination of fungal virulence, in this study, we have investigated the importance of SUMOylation in the human pathogen, Candida glabrata We identified the enzymes involved in small ubiquitin-like modifier conjugation and show that there is strong conservation between Saccharomyces cerevisiae and C. glabrata We demonstrate that SUMOylation is an essential process and that adaptation to stress involves changes in global SUMOylation in C. glabrata Importantly, loss of the deSUMOylating enzyme CgUlp2 leads to highly reduced small ubiquitin-like modifier protein levels, and impaired growth, sensitivity to multiple stress conditions, reduced adherence to epithelial cells, and poor colonization of specific tissues in mice. Our study thus demonstrates a key role for protein SUMOylation in the life cycle and pathobiology of C. glabrata.
Assuntos
Candida glabrata/enzimologia , Candida glabrata/patogenicidade , Candidíase/enzimologia , Endopeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Sumoilação , Fatores de Virulência/metabolismo , Animais , Candida glabrata/genética , Candidíase/genética , Candidíase/patologia , Linhagem Celular Tumoral , Endopeptidases/genética , Feminino , Proteínas Fúngicas/genética , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Chitinases have recently gained attention in the field of pulmonary diseases, particularly in asthma and chronic obstructive pulmonary disease, but their potential role in patients with cystic fibrosis (CF)-associated lung disease remains unclear. OBJECTIVE: The aim of this study was to assess chitinase activity systemically and in the airways of patients with CF and asthma compared with healthy subjects. Additionally, we assessed factors that regulate chitinase activity within the lungs of patients with CF. METHODS: Chitinase activities were quantified in serum and bronchoalveolar lavage fluid from patients with CF, asthmatic patients, and healthy control subjects. Mechanistically, the role of CF airway proteases and genetic chitinase deficiency was assessed. RESULTS: Chitinase activity was systemically increased in patients with CF compared with that in healthy control subjects and asthmatic patients. Further stratification showed that chitinase activity was enhanced in patients with CF colonized with Candida albicans compared with that in noncolonized patients. CF proteases degraded chitinases in the airway microenvironment of patients with CF. Genetic chitinase deficiency was associated with C albicans colonization in patients with CF. CONCLUSION: Patients with CF have enhanced chitinase activation associated with C albicans colonization. Therefore chitinases might represent a novel biomarker and therapeutic target for CF-associated fungal disease.
Assuntos
Candidíase/complicações , Quitinases/metabolismo , Fibrose Cística/complicações , Fibrose Cística/microbiologia , Adolescente , Adulto , Asma/complicações , Candida albicans/isolamento & purificação , Candida albicans/metabolismo , Candidíase/enzimologia , Quitinases/sangue , Quitinases/deficiência , Quitinases/genética , Feminino , Humanos , Masculino , Regulação para Cima , Adulto JovemRESUMO
The major fungal pathogen of humans, Candida albicans, is exposed to reactive nitrogen and oxygen species following phagocytosis by host immune cells. In response to these toxins, this fungus activates potent anti-stress responses that include scavenging of reactive nitrosative and oxidative species via the glutathione system. Here we examine the differential roles of two glutathione recycling enzymes in redox homeostasis, stress adaptation and virulence in C. albicans: glutathione reductase (Glr1) and the S-nitrosoglutathione reductase (GSNOR), Fdh3. We show that the NADPH-dependent Glr1 recycles GSSG to GSH, is induced in response to oxidative stress and is required for resistance to macrophage killing. GLR1 deletion increases the sensitivity of C. albicans cells to H2O2, but not to formaldehyde or NO. In contrast, Fdh3 detoxifies GSNO to GSSG and NH3, and FDH3 inactivation delays NO adaptation and increases NO sensitivity. C. albicans fdh3â cells are also sensitive to formaldehyde, suggesting that Fdh3 also contributes to formaldehyde detoxification. FDH3 is induced in response to nitrosative, oxidative and formaldehyde stress, and fdh3Δ cells are more sensitive to killing by macrophages. Both Glr1 and Fdh3 contribute to virulence in the Galleria mellonella and mouse models of systemic infection. We conclude that Glr1 and Fdh3 play differential roles during the adaptation of C. albicans cells to oxidative, nitrosative and formaldehyde stress, and hence during the colonisation of the host. Our findings emphasise the importance of the glutathione system and the maintenance of intracellular redox homeostasis in this major pathogen.
Assuntos
Adaptação Fisiológica , Aldeído Oxirredutases , Candida albicans , Proteínas Fúngicas , Glutationa Redutase , Estresse Oxidativo , Aldeído Oxirredutases/genética , Aldeído Oxirredutases/metabolismo , Animais , Candida albicans/enzimologia , Candida albicans/genética , Candida albicans/patogenicidade , Candidíase/enzimologia , Candidíase/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Humanos , Macrófagos/metabolismo , Macrófagos/microbiologia , Camundongos , Óxido Nítrico/metabolismoRESUMO
The cyclic AMP (cAMP)-protein kinase A (PKA) signaling activates virulence expression during hyphal development in the fungal human pathogen Candida albicans. The hyphal growth is characterized by Golgi polarization toward the hyphal tips, which is thought to enhance directional vesicle transport. However, how the hypha-induction signal regulates Golgi polarization is unknown. Gyp1, a Golgi-associated protein and the first GTPase-activating protein (GAP) in the Rab GAP cascade, critically regulates membrane trafficking from the endoplasmic reticulum to the plasma membrane. Here, we report a novel pathway by which the cAMP-PKA signaling triggers Golgi polarization during hyphal growth. We demonstrate that Gyp1 plays a crucial role in actin-dependent Golgi polarization. Hyphal induction activates PKA, which in turn phosphorylates Gyp1. Phosphomimetic mutation of four PKA sites identified by mass spectrometry (Gyp1(4E)) caused strong Gyp1 polarization to hyphal tips, whereas nonphosphorylatable mutations (Gyp1(4A)) abolished it. Gyp1(4E) exhibited enhanced association with the actin motor Myo2, while Gyp1(4A) showed the opposite effect, providing a possible mechanism for Golgi polarization. A GAP-dead Gyp1 (Gyp1(R292K)) showed strong polarization similar to that seen with Gyp1(4E), indicating a role for the GAP activity. Mutating the PKA sites on Gyp1 also impaired the recruitment of a late Golgi marker, Sec7. Furthermore, proper PKA phosphorylation and GAP activity of Gyp1 are required for virulence in mice. We propose that the cAMP-PKA signaling directly targets Gyp1 to promote Golgi polarization in the yeast-to-hypha transition, an event crucial for C. albicans infection.
Assuntos
Candida albicans/enzimologia , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Complexo de Golgi/enzimologia , Animais , Candida albicans/patogenicidade , Candidíase/enzimologia , Candidíase/microbiologia , Hifas/enzimologia , Hifas/patogenicidade , Rim/microbiologia , Rim/patologia , Camundongos , Fosforilação , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transdução de Sinais , VirulênciaRESUMO
In terms of infection incidence, the yeast Candida parapsilosis is the second after Candida albicans as causative agent of candidiases in humans. The major virulence factors of C. parapsilosis are secreted aspartic proteases (SAPPs) which help the pathogen to disseminate, acquire nutrients and dysregulate the mechanisms of innate immunity of the host. In the current work we characterized the action of two major extracellular proteases of C. parapsilosis, SAPP1 and SAPP2, on human kininogens, proteinaceous precursors of vasoactive and proinflammatory bradykinin-related peptides, collectively called the kinins. The kininogens, preferably the form with lower molecular mass, were effectively cleaved by SAPPs, with the release of two uncommon kinins, Met-Lys-bradykinin and Leu-Met-Lys-bradykinin. While optimal at acidic pH (4-5), the kinin release yield was only 2-3-fold lower at neutral pH. These peptides were able to interact with cellular kinin receptors of B2 subtype and to stimulate the human endothelial cells HMEC-1 to increased secretion of proinflammatory interleukins (ILs), IL-1ß and IL-6. The analysis of the stability of SAPP-generated kinins in plasma suggested that they are biologically equivalent to bradykinin, the best agonist of B2 receptor subtype and can be quickly converted to des-Arg(9)-bradykinin, the agonist of inflammation-inducible B1 receptors.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Bradicinina/análogos & derivados , Candidíase/enzimologia , Proteínas Fúngicas/metabolismo , Cininogênios/metabolismo , Ácido Aspártico Proteases , Bradicinina/química , Bradicinina/metabolismo , Candida/enzimologia , Candida/patogenicidade , Candidíase/microbiologia , Candidíase/patologia , Células Endoteliais , Humanos , Cininogênios/química , Oligopeptídeos , Peptídeos/química , Peptídeos/metabolismoRESUMO
Systemic infection by the pathogenic yeast Candida albicans produces high mortality in immune-compromised people. Such infection starts with the penetration of the organism at the mucosal surfaces, facilitated by the secreted aspartic proteases (Saps) 4, 5, and 6. The functional mechanism of these virulence factors is unclear. We discovered that Saps 4-6 each contains amino acid motifs RGD/KGD to bind integrins on epithelial cell A549 and are internalized to endosomes and lysosomes. These processes are inhibited by RGD-containing peptides or by substituting RGD motifs of these Saps. The internalization of Saps 4-6 results in partial permeabilization of lysosomal membranes, measured by the redistribution of the lysosomal tropic dye acridine orange to the cytosol, and the triggering of apoptosis via caspase activation. Sap 2 and mutated Saps 4-6 contain no RGD motif, are ineffective in these processes, and a proteolytic inhibitor abolished Sap 4 activity in lysosome permeabilization. Same results were also seen for human tongue keratinocyte SCC-15 cells. Mucosal lesions from this fundamental new mechanism may permit C. albicans to enter the body and may be used to attack cells in immune defense during systemic infections. RGD-motif may also be incorporated in Sap inhibitors for Candidiasis drugs targeting to lysosomes.
Assuntos
Apoptose , Ácido Aspártico Endopeptidases/fisiologia , Candida albicans/enzimologia , Candida albicans/patogenicidade , Proteínas Fúngicas/fisiologia , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/química , Ácido Aspártico Endopeptidases/genética , Candida albicans/genética , Candidíase/enzimologia , Candidíase/etiologia , Linhagem Celular , Células Epiteliais/enzimologia , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno , Humanos , Integrinas/metabolismo , Lisossomos/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/genética , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , VirulênciaRESUMO
Tumor necrosis factor receptor-associated factor 6 (TRAF6) and TGFß-activated kinase 1 (TAK1) are considered as key intermediates in Toll-like receptor (TLR) signaling. However, the role of TRAF6 and TAK1 in C-type lectin receptors (CLRs) in response to fungal infection has not been studied. In this study, we have utilized macrophages derived from TRAF6 knock-out mice and myeloid-specific TAK1-deficient mice and determined the role of TRAF6 and TAK1 in CLR-induced signal transduction events. We demonstrate that TRAF6 and TAK1 are required for NF-κB and JNK activation, and expression of proinflammatory cytokines in response to Candida albicans infection. Our results highlight TRAF6 and TAK1 as key components in the signaling cascade downstream of C-type lectin receptors and as critical mediators of the anti-fungal immune response. Therefore, our studies provide a mechanistic understanding of the host immune response to C. albicans, which has a significant impact for the development of anti-fungal therapeutics and in understanding risk-factors and determining susceptibility to C. albicans infection.
Assuntos
Candida albicans/fisiologia , Candidíase/imunologia , Lectinas Tipo C/imunologia , MAP Quinase Quinase Quinases/imunologia , Transdução de Sinais , Fator 6 Associado a Receptor de TNF/imunologia , Animais , Candida albicans/imunologia , Candidíase/enzimologia , Candidíase/genética , Células Cultivadas , Feminino , Humanos , Lectinas Tipo C/genética , MAP Quinase Quinase Quinases/genética , Macrófagos/imunologia , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 6 Associado a Receptor de TNF/genéticaRESUMO
Bradykinin-related peptides, universal mediators of inflammation collectively referred to as the kinins, are often produced in excessive amounts during microbial infections. We have recently shown that the yeast Candida albicans, the major fungal pathogen to humans, can exploit two mechanisms to enhance kinin levels at the sites of candidial infection, one depending on adsorption and activation of the endogenous kinin-generating system of the host on the fungal cell wall and the other relying on cleavage of kinin precursors, the kininogens, by pathogen-secreted proteases. This work aimed at assigning this kininogenase activity to the major secreted aspartic protease of C. albicans (SAP2). The purified SAP2 was shown to cleave human kininogens, preferably the low molecular mass form (LK) and optimally in an acidic environment (pH 3.5-4.0), and to produce two kinins, Met-Lys-bradykinin and its derivative, [Hydroxyproline(3)]-Met-Lys-bradykinin, both of which are capable of interacting with cellular bradykinin receptors of the B2 subtype. Additionally, albeit with a lower yield, des-Arg(9)-Met-Lys-bradykinin, an effective agonist of B1-subtype receptors, was released. The pathophysiological potential of these kinins and des-Arg-kinin was also proven by presenting their ability to stimulate human promonocytic cells U937 to release proinflammatory interleukin 1ß (IL-1ß) and IL-6.
Assuntos
Ácido Aspártico Endopeptidases/imunologia , Bradicinina/análogos & derivados , Candida albicans/imunologia , Candidíase/imunologia , Proteínas Fúngicas/imunologia , Cininogênios/imunologia , Sequência de Aminoácidos , Ácido Aspártico Endopeptidases/metabolismo , Bradicinina/química , Bradicinina/imunologia , Bradicinina/metabolismo , Candida albicans/enzimologia , Candidíase/enzimologia , Candidíase/metabolismo , Linhagem Celular , Proteínas Fúngicas/metabolismo , Humanos , Interleucina-1beta/imunologia , Interleucina-6/imunologia , Cininogênios/metabolismo , Dados de Sequência Molecular , Receptores da Bradicinina/imunologiaRESUMO
Binding between vacuolar H+-ATPases (V-ATPases) and microfilaments is mediated by an actin binding domain in the B-subunit. Both isoforms of mammalian B-subunit bind microfilaments with high affinity. A similar actinbinding activity has been demonstrated in the B-subunit of yeast. A conserved "profilin-like" domain in the B-subunit mediates this actin-binding activity, named due to its sequence and structural similarity to an actin-binding surface of the canonical actin binding protein profilin. Subtle mutations in the "profilin-like" domain eliminate actin binding activity without disrupting the ability of the altered protein to associate with the other subunits of V-ATPase to form a functional proton pump. Analysis of these mutated B-subunits suggests that the actin-binding activity is not required for the "housekeeping" functions of V-ATPases, but is important for certain specialized roles. In osteoclasts, the actin-binding activity is required for transport of V-ATPases to the plasma membrane, a prerequisite for bone resorption. A virtual screen led to the identification of enoxacin as a small molecule that bound to the actin-binding surface of the B2-subunit and competitively inhibited B2-subunit and actin interaction. Enoxacin disrupted osteoclastic bone resorption in vitro, but did not affect osteoblast formation or mineralization. Recently, enoxacin was identified as an inhibitor of the virulence of Candida albicans and more importantly of cancer growth and metastasis. Efforts are underway to determine the mechanisms by which enoxacin and other small molecule inhibitors of B2 and microfilament binding interaction selectively block bone resorption, the virulence of Candida, cancer growth, and metastasis.
Assuntos
Anti-Infecciosos/farmacologia , Antineoplásicos/farmacologia , Candidíase/tratamento farmacológico , Descoberta de Drogas/métodos , Enoxacino/farmacologia , Neoplasias/tratamento farmacológico , Osteoclastos/efeitos dos fármacos , ATPases Vacuolares Próton-Translocadoras/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Animais , Candida/efeitos dos fármacos , Candida/enzimologia , Candidíase/enzimologia , Humanos , Neoplasias/enzimologia , Osteoclastos/enzimologia , Ligação Proteica/efeitos dos fármacos , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Candida albicans is an increasingly important pulmonary fungal pathogen. Resident alveolar macrophages are important in host defense against opportunistic fungal infections. Activation of Group IVA cytosolic phospholipase A(2)alpha (cPLA(2)alpha) in macrophages initiates arachidonic acid (AA) release for production of eicosanoids, which regulate inflammation and immune responses. We investigated the ability of C. albicans to activate cPLA(2)alpha in unprimed alveolar macrophages and after priming with granulocyte macrophage colony-stimulating factor (GM-CSF), which regulates alveolar macrophage maturation. AA was released within minutes by GM-CSF-primed but not unprimed alveolar macrophages in response to C. albicans, and was blocked by soluble glucan phosphate (S-GP). The expression of the beta-glucan receptor dectin-1 was increased in GM-CSF-primed macrophages, and AA release from GM-CSF-primed dectin-1(-/-) alveolar macrophages was reduced to basal levels. The enhanced activation of extracellular signal-regulated kinases and phosphorylation of cPLA(2)alpha on Ser-505 that occurred in GM-CSF-primed macrophages were reduced by MEK1 and Syk inhibitors, which also suppressed AA release. At later times after C. albicans infection (6 h), unprimed and GM-CSF-primed macrophages released similar levels of AA. The expression of cyclooxygenase 2 and prostanoid production at 6 hours was higher in GM-CSF-primed macrophages, but the responses were not dependent on dectin-1. However, dectin-1 contributed to the C. albicans-stimulated increase in TNF-alpha production that occurred in GM-CSF-primed macrophages. The results demonstrate that dectin-1 mediates the acute activation of cPLA(2)alpha in GM-CSF-primed alveolar macrophages, but not in the more delayed phase of AA release and GM-CSF-dependent prostanoid production.
Assuntos
Ácido Araquidônico/imunologia , Candida albicans/imunologia , Candidíase/imunologia , Fosfolipases A2 do Grupo IV/imunologia , Macrófagos Alveolares/imunologia , Proteínas de Membrana/imunologia , Proteínas do Tecido Nervoso/imunologia , Animais , Ácido Araquidônico/metabolismo , Candidíase/enzimologia , Candidíase/genética , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/genética , Ativação Enzimática/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/imunologia , Fator Estimulador de Colônias de Granulócitos e Macrófagos/metabolismo , Fosfolipases A2 do Grupo IV/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lectinas Tipo C , MAP Quinase Quinase 1/imunologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Sistema de Sinalização das MAP Quinases/imunologia , Macrófagos Alveolares/enzimologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos ICR , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Fosforilação/imunologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Tirosina Quinases/imunologia , Proteínas Tirosina Quinases/metabolismo , Quinase Syk , Fatores de Tempo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Cytosolic fungal fatty acid synthase is composed of two subunits alpha and beta, which are encoded by Fas1 and Fas2 genes. In this study, the Fas2 genes of the human pathogen Candida parapsilosis were deleted using a modified SAT1 flipper technique. CpFas2 was essential in media lacking exogenous fatty acids and the growth of Fas2 disruptants (Fas2 KO) was regulated by the supplementation of different long chain fatty acids, such as myristic acid (14ratio0), palmitic acid (16ratio0), and Tween 80, in a dose-specific manner. Lipidomic analysis revealed that Fas2 KO cells were severely restricted in production of unsaturated fatty acids. The Fas2 KO strains were unable to form normal biofilms and were more efficiently killed by murine-like macrophages, J774.16, than the wild type, heterozygous and reconstituted strains. Furthermore, Fas2 KO yeast were significantly less virulent in a systemic murine infection model. The Fas2 KO cells were also hypersensitive to human serum, and inhibition of CpFas2 in WT C. parapsilosis by cerulenin significantly decreased fungal growth in human serum. This study demonstrates that CpFas2 is essential for C. parapsilosis growth in the absence of exogenous fatty acids, is involved in unsaturated fatty acid production, influences fungal virulence, and represents a promising antifungal drug target.
Assuntos
Candida/enzimologia , Candida/patogenicidade , Candidíase/enzimologia , Candidíase/microbiologia , Ácido Graxo Sintases/metabolismo , Animais , Biofilmes/efeitos dos fármacos , Candida/efeitos dos fármacos , Candida/genética , Carbono/farmacologia , Meios de Cultura , Ácido Graxo Sintases/genética , Ácidos Graxos/farmacologia , Feminino , Deleção de Genes , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Homozigoto , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/microbiologia , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Soro , Estresse Fisiológico/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Candida albicans secretes various hydrolytic enzymes which are considered to be an integral part in the pathogenesis. However, the role of lipases is far from being completely understood and the direct effects of these fungal enzymes during the host-pathogen interaction remain to be established. We recently isolated and characterized an extracellular C. albicans lipase (CaLIP), and demonstrated the ability of this fungal enzyme to interact directly with macrophages (Mvarphi) and hepatocytes and to operate as a virulence factor. Herein, we explored the effects of CaLIP on Mvarphi functions such as oxidative burst and l-arginine metabolism. The study was performed in cells with different activation status: normal-resting Mvarphis and Mvarphis primed in vivo or in vitro with C. albicans. The ability of this fungal factor to modulate the above-mentioned parameters was dependent on cells status, dose, and microenvironment, where the interaction took place. These results constitute a new finding in the biology of candidiasis and could illustrate an additional evolutive advantage for the fungus in the framework of the bidirectional host-pathogen interaction.
Assuntos
Arginina/metabolismo , Candida albicans/patogenicidade , Lipase/metabolismo , Macrófagos/metabolismo , NADPH Oxidases/metabolismo , Animais , Arginase/metabolismo , Candida albicans/enzimologia , Candidíase/enzimologia , Candidíase/metabolismo , Candidíase/microbiologia , Feminino , Interações Hospedeiro-Patógeno , Humanos , Lipase/farmacologia , Ativação de Macrófagos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Óxido Nítrico Sintase Tipo II/biossíntese , Ratos , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismoRESUMO
Infecções por leveduras são freqüentes em imunocomprometidos, contudo espécies emergentes têm alterado o perfil epidemiológico. A habilidade de secretar proteases tem sido associada à patogenicidade do gênero Candida. Esta pesquisa teve como objetivos diagnosticar leveduroses em pacientes imunocomprometidos e avaliar a virulência dos agentes etiológicos baseado em teste de secreção de protease utilizando soro de albumina bovina como substrato. Do total de 104 pacientes estudados, 19 apresentaram episódios de leveduroses. O trato respiratório (63,2 por cento), seguido pelo trato urinário (10,5 por cento) foram os locais mais comuns de infecção. Candida albicans, Candida parapsilosis, Candida tropicalis e espécies emergentes como Candida krusei e Candida guilliermondii foram isoladas. Cinco isolados de Candida parapsilosis e um de Candida albicans e Candida guilliermondii exibiram alta atividade enzimática. Concluímos que a caracterização enzimática de isolados de Candida pode ser um útil marcador prognóstico, especialmente em imunocomprometidos, uma vez que leveduroses nestes pacientes são geralmente graves.
Yeast infections are common in immunocompromised patients, although emerging species have been changing the epidemiological profile. The ability to secrete proteinases has been associated with pathogenicity within the genus Candida. This study had the aims of diagnosing yeast infections in immunocompromised patients and evaluating the virulence of the etiological agents, based on a proteinase secretion assay using bovine serum albumin as a substrate. Out of a total of 104 patients studied, 19 presented episodes of yeast infection. The respiratory tract (63.2 percent), followed by the urinary tract (10.5 percent), were the most common sites of infection. Candida albicans, Candida parapsilosis, Candida tropicalis and emerging species such as Candida krusei and Candida guilliermondii were isolated. Five isolates of Candida parapsilosis and one of Candida albicans and Candida guilliermondii exhibited high enzymatic activity. We conclude that enzymatic characterization of Candida isolates may be a useful prognostic marker, especially among immunocompromised individuals, since yeast infections in such patients are generally serious.
Assuntos
Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Bovinos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Candida/enzimologia , Candidíase/enzimologia , Peptídeo Hidrolases/biossíntese , Candida/classificação , Candida/patogenicidade , Candidíase/imunologia , Candidíase/microbiologia , Hospedeiro Imunocomprometido , Virulência , Adulto JovemRESUMO
PURPOSES: To investigate the early molecular events in oral keratinocytes induced by Candida albicans challenge. METHODS: The oral keratinocyte cell line, Tca8113, was used to study the molecular events induced by C. albicans challenge in oral keratinocytes. The phosphorylation statuses of extracellular signal-regulated protein kinase (ERK) and focal adhesion kinase (FAK) upon C. albicans challenge were assessed using specific antibodies and western blotting. Specific inhibitors for ERK and FAK were used to validate the involvement of ERK-FAK signaling cascade. A Transwell insert system-based migration study was performed to evaluate the involvement of the C. albicans-dependent ERK-FAK activation with cell migration. RESULTS: Following the stimulation with C. albicans, a transient activation of ERK was observed, which reached a peak at 10 min post stimulation. Similarly, a transient activation of FAK, the downstream substrate of ERK, was also observed upon C. albicans challenges, which reach the maximum at 20 min. Specific inhibitors for ERK and FAK abolished the C. albicans-induced ERK and FAK activations. The elevated migratory ability of oral keratinocyte was observed upon stimulation with C. albicans, and was synchronous with the ERK-FAK activation. CONCLUSION: ERK-FAK signaling cascades are involved in the early interaction between the oral keratinocytes and C. albicans, which appears to be linked with the enhanced cell migration.
Assuntos
Candida albicans , Candidíase , Movimento Celular/efeitos dos fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Queratinócitos/enzimologia , Queratinócitos/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Candidíase/enzimologia , Candidíase/fisiopatologia , Linhagem Celular Tumoral , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Humanos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Boca/microbiologia , Boca/patologia , Inibidores de Proteínas Quinases/farmacologia , Transdução de Sinais , Fatores de TempoRESUMO
With the increase in prevalence of fungal infections, newer antifungal agents are needed to effectively treat invasive disease, and at the same time minimize adverse effects from therapy. The echinocandins comprise a novel class of antifungals; their mechanism of action involves inhibiting 1,3-beta-D-glucan synthase, which is essential in cell wall synthesis for certain fungi. All three echinocandins are US FDA-approved for the treatment of esophageal candidiasis. Caspofungin and anidulafungin are licensed for the treatment of candidemia, and other select forms of invasive candidiasis. Micafungin is at present the only echinocandin approved for prophylaxis of fungal infections in hematopoietic stem cell transplants; whereas caspofungin is approved for empiric therapy of febrile neutropenia. Although all three echinocandins are active against Aspergillus, only caspofungin is presently approved for salvage therapy in invasive aspergillosis. Combination therapy with echinocandins plus other licensed antifungal therapy shows promise in treating invasive aspergillosis. This article will explore the similarities and differences among the echinocandins.
Assuntos
Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacologia , Glucosiltransferases/antagonistas & inibidores , Micoses/tratamento farmacológico , Anidulafungina , Animais , Antifúngicos/administração & dosagem , Antifúngicos/efeitos adversos , Antifúngicos/farmacocinética , Aspergilose/tratamento farmacológico , Aspergilose/enzimologia , Aspergillus/efeitos dos fármacos , Aspergillus/enzimologia , Aspergillus/crescimento & desenvolvimento , Candida/efeitos dos fármacos , Candida/enzimologia , Candida/crescimento & desenvolvimento , Candidíase/tratamento farmacológico , Candidíase/enzimologia , Caspofungina , Esquema de Medicação , Interações Medicamentosas , Quimioterapia Combinada , Equinocandinas , Inibidores Enzimáticos/efeitos adversos , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Glucosiltransferases/metabolismo , Humanos , Lipopeptídeos , Lipoproteínas/farmacologia , Lipoproteínas/uso terapêutico , Micafungina , Micoses/enzimologia , Micoses/microbiologia , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Resultado do TratamentoRESUMO
BACKGROUND: Candida sp are frequently isolated from the ascitic fluid of patients with perforated ulcers. The present study was performed to examine whether Candida infection may be involved in the process of ulcer perforation. METHODS: Male Wistar rats were divided into a saline group (n = 15) and a Candida group (n = 17). Cysteamine-HCl (Sigma; 31 mg/100 g) was administered thrice on day 1 to both groups of animals. Candida albicans at a density of 10(8) in 0.5 mL of saline was administered 1 h before, and 12 h and 24 h after the first administration of cysteamine in the Candida group. RESULTS: Perforated duodenal ulcers were observed in 94.1% of the rats in the Candida group, but only 26.7% of the rats in the saline group (P < 0.01). The area of the duodenal ulcers in the Candida group was 40.89 +/- 33.07 mm2, whereas that in the saline group was 16.53 +/- 20.4 mm2 (P < 0.05). The mortality rate was significantly higher in the Candida group than in the saline group. In the Candida group, colonization by C. albicans was recognized at the ulcer base, surrounded by marked granulocytic infiltration. The number of eosinophils infiltrating the ulcer base was also significantly greater in the Candida group than in the saline group. Immunohistochemical analysis revealed the expression of secretory aspartyl protease (SAP) in the region of the ulcer showing colonization by C. albicans in the Candida group. CONCLUSION: Candida albicans aggravates duodenal ulcer perforation in the experimental model of cysteamine-induced duodenal ulcer perforation. The present findings suggest that SAP and host-parasite relationships, including granulocyte-dependent mechanisms, may be involved in the aggravation of ulcer perforation by C. albicans.
Assuntos
Candida albicans/isolamento & purificação , Candidíase/complicações , Úlcera Duodenal/complicações , Duodeno/microbiologia , Úlcera Péptica Perfurada/etiologia , Animais , Ácido Aspártico Endopeptidases/metabolismo , Candida albicans/enzimologia , Candidíase/enzimologia , Candidíase/microbiologia , Candidíase/patologia , Cisteamina , Úlcera Duodenal/induzido quimicamente , Úlcera Duodenal/enzimologia , Úlcera Duodenal/patologia , Duodeno/enzimologia , Duodeno/patologia , Ensaio de Imunoadsorção Enzimática , Eosinófilos/microbiologia , Granulócitos/microbiologia , Imuno-Histoquímica , Masculino , Úlcera Péptica Perfurada/enzimologia , Úlcera Péptica Perfurada/microbiologia , Úlcera Péptica Perfurada/patologia , Ratos , Ratos Wistar , Fatores de TempoRESUMO
Candida glabrata is a yeast pathogen of humans. We have established a tissue culture model to analyze the interaction of C. glabrata with macrophages. Transcript profiling of yeast ingested by macrophages reveals global changes in metabolism as well as increased expression of a gene family (YPS genes) encoding extracellular glycosylphosphatidylinositol-linked aspartyl proteases. Eight of these YPS genes are found in a cluster that is unique to C. glabrata. Genetic analysis shows that the C. glabrata YPS genes are required for cell wall integrity, adherence to mammalian cells, survival in macrophages and virulence. By monitoring the processing of a cell wall adhesin, Epa1, we also show that Yps proteases play an important role in cell wall re-modeling by removal and release of glycosylphosphatidylinositol-anchored cell wall proteins.