RESUMO
Ergosterol is essential for fungal cell membrane integrity and growth, and numerous antifungal drugs target ergosterol. Inactivation or modification of ergosterol biosynthetic genes can lead to changes in antifungal drug susceptibility, filamentation and stress response. Here, we found that the ergosterol biosynthesis gene ERG251 is a hotspot for point mutations during adaptation to antifungal drug stress within two distinct genetic backgrounds of Candida albicans. Heterozygous point mutations led to single allele dysfunction of ERG251 and resulted in azole tolerance in both genetic backgrounds. This is the first known example of point mutations causing azole tolerance in C. albicans. Importantly, single allele dysfunction of ERG251 in combination with recurrent chromosome aneuploidies resulted in bona fide azole resistance. Homozygous deletions of ERG251 caused increased fitness in low concentrations of fluconazole and decreased fitness in rich medium, especially at low initial cell density. Homozygous deletions of ERG251 resulted in accumulation of ergosterol intermediates consistent with the fitness defect in rich medium. Dysfunction of ERG251, together with FLC exposure, resulted in decreased accumulation of the toxic sterol (14-É-methylergosta-8,24(28)-dien-3ß,6α-diol) and increased accumulation of non-toxic alternative sterols. The altered sterol composition of the ERG251 mutants had pleiotropic effects on transcription, filamentation, and stress responses including cell membrane, osmotic and oxidative stress. Interestingly, while dysfunction of ERG251 resulted in azole tolerance, it also led to transcriptional upregulation of ZRT2, a membrane-bound Zinc transporter, in the presence of FLC, and overexpression of ZRT2 is sufficient to increase azole tolerance in wild-type C. albicans. Finally, in a murine model of systemic infection, homozygous deletion of ERG251 resulted in decreased virulence while the heterozygous deletion mutants maintain their pathogenicity. Overall, this study demonstrates that single allele dysfunction of ERG251 is a recurrent and effective mechanism of acquired azole tolerance. We propose that altered sterol composition resulting from ERG251 dysfunction mediates azole tolerance as well as pleiotropic effects on stress response, filamentation and virulence.
Assuntos
Antifúngicos , Candida albicans , Candidíase , Farmacorresistência Fúngica , Ergosterol , Proteínas Fúngicas , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/metabolismo , Antifúngicos/farmacologia , Camundongos , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , Animais , Candidíase/microbiologia , Candidíase/metabolismo , Candidíase/tratamento farmacológico , Ergosterol/metabolismo , Azóis/farmacologia , Esteróis/metabolismo , Fenótipo , Estresse Fisiológico , Testes de Sensibilidade Microbiana , Fluconazol/farmacologiaRESUMO
Fungal invasion of the oral epithelium is central to the pathogenesis of oropharyngeal candidiasis (OPC). Candida albicans invades the oral epithelium by receptor-induced endocytosis but this process is incompletely understood. We found that C. albicans infection of oral epithelial cells induces c-Met to form a multi-protein complex with E-cadherin and the epidermal growth factor receptor (EGFR). E-cadherin is necessary for C. albicans to activate both c-Met and EGFR and to induce the endocytosis of C. albicans. Proteomics analysis revealed that c-Met interacts with C. albicans Hyr1, Als3 and Ssa1. Both Hyr1 and Als3 are required for C. albicans to stimulate c-Met and EGFR in oral epithelial cells in vitro and for full virulence during OPC in mice. Treating mice with small molecule inhibitors of c-Met and EGFR ameliorates OPC, demonstrating the potential therapeutic efficacy of blocking these host receptors for C. albicans.
Assuntos
Candida albicans , Candidíase Bucal , Animais , Camundongos , Membrana Celular , Receptores ErbB , Caderinas , Células EpiteliaisRESUMO
Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA-approved small molecule inhibitors to identify those that prevent NDU1-associated functions. We identified an antihelminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug-resistant C. albicans and Candida auris from their growth surface. To overcome the suboptimal solubility and permeability of NCL that is well known to affect its in vivo efficacy, we developed NCL-encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, which also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole-resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms and uses NPs of an FDA-approved nontoxic drug to improve biofilm penetrability and microbial killing.
Assuntos
Candidíase , Nanopartículas , Animais , Antifúngicos/farmacologia , Biofilmes , Candida , Candida albicans , Candidíase/microbiologia , Fluconazol/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Niclosamida/farmacologia , Niclosamida/uso terapêuticoRESUMO
Candida albicans is a major opportunistic pathogen of humans. It can grow as morphologically distinct yeast, pseudohyphae and hyphae, and the ability to switch reversibly among different forms is critical for its virulence. The relationship between morphogenesis and innate immune recognition is not quite clear. Dectin-1 is a major C-type lectin receptor that recognizes ß-glucan in the fungal cell wall. C. albicans ß-glucan is usually masked by the outer mannan layer of the cell wall. Whether and how ß-glucan masking is differentially regulated during hyphal morphogenesis is not fully understood. Here we show that the endo-1,3-glucanase Eng1 is differentially expressed in yeast, and together with Yeast Wall Protein 1 (Ywp1), regulates ß-glucan exposure and Dectin-1-dependent immune activation of macrophage by yeast cells. ENG1 deletion results in enhanced Dectin-1 binding at the septa of yeast cells; while eng1 ywp1 yeast cells show strong overall Dectin-1 binding similar to hyphae of wild-type and eng1 mutants. Correlatively, hyphae of wild-type and eng1 induced similar levels of cytokines in macrophage. ENG1 expression and Eng1-mediated ß-glucan trimming are also regulated by antifungal drugs, lactate and N-acetylglucosamine. Deletion of ENG1 modulates virulence in the mouse model of hematogenously disseminated candidiasis in a Dectin-1-dependent manner. The eng1 mutant exhibited attenuated lethality in male mice, but enhanced lethality in female mice, which was associated with a stronger renal immune response and lower fungal burden. Thus, Eng1-regulated ß-glucan exposure in yeast cells modulates the balance between immune protection and immunopathogenesis during disseminated candidiasis.
Assuntos
Candida albicans/patogenicidade , Candidíase/imunologia , Glucana Endo-1,3-beta-D-Glucosidase/metabolismo , Virulência/fisiologia , beta-Glucanas/imunologia , Animais , Candida albicans/imunologia , Candida albicans/metabolismo , Candidíase/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , beta-Glucanas/metabolismoRESUMO
During hematogenously disseminated candidiasis, blood borne fungi must invade the endothelial cells that line the blood vessels to infect the deep tissues. Although Candida albicans, which forms hyphae, readily invades endothelial cells, other medically important species of Candida are poorly invasive in standard in vitro assays and have low virulence in immunocompetent mouse models of disseminated infection. Here, we show that Candida glabrata, Candida tropicalis, Candida parapsilosis, and Candida krusei can bind to vitronectin and high molecular weight kininogen present in human serum. Acting as bridging molecules, vitronectin and kininogen bind to αv integrins and the globular C1q receptor (gC1qR), inducing human endothelial cells to endocytose the fungus. This mechanism of endothelial cell invasion is poorly supported by mouse endothelial cells but can be restored when mouse endothelial cells are engineered to express human gC1qR or αv integrin. Overall, these data indicate that bridging molecule-mediated endocytosis is a common pathogenic strategy used by many medically important Candida spp. to invade human vascular endothelial cells.
Assuntos
Candidíase , Células Endoteliais , Animais , Candida , Candida albicans , Candidíase/microbiologia , Células Endoteliais/microbiologia , Humanos , Camundongos , VitronectinaRESUMO
To gain a better understanding of the transcriptional response of Aspergillus fumigatus during invasive pulmonary infection, we used a NanoString nCounter to assess the transcript levels of 467 A. fumigatus genes during growth in the lungs of immunosuppressed mice. These genes included ones known to respond to diverse environmental conditions and those encoding most transcription factors in the A. fumigatus genome. We found that invasive growth in vivo induces a unique transcriptional profile as the organism responds to nutrient limitation and attack by host phagocytes. This in vivo transcriptional response is largely mimicked by in vitro growth in Aspergillus minimal medium that is deficient in nitrogen, iron, and/or zinc. From the transcriptional profiling data, we selected 9 transcription factor genes that were either highly expressed or strongly up-regulated during in vivo growth. Deletion mutants were constructed for each of these genes and assessed for virulence in mice. Two transcription factor genes were found to be required for maximal virulence. One was rlmA, which is required for the organism to achieve maximal fungal burden in the lung. The other was sltA, which regulates of the expression of multiple secondary metabolite gene clusters and mycotoxin genes independently of laeA. Using deletion and overexpression mutants, we determined that the attenuated virulence of the ΔsltA mutant is due in part to decreased expression aspf1, which specifies a ribotoxin, but is not mediated by reduced expression of the fumigaclavine gene cluster or the fumagillin-pseruotin supercluster. Thus, in vivo transcriptional profiling focused on transcription factors genes provides a facile approach to identifying novel virulence regulators.
Assuntos
Aspergillus fumigatus/genética , Regulação Fúngica da Expressão Gênica/genética , Pulmão/virologia , Fatores de Transcrição/metabolismo , Animais , Aspergilose/microbiologia , Aspergillus fumigatus/patogenicidade , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica/métodos , Ferro/metabolismo , Pulmão/metabolismo , Camundongos , Virulência/genéticaRESUMO
During oropharyngeal candidiasis (OPC), Candida albicans invades and damages oral epithelial cells, which respond by producing proinflammatory mediators that recruit phagocytes to foci of infection. The ephrin type-A receptor 2 (EphA2) detects ß-glucan and plays a central role in stimulating epithelial cells to release proinflammatory mediators during OPC. The epidermal growth factor receptor (EGFR) also interacts with C. albicans and is known to be activated by the Als3 adhesin/invasin and the candidalysin pore-forming toxin. Here, we investigated the interactions among EphA2, EGFR, Als3 and candidalysin during OPC. We found that EGFR and EphA2 constitutively associate with each other as part of a heteromeric physical complex and are mutually dependent for C. albicans-induced activation. Als3-mediated endocytosis of a C. albicans hypha leads to the formation of an endocytic vacuole where candidalysin accumulates at high concentration. Thus, Als3 potentiates targeting of candidalysin, and both Als3 and candidalysin are required for C. albicans to cause maximal damage to oral epithelial cells, sustain activation of EphA2 and EGFR, and stimulate pro-inflammatory cytokine and chemokine secretion. In the mouse model of OPC, C. albicans-induced production of CXCL1/KC and CCL20 is dependent on the presence of candidalysin and EGFR, but independent of Als3. The production of IL-1α and IL-17A also requires candidalysin but is independent of Als3 and EGFR. The production of TNFα requires Als1, Als3, and candidalysin. Collectively, these results delineate the complex interplay among host cell receptors EphA2 and EGFR and C. albicans virulence factors Als1, Als3 and candidalysin during the induction of OPC and the resulting oral inflammatory response.
Assuntos
Candida albicans/fisiologia , Candidíase Bucal/patologia , Efrina-A2/metabolismo , Células Epiteliais/patologia , Orofaringe/patologia , Fatores de Virulência/metabolismo , Animais , Candidíase Bucal/genética , Candidíase Bucal/metabolismo , Candidíase Bucal/microbiologia , Citocinas/metabolismo , Modelos Animais de Doenças , Efrina-A2/genética , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Receptores ErbB/genética , Receptores ErbB/metabolismo , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Orofaringe/metabolismo , Orofaringe/microbiologia , Receptor EphA2 , Fatores de Virulência/genéticaRESUMO
Iron is an essential nutrient required as a cofactor for many biological processes. As a fungal commensal-pathogen of humans, Candida albicans encounters a range of bioavailable iron levels in the human host and maintains homeostasis with a conserved regulatory circuit. How C. albicans senses and responds to iron availability is unknown. In model yeasts, regulation of the iron homeostasis circuit requires monothiol glutaredoxins (Grxs), but their functions beyond the regulatory circuit are unclear. Here, we show Grx3 is required for virulence and growth on low iron for C. albicans. To explore the global roles of Grx3, we applied a proteomic approach and performed in vivo cross-linked tandem affinity purification coupled with mass spectrometry. We identified a large number of Grx3 interacting proteins that function in diverse biological processes. This included Fra1 and Bol2/Fra2, which function with Grxs in intracellular iron trafficking in other organisms. Grx3 interacts with and regulates the activity of Sfu1 and Hap43, components of the C. albicans iron regulatory circuit. Unlike the regulatory circuit, which determines expression or repression of target genes in response to iron availability, Grx3 amplifies levels of gene expression or repression. Consistent with the proteomic data, the grx3 mutant is sensitive to heat shock, oxidative, nitrosative, and genotoxic stresses, and shows growth dependence on histidine, leucine, and tryptophan. We suggest Grx3 is a conserved global regulator of iron-dependent processes occurring within the cell.
Assuntos
Candida albicans/fisiologia , Candidíase Invasiva/microbiologia , Proteínas Fúngicas/metabolismo , Glutarredoxinas/metabolismo , Ferro/metabolismo , Animais , Candida albicans/patogenicidade , Modelos Animais de Doenças , Proteínas Fúngicas/genética , Proteínas Fúngicas/isolamento & purificação , Fatores de Transcrição GATA/metabolismo , Regulação Fúngica da Expressão Gênica , Glutarredoxinas/genética , Glutarredoxinas/isolamento & purificação , Homeostase , Humanos , Hifas , Masculino , Camundongos , Mutação , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/genética , Proteômica , Virulência/genéticaRESUMO
Metabolic adaptation is linked to the ability of the opportunistic pathogen Candida albicans to colonize and cause infection in diverse host tissues. One way that C. albicans controls its metabolism is through the glucose repression pathway, where expression of alternative carbon source utilization genes is repressed in the presence of its preferred carbon source, glucose. Here we carry out genetic and gene expression studies that identify transcription factors Mig1 and Mig2 as mediators of glucose repression in C. albicans. The well-studied Mig1/2 orthologs ScMig1/2 mediate glucose repression in the yeast Saccharomyces cerevisiae; our data argue that C. albicans Mig1/2 function similarly as repressors of alternative carbon source utilization genes. However, Mig1/2 functions have several distinctive features in C. albicans. First, Mig1 and Mig2 have more co-equal roles in gene regulation than their S. cerevisiae orthologs. Second, Mig1 is regulated at the level of protein accumulation, more akin to ScMig2 than ScMig1. Third, Mig1 and Mig2 are together required for a unique aspect of C. albicans biology, the expression of several pathogenicity traits. Such Mig1/2-dependent traits include the abilities to form hyphae and biofilm, tolerance of cell wall inhibitors, and ability to damage macrophage-like cells and human endothelial cells. Finally, Mig1 is required for a puzzling feature of C. albicans biology that is not shared with S. cerevisiae: the essentiality of the Snf1 protein kinase, a central eukaryotic carbon metabolism regulator. Our results integrate Mig1 and Mig2 into the C. albicans glucose repression pathway and illuminate connections among carbon control, pathogenicity, and Snf1 essentiality.
Assuntos
Candida albicans/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Glucose/metabolismo , Fatores de Transcrição/metabolismo , Animais , Biofilmes , Candida albicans/efeitos dos fármacos , Candida albicans/patogenicidade , Linhagem Celular , Farmacorresistência Fúngica , Células Endoteliais/microbiologia , Proteínas Fúngicas/genética , Humanos , Macrófagos/microbiologia , Camundongos , Proteínas Serina-Treonina Quinases/metabolismo , Fatores de Transcrição/genéticaRESUMO
When the fungus Candida albicans proliferates in the oropharyngeal cavity during experimental oropharyngeal candidiasis (OPC), it undergoes large-scale genome changes at a much higher frequency than when it grows in vitro. Previously, we identified a specific whole chromosome amplification, trisomy of Chr6 (Chr6x3), that was highly overrepresented among strains recovered from the tongues of mice with OPC. To determine the functional significance of this trisomy, we assessed the virulence of two Chr6 trisomic strains and a Chr5 trisomic strain in the mouse model of OPC. We also analyzed the expression of virulence-associated traits in vitro. All three trisomic strains exhibited characteristics of a commensal during OPC in mice. They achieved the same oral fungal burden as the diploid progenitor strain but caused significantly less weight loss and elicited a significantly lower inflammatory host response. In vitro, all three trisomic strains had reduced capacity to adhere to and invade oral epithelial cells and increased susceptibility to neutrophil killing. Whole genome sequencing of pre- and post-infection isolates found that the trisomies were usually maintained. Most post-infection isolates also contained de novo point mutations, but these were not conserved. While in vitro growth assays did not reveal phenotypes specific to de novo point mutations, they did reveal novel phenotypes specific to each lineage. These data reveal that during OPC, clones that are trisomic for Chr5 or Chr6 are selected and they facilitate a commensal-like phenotype.
Assuntos
Candida albicans/genética , Candidíase Bucal/genética , Orofaringe/microbiologia , Animais , Candida albicans/metabolismo , Candidíase/genética , Modelos Animais de Doenças , Células Epiteliais , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Neutrófilos , Fenótipo , Trissomia/genética , VirulênciaRESUMO
The mechanisms by which Candida glabrata resists host defense peptides and caspofungin are incompletely understood. To identify transcriptional regulators that enable C. glabrata to withstand these classes of stressors, a library of 215 C. glabrata transcriptional regulatory deletion mutants was screened for susceptibility to both protamine and caspofungin. We identified eight mutants that had increased susceptibility to both host defense peptides and caspofungin. Of these mutants, six were deleted for genes that were predicted to specify proteins involved in histone modification. These genes were ADA2, GCN5, SPT8, HOS2, RPD3, and SPP1 Deletion of ADA2, GCN5, and RPD3 also increased susceptibility to mammalian host defense peptides. The Δada2 and Δgcn5 mutants had increased susceptibility to other stressors, such as H2O2 and SDS. In the Galleria mellonella model of disseminated infection, the Δada2 and Δgcn5 mutants had attenuated virulence, whereas in neutropenic mice, the virulence of the Δada2 and Δrpd3 mutants was decreased. Thus, histone modification plays a central role in enabling C. glabrata to survive host defense peptides and caspofungin, and Ada2 and Rpd3 are essential for the maximal virulence of this organism during disseminated infection.
Assuntos
Candida glabrata/genética , Candida glabrata/patogenicidade , Proteínas Fúngicas/genética , Interações Hospedeiro-Patógeno/genética , Fatores de Transcrição/genética , Virulência/genética , Deleção de Genes , Variação Genética , Humanos , MutaçãoRESUMO
BACKGROUND & AIMS: A high-fat diet has been associated with an increased risk of ulcerative colitis (UC). We studied the effects of a low-fat, high-fiber diet (LFD) vs an improved standard American diet (iSAD, included higher quantities of fruits, vegetables, and fiber than a typical SAD). We collected data on quality of life, markers of inflammation, and fecal markers of intestinal dysbiosis in patients with UC. METHODS: We analyzed data from a parallel-group, cross-over study of 17 patients with UC in remission or with mild disease (with a flare within the past 18 mo), from February 25, 2015, through September 11, 2018. Participants were assigned randomly to 2 groups and received a LFD (10% of calories from fat) or an iSAD (35%-40% of calories from fat) for the first 4-week period, followed by a 2-week washout period, and then switched to the other diet for 4 weeks. All diets were catered and delivered to patients' homes, and each participant served as her or his own control. Serum and stool samples were collected at baseline and week 4 of each diet and analyzed for markers of inflammation. We performed 16s ribosomal RNA sequencing and untargeted and targeted metabolomic analyses on stool samples. The primary outcome was quality of life, which was measured by the short inflammatory bowel disease (IBD) questionnaire at baseline and week 4 of the diets. Secondary outcomes included changes in the Short-Form 36 health survey, partial Mayo score, markers of inflammation, microbiome and metabolome analysis, and adherence to the diet. RESULTS: Participants' baseline diets were unhealthier than either study diet. All patients remained in remission throughout the study period. Compared with baseline, the iSAD and LFD each increased quality of life, based on the short IBD questionnaire and Short-Form 36 health survey scores (baseline short IBD questionnaire score, 4.98; iSAD, 5.55; LFD, 5.77; baseline vs iSAD, P = .02; baseline vs LFD, P = .001). Serum amyloid A decreased significantly from 7.99 mg/L at baseline to 4.50 mg/L after LFD (P = .02), but did not decrease significantly compared with iSAD (7.20 mg/L; iSAD vs LFD, P = .07). The serum level of C-reactive protein decreased numerically from 3.23 mg/L at baseline to 2.51 mg/L after LFD (P = .07). The relative abundance of Actinobacteria in fecal samples decreased from 13.69% at baseline to 7.82% after LFD (P = .017), whereas the relative abundance of Bacteroidetes increased from 14.6% at baseline to 24.02% on LFD (P = .015). The relative abundance of Faecalibacterium prausnitzii was higher after 4 weeks on the LFD (7.20%) compared with iSAD (5.37%; P = .04). Fecal levels of acetate (an anti-inflammatory metabolite) increased from a relative abundance of 40.37 at baseline to 42.52 on the iSAD and 53.98 on the LFD (baseline vs LFD, P = .05; iSAD vs LFD, P = .09). The fecal level of tryptophan decreased from a relative abundance of 1.33 at baseline to 1.08 on the iSAD (P = .43), but increased to a relative abundance of 2.27 on the LFD (baseline vs LFD, P = .04; iSAD vs LFD, P = .08); fecal levels of lauric acid decreased after LFD (baseline, 203.4; iSAD, 381.4; LFD, 29.91; baseline vs LFD, P = .04; iSAD vs LFD, P = .02). CONCLUSIONS: In a cross-over study of patients with UC in remission, we found that a catered LFD or iSAD were each well tolerated and increased quality of life. However, the LFD decreased markers of inflammation and reduced intestinal dysbiosis in fecal samples. Dietary interventions therefore might benefit patients with UC in remission. ClinicalTrials.gov no: NCT04147598.
Assuntos
Colite Ulcerativa , Qualidade de Vida , Estudos Cross-Over , Dieta , Disbiose , Fezes , Feminino , Humanos , Inflamação , MasculinoRESUMO
Phosphate is an essential macronutrient required for cell growth and division. Pho84 is the major high-affinity cell-surface phosphate importer of Saccharomyces cerevisiae and a crucial element in the phosphate homeostatic system of this model yeast. We found that loss of Candida albicans Pho84 attenuated virulence in Drosophila and murine oropharyngeal and disseminated models of invasive infection, and conferred hypersensitivity to neutrophil killing. Susceptibility of cells lacking Pho84 to neutrophil attack depended on reactive oxygen species (ROS): pho84-/- cells were no more susceptible than wild type C. albicans to neutrophils from a patient with chronic granulomatous disease, or to those whose oxidative burst was pharmacologically inhibited or neutralized. pho84-/- mutants hyperactivated oxidative stress signalling. They accumulated intracellular ROS in the absence of extrinsic oxidative stress, in high as well as low ambient phosphate conditions. ROS accumulation correlated with diminished levels of the unique superoxide dismutase Sod3 in pho84-/- cells, while SOD3 overexpression from a conditional promoter substantially restored these cells' oxidative stress resistance in vitro. Repression of SOD3 expression sharply increased their oxidative stress hypersensitivity. Neither of these oxidative stress management effects of manipulating SOD3 transcription was observed in PHO84 wild type cells. Sod3 levels were not the only factor driving oxidative stress effects on pho84-/- cells, though, because overexpressing SOD3 did not ameliorate these cells' hypersensitivity to neutrophil killing ex vivo, indicating Pho84 has further roles in oxidative stress resistance and virulence. Measurement of cellular metal concentrations demonstrated that diminished Sod3 expression was not due to decreased import of its metal cofactor manganese, as predicted from the function of S. cerevisiae Pho84 as a low-affinity manganese transporter. Instead of a role of Pho84 in metal transport, we found its role in TORC1 activation to impact oxidative stress management: overexpression of the TORC1-activating GTPase Gtr1 relieved the Sod3 deficit and ROS excess in pho84-/- null mutant cells, though it did not suppress their hypersensitivity to neutrophil killing or hyphal growth defect. Pharmacologic inhibition of Pho84 by small molecules including the FDA-approved drug foscarnet also induced ROS accumulation. Inhibiting Pho84 could hence support host defenses by sensitizing C. albicans to oxidative stress.
Assuntos
Candida albicans/patogenicidade , Candidíase/metabolismo , Estresse Oxidativo/fisiologia , Simportadores de Próton-Fosfato/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Animais , Transporte Biológico/fisiologia , Drosophila , Proteínas Fúngicas/metabolismo , Humanos , Camundongos , Fosfatos/metabolismo , Transdução de Sinais/fisiologia , VirulênciaRESUMO
The development of effective antifungal therapeutics remains a formidable challenge because of the close evolutionary relationship between humans and fungi. Mitochondrial function may present an exploitable vulnerability because of its differential utilization in fungi and its pivotal roles in fungal morphogenesis, virulence, and drug resistance already demonstrated by others. We now report mechanistic characterization of ML316, a thiohydantoin that kills drug-resistant Candida species at nanomolar concentrations through fungal-selective inhibition of the mitochondrial phosphate carrier Mir1. Using genetic, biochemical, and metabolomic approaches, we established ML316 as the first Mir1 inhibitor. Inhibition of Mir1 by ML316 in respiring yeast diminished mitochondrial oxygen consumption, resulting in an unusual metabolic catastrophe marked by citrate accumulation and death. In a mouse model of azole-resistant oropharyngeal candidiasis, ML316 reduced fungal burden and enhanced azole activity. Targeting Mir1 could provide a new, much-needed therapeutic strategy to address the rapidly rising burden of drug-resistant fungal infection.
Assuntos
Candidíase/tratamento farmacológico , Mitocôndrias/metabolismo , Fosfatos/metabolismo , Animais , Antifúngicos/farmacologia , Transporte Biológico/efeitos dos fármacos , Candida/efeitos dos fármacos , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Farmacorresistência Fúngica , Feminino , Células Hep G2 , Humanos , Imunossupressores , Camundongos , Camundongos Endogâmicos BALB C , Testes de Sensibilidade Microbiana , Consumo de Oxigênio , Tioidantoínas/farmacologiaRESUMO
BACKGROUND: Candidalysin is a cytolytic peptide toxin secreted by Candida albicans hyphae and has significantly advanced our understanding of fungal pathogenesis. Candidalysin is critical for mucosal C albicans infections and is known to activate epithelial cells to induce downstream innate immune responses that are associated with protection or immunopathology during oral or vaginal infections. Furthermore, candidalysin activates the NLRP3 inflammasome and causes cytolysis in mononuclear phagocytes. However, the role of candidalysin in driving systemic infections is unknown. METHODS: In this study, using candidalysin-producing and candidalysin-deficient C albicans strains, we show that candidalysin activates mitogen-activated protein kinase (MAPK) signaling and chemokine secretion in endothelial cells in vitro. RESULTS: Candidalysin induces immune activation and neutrophil recruitment in vivo, and it promotes mortality in zebrafish and murine models of systemic fungal infection. CONCLUSIONS: The data demonstrate a key role for candidalysin in neutrophil recruitment and fungal virulence during disseminated systemic C albicans infections.
Assuntos
Candida albicans/imunologia , Candida albicans/metabolismo , Candidíase Invasiva/microbiologia , Candidíase Invasiva/patologia , Proteínas Fúngicas/metabolismo , Infiltração de Neutrófilos , Fatores de Virulência/metabolismo , Animais , Candida albicans/crescimento & desenvolvimento , Citocinas/metabolismo , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/imunologia , Células Endoteliais/metabolismo , Feminino , Masculino , Camundongos Endogâmicos BALB C , Transdução de Sinais , Análise de Sobrevida , Virulência , Peixe-ZebraRESUMO
Virulence of the human fungal pathogen Candida albicans depends on the switch from budding to filamentous growth, which requires sustained membrane traffic and polarized growth. In many organisms, small GTPases of the Arf (ADP-ribosylation factor) family regulate membrane/protein trafficking, yet little is known about their role in fungal filamentous growth. To investigate these GTPases in C. albicans, we generated loss of function mutants in all 3 Arf proteins, Arf1-Arf3, and 2 Arf-like proteins, Arl1 and Arl3. Our results indicate that of these proteins, Arf2 is required for viability and sensitivity to antifungal drugs. Repressible ARF2 expression results in defects in filamentous growth, cell wall integrity and virulence, likely due to alteration of the Golgi. Arl1 is also required for invasive filamentous growth and, although arl1/arl1 cells can initiate hyphal growth, hyphae are substantially shorter than that of the wild-type, due to the inability of this mutant to maintain hyphal growth at a single site. We show that this defect does not result from an alteration of phospholipid distribution and is unlikely to result from the sole Golgin Imh1 mislocalization, as Imh1 is not required for invasive filamentous growth. Rather, our results suggest that the arl1/arl1 hyphal growth defect results from increased secretion in this mutant. Strikingly, the arl1/arl1 mutant is drastically reduced in virulence during oropharyngeal candidiasis. Together, our results highlight the importance of Arl1 and Arf2 as key regulators of hyphal growth and virulence in C. albicans and identify a unique function of Arl1 in secretion.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Candida albicans/enzimologia , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Animais , Western Blotting , Candidíase , Modelos Animais de Doenças , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos BALB C , Morfogênese , VirulênciaRESUMO
The capacity of Candida albicans to switch reversibly between the white phenotype and the opaque phenotype is required for the fungus to mate. It also influences virulence during hematogenously disseminated candidiasis. We investigated the roles of the mating type loci (MTL) and white-opaque switching in the capacity of C. albicans to mate in the oropharynx and cause oropharyngeal candidiasis (OPC). When immunosuppressed mice were orally infected with mating-competent opaque a/a and α/α cells either alone or mixed with white cells, no detectable mating occurred, indicating that the mating frequency was less than 1.6 × 10-6 Opaque cells were also highly attenuated in virulence; they either were cleared from the oropharynx or switched to the white phenotype during OPC. Although there were strain-to-strain differences in the virulence of white cells, they were consistently more virulent than opaque cells. In vitro studies indicated that relative to white cells, opaque cells had decreased capacity to invade and damage oral epithelial cells. The reduced invasion of at least one opaque strain was due to reduced surface expression of the Als3 invasin and inability to activate the epidermal growth factor receptor, which is required to stimulate the epithelial cell endocytic machinery. These results suggest that mating is a rare event during OPC because opaque cells have reduced capacity to invade and damage the epithelial cells of the oral mucosa.
Assuntos
Candida albicans/fisiologia , Candidíase Bucal/microbiologia , Genes Fúngicos Tipo Acasalamento/fisiologia , Animais , Candida albicans/classificação , Candidíase Bucal/imunologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Hospedeiro Imunocomprometido , Camundongos , Orofaringe/microbiologia , VirulênciaRESUMO
Candida albicans mutants for phosphatidylserine (PS) synthase (cho1ΔΔ) and PS decarboxylase (psd1ΔΔ psd2ΔΔ) are compromised for virulence in mouse models of systemic infection and oropharyngeal candidiasis (OPC). Both of these enzymes are necessary to synthesize phosphatidylethanolamine (PE) by the de novo pathway, but these mutants are still capable of growth in culture media, as they can import ethanolamine from media to synthesize PE through the Kennedy pathway. Given that the host has ethanolamine in its serum, the exact mechanism by which virulence is lost in these mutants is not clear. There are two competing hypotheses to explain their loss of virulence. (i) PE from the Kennedy pathway cannot substitute for de novo-synthesized PE. (ii) The mutants cannot acquire sufficient ethanolamine from the host to support adequate PE synthesis. These hypotheses can be simultaneously tested if ethanolamine availability is increased for Candida while it is inside the host. We accomplish this by transcomplementation of C. albicans with the Arabidopsis thaliana serine decarboxylase gene (AtSDC), which converts cytoplasmic serine to ethanolamine. Expression of AtSDC in either mutant restores PE synthesis, even in the absence of exogenous ethanolamine. AtSDC also restores virulence to cho1ΔΔ and psd1ΔΔ psd2ΔΔ strains in systemic and OPC infections. Thus, in the absence of de novo PE synthesis, C. albicans cannot acquire sufficient ethanolamine from the host to support virulence. In addition, expression of AtSDC restores PS synthesis in the cho1ΔΔ mutant, which may be due to causing PS decarboxylase to run backwards and convert PE to PS.
Assuntos
Candida albicans/genética , Candida albicans/metabolismo , Carboxiliases/metabolismo , Etanolamina/metabolismo , Fosfatidiletanolaminas/metabolismo , Virulência/genética , Virulência/fisiologia , Animais , Candida albicans/crescimento & desenvolvimento , Candida albicans/patogenicidade , Variação Genética , Interações Hospedeiro-Patógeno/fisiologia , CamundongosRESUMO
Gene expression dynamics have provided foundational insight into almost all biological processes. Here, we analyze expression of environmentally responsive genes and transcription factor genes to infer signals and pathways that drive pathogen gene regulation during invasive Candida albicans infection of a mammalian host. Environmentally responsive gene expression shows that there are early and late phases of infection. The early phase includes induction of zinc and iron limitation genes, genes that respond to transcription factor Rim101, and genes characteristic of invasive hyphal cells. The late phase includes responses related to phagocytosis by macrophages. Transcription factor gene expression also reflects early and late phases. Transcription factor genes that are required for virulence or proliferation in vivo are enriched among highly expressed transcription factor genes. Mutants defective in six transcription factor genes, three previously studied in detail (Rim101, Efg1, Zap1) and three less extensively studied (Rob1, Rpn4, Sut1), are profiled during infection. Most of these mutants have distinct gene expression profiles during infection as compared to in vitro growth. Infection profiles suggest that Sut1 acts in the same pathway as Zap1, and we verify that functional relationship with the finding that overexpression of either ZAP1 or the Zap1-dependent zinc transporter gene ZRT2 restores pathogenicity to a sut1 mutant. Perturbation with the cell wall inhibitor caspofungin also has distinct gene expression impact in vivo and in vitro. Unexpectedly, caspofungin induces many of the same genes that are repressed early during infection, a phenomenon that we suggest may contribute to drug efficacy. The pathogen response circuitry is tailored uniquely during infection, with many relevant regulatory relationships that are not evident during growth in vitro. Our findings support the principle that virulence is a property that is manifested only in the distinct environment in which host-pathogen interaction occurs.
Assuntos
Candida albicans/metabolismo , Candida albicans/patogenicidade , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Animais , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candidíase/tratamento farmacológico , Candidíase/metabolismo , Candidíase/microbiologia , Candidíase/patologia , Caspofungina , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Equinocandinas/farmacologia , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Injeções Intravenosas , Rim/metabolismo , Rim/microbiologia , Rim/patologia , Lipopeptídeos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Anotação de Sequência Molecular , Mutação , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , VirulênciaRESUMO
BACKGROUND: The renin-angiotensin system is key in the physiopathology of arterial hypertension because it converts angiotensin I, via angiotensin I-converting enzyme (ACE), into angiotensin II. In vitro analyses were done of the ACE-inhibitory and renin-inhibitory activities of peptide fractions isolated by enzymatic hydrolysis of lima bean (Phaseolus lunatus) protein. Antihypertensive activity was confirmed in vivo using a rat model. RESULTS: Lima bean protein was hydrolyzed with one of two sequential enzymatic systems (pepsin-pancreatin or Alcalase®-Flavourzyme®). Ultrafiltration of the hydrolysates produced fractions of different molecular weights. The >3 kDa fraction of the pepsin-pancreatin hydrolysate had the highest ACE-inhibitory activity (60.15%, IC50: 172.62 µg mL-1 ), while the >3 KDa fraction of the Alcalase®-Flavourzyme® hydrolysate had the highest in vitro renin-inhibitory activity. A weak correlation (r = 0.44) was found between ACE-inhibitory and renin-inhibitory activities. When tested in vivo, the latter fraction lowered systolic blood pressure by 64% and diastolic blood pressure by 51%. CONCLUSION: Peptide fractions from lima bean Phaseolus lunatus protein hydrolysates exhibit both in vitro and in vivo antihypertensive activity. Bioactive peptides from lima bean have potential applications as ingredients in functional foods. © 2017 Society of Chemical Industry.