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1.
Nat Commun ; 11(1): 2331, 2020 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-32393780

RESUMO

Extracellular vesicles have an important function in cellular communication. Here, we show that human and mouse monocytes release TGF-ß1-transporting vesicles in response to the pathogenic fungus Candida albicans. Soluble ß-glucan from C. albicans binds to complement receptor 3 (CR3, also known as CD11b/CD18) on monocytes and induces the release of TGF-ß1-transporting vesicles. CR3-dependence is demonstrated using CR3-deficient (CD11b knockout) monocytes generated by CRISPR-CAS9 genome editing and isolated from CR3-deficient (CD11b knockout) mice. These vesicles reduce the pro-inflammatory response in human M1-macrophages as well as in whole blood. Binding of the vesicle-transported TGF-ß1 to the TGF-ß receptor inhibits IL1B transcription via the SMAD7 pathway in whole blood and induces TGFB1 transcription in endothelial cells, which is resolved upon TGF-ß1 inhibition. Notably, human complement-opsonized apoptotic bodies induce production of similar TGF-ß1-transporting vesicles in monocytes, suggesting that the early immune response might be suppressed through this CR3-dependent anti-inflammatory vesicle pathway.


Assuntos
Imunomodulação , Antígeno de Macrófago 1/metabolismo , Monócitos/metabolismo , Fator de Crescimento Transformador beta1/metabolismo , Vesículas Transportadoras/metabolismo , Animais , Apoptose , Candida albicans/metabolismo , Candida albicans/ultraestrutura , Regulação para Baixo , Difusão Dinâmica da Luz , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Inflamação/patologia , Interleucina-6/genética , Interleucina-6/metabolismo , Macrófagos/metabolismo , Camundongos Endogâmicos C57BL , Modelos Biológicos , Monócitos/microbiologia , Monócitos/ultraestrutura , Transporte Proteico , Solubilidade , Transcrição Genética , Regulação para Cima , beta-Glucanas/metabolismo
3.
Biofouling ; 36(2): 210-221, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32292058

RESUMO

Surfactin is a type of cyclic lipopeptide biosurfactant implicated in a wide range of applications. Although its antimicrobial activity has been characterized, its effect on Candida albicans physiology remains to be elucidated. The present study evaluated the influence of surfactin-C15 (SF) and its complexes with divalent counterions on C. albicans biofilm formation and preformed biofilms. The SF and metal(II)-SF complexes inhibited biofilm formation and reduced the metabolic activity of mature biofilms in a concentration-dependent manner. The same concentrations of the compounds studied dislodged preexisting biofilms grown on polystyrene plates. Moreover, SF and its metal(II) complexes reduced the mRNA expression of hypha-specific genes HWP1, ALS1, ALS3, ECE1 and SAP4 without exhibiting significant growth inhibition. Further research showed that the compounds tested reduced cellular surface hydrophobicity (CSH). These results suggest that SF and metal(II)-SF complexes could be used as anti-biofilm agents against C. albicans hypha-related infections in clinical practice.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Complexos de Coordenação/farmacologia , Hifas/efeitos dos fármacos , Tensoativos/metabolismo , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Proteínas Fúngicas/genética , Proteínas de Fluorescência Verde/genética , Hifas/crescimento & desenvolvimento
4.
J Biosci Bioeng ; 130(1): 6-13, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32192842

RESUMO

Defensins are antibacterial peptides that function in the innate immune system. OsAFP1, a defensin identified from Oryza sativa (rice), exhibits antimicrobial activity against rice pathogens. Intriguingly, OsAFP1 was also shown to demonstrate potent antifungal activity against the human pathogenic fungus Candida albicans by inducing apoptosis in target cells, suggesting that OsAFP1 represents a potential new antibiotic candidate; however, further analyses, particularly at the structural level, are required to elucidate the mechanistic underpinnings of OsAFP1 antifungal activity. Here, we determined the three-dimensional structure of OsAFP1 using X-ray crystallography. OsAFP1 features the cysteine-stabilized αß structure highly conserved in plant defensins and presents a dimeric structure that appears necessary for antifungal activity. Superimposition of the OsAFP1 structure with that of Nicotiana alata NaD1 complexed with phosphatidic acid indicated that the target molecule is likely trapped between the S2-S3 loops of each OsAFP1 dimer. In lipid-binding analyses performed using nitrocellulose membranes immobilized with various membrane lipid components, OsAFP1 was found to bind to phosphatidylinositols (PIPs) harboring phosphate groups, particularly PI(3)P. These results indicate that OsAFP1 exerts antifungal activity by binding to PI(3)P contained in the C. albicans cell membrane, thereby applying cellular stress and inducing apoptosis. Furthermore, the OsAFP1 structure and site-specific-mutation analyses revealed that Arg1, His2, Leu4, Arg9, and Phe10 play critical roles in OsAFP1 dimer formation. Thus, our study provides novel insights into the antifungal mechanism of OsAFP1.


Assuntos
Defensinas/química , Defensinas/metabolismo , Oryza/metabolismo , Fosfatidilinositóis/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Antifúngicos/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Cristalização , Defensinas/genética , Defensinas/farmacologia , Oryza/química , Oryza/genética , Fosfatidilinositóis/química , Proteínas de Plantas/genética , Proteínas de Plantas/farmacologia
5.
Nucleic Acids Res ; 48(5): 2312-2331, 2020 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-32020195

RESUMO

Eukaryotic protein synthesis generally initiates at a start codon defined by an AUG and its surrounding Kozak sequence context, but the quantitative importance of this context in different species is unclear. We tested this concept in two pathogenic Cryptococcus yeast species by genome-wide mapping of translation and of mRNA 5' and 3' ends. We observed thousands of AUG-initiated upstream open reading frames (uORFs) that are a major contributor to translation repression. uORF use depends on the Kozak sequence context of its start codon, and uORFs with strong contexts promote nonsense-mediated mRNA decay. Transcript leaders in Cryptococcus and other fungi are substantially longer and more AUG-dense than in Saccharomyces. Numerous Cryptococcus mRNAs encode predicted dual-localized proteins, including many aminoacyl-tRNA synthetases, in which a leaky AUG start codon is followed by a strong Kozak context in-frame AUG, separated by mitochondrial-targeting sequence. Analysis of other fungal species shows that such dual-localization is also predicted to be common in the ascomycete mould, Neurospora crassa. Kozak-controlled regulation is correlated with insertions in translational initiation factors in fidelity-determining regions that contact the initiator tRNA. Thus, start codon context is a signal that quantitatively programs both the expression and the structures of proteins in diverse fungi.


Assuntos
Códon de Iniciação/química , Cryptococcus/genética , Regulação Fúngica da Expressão Gênica , Genoma Fúngico , Iniciação Traducional da Cadeia Peptídica , Aminoacil-tRNA Sintetases/genética , Aminoacil-tRNA Sintetases/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Mapeamento Cromossômico , Códon de Iniciação/metabolismo , Cryptococcus/metabolismo , Neurospora crassa/genética , Neurospora crassa/metabolismo , Fases de Leitura Aberta , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Especificidade da Espécie
6.
J Mycol Med ; 30(1): 100921, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31937429

RESUMO

AIM: Among the most common mechanisms of multidrug resistance (MDR) in prevalent human fungal pathogen, Candida albicans, overexpression of drug efflux pumps remains the predominant mechanism. Hence to inhibit efflux pumps and chemosensitize C. albicans against traditional antifungal drugs still represents an attractive approach. The present study aimed to analyze the effect of Vanillin (Van), a natural food flavoring agent, on drug efflux pump activity of Candida albicans. METHODS AND RESULTS: We observed that Van specifically inhibits Candida drug resistance protein 2 (CaCdr2p) activity belonging to ATP Binding Cassette (ABC) superfamily as revealed by abrogated rhodamine 6G efflux and nile red accumulation assay with CaCdr2p over expressing strain. Insight studies into the mechanisms suggested that abrogated efflux by CaCdr2p is due to competitive mode of inhibition by Van as depicted by Lineweaver-Burk plot. RT-PCR, western blot and confocal microscopy further unraveled that Van leads to reduced expression of CDR2 and CaCdr2p mislocalization respectively. Furthermore, Van sensitizes the azole sensitive and resistant clinical matched pair of isolates Gu4/Gu5 and led to abrogated rhodamine 6G efflux and depleted ergosterol. Furthermore, Van synergizes with membrane targeting drugs fluconazole and amphotericin B as their fractional inhibitory coefficient index was less than 0.5. CONCLUSION: Van being a potent inhibitor of CaCdr2p and chemosensitizing of drug resistant C. albicans warrants further studies to be exploited as effective antifungal agent.


Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Antifúngicos/farmacologia , Benzaldeídos/farmacologia , Candida albicans , Farmacorresistência Fúngica/genética , Transportadores de Cassetes de Ligação de ATP/efeitos dos fármacos , Transportadores de Cassetes de Ligação de ATP/genética , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/genética , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candida albicans/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Sinergismo Farmacológico , Proteínas Fúngicas/efeitos dos fármacos , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Testes de Sensibilidade Microbiana
7.
Biofouling ; 36(1): 56-72, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31997658

RESUMO

The effects of two prominent copper oxide nanoparticles (CuO-NP and Cu2O-NP), with the oxidation state of Cu++ (cupric) and Cu+ (cuprous), on Candida albicans were evaluated. CuO-NP and Cu2O-NP were synthesized and characterized by XRD, FESEM, HR-TEM and Zeta potential. At sub-MIC (50 µg ml-1), both cupric and cuprous oxide NPs prevented yeast-to-hyphae switching and wrinkling behaviour in C. albicans. The mechanism for the antifungal action of the two NPs differed; CuO-NP significantly elicited reactive oxygen species, whereas membrane damage was more pronounced with Cu2O-NP. Real time PCR analysis revealed that CuO-NP suppressed the morphological switching of yeast-to-hyphae by down-regulating cph1, hst7 and ras1 and by up-regulation of the negative regulator tup1. In comparison, Cu2O-NP resulted in down-regulation of ras1 and up-regulation of the negative regulators nrg1 and tup1. Between the two NPs, CuO exhibited increased antifungal activity due to its stable oxidation state (Cu++) and its smaller dimensions compared with Cu2O-NP.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Cobre/farmacologia , Hifas/efeitos dos fármacos , Nanopartículas Metálicas/química , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Membrana Celular/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Testes de Sensibilidade Microbiana , Espécies Reativas de Oxigênio/metabolismo
8.
Ann Clin Microbiol Antimicrob ; 19(1): 5, 2020 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-31992328

RESUMO

Recurrent vulvovaginal infections (RVVI) has not only become an epidemiological and clinical problem but also include large social and psychological consequences. Understanding the mechanisms of both commensalism and pathogenesis are necessary for the development of efficient diagnosis and treatment strategies for these enigmatic vaginal infections. Through this review, an attempt has been made to analyze vaginal microbiota (VMB) from scratch and to provide an update on its current understanding in relation to health and common RVVI i.e. bacterial vaginosis, vulvovaginal candidiaisis and Trichomoniasis, making the present review first of its kind. For this, potentially relevant studies were retrieved from data sources and critical analysis of the literature was made. Though, culture-independent methods have greatly unfolded the mystery regarding vaginal bacterial microbiome, there are only a few studies regarding the composition and diversity of vaginal mycobiome and different Trichomonas vaginalis strains. This scenario suggests a need of further studies based on comparative genomics of RVVI pathogens to improve our perceptive of RVVI pathogenesis that is still not clear (Fig. 5). Besides this, the review details the rationale for Lactobacilli dominance and changes that occur in healthy VMB throughout a women's life. Moreover, the list of possible agents continues to expand and new species recognised in both health and VVI are updated in this review. The review concludes with the controversies challenging the widely accepted dogma i.e. "VMB dominated with Lactobacilli is healthier than a diverse VMB". These controversies, over the past decade, have complicated the definition of vaginal health and vaginal infections with no definite conclusion. Thus, further studies on newly recognised microbial agents may reveal answers to these controversies. Conversely, VMB of women could be an answer but it is not enough to just look at the microbiology. We have to look at the woman itself, as VMB which is fine for one woman may be troublesome for others. These differences in women's response to the same VMB may be determined by a permutation of behavioural, cultural, genetic and various other anonymous factors, exploration of which may lead to proper definition of vaginal health and disease.


Assuntos
Candidíase Vulvovaginal , Microbiota , Vaginite por Trichomonas , Vagina/microbiologia , Vaginose Bacteriana , Biofilmes/crescimento & desenvolvimento , Candida/isolamento & purificação , Candida/metabolismo , Candida albicans/isolamento & purificação , Candida albicans/metabolismo , Candidíase Vulvovaginal/microbiologia , Candidíase Vulvovaginal/patologia , Candidíase Vulvovaginal/transmissão , Coinfecção/microbiologia , Coinfecção/parasitologia , Feminino , Gardnerella vaginalis/isolamento & purificação , Interações entre Hospedeiro e Microrganismos , Humanos , Lactobacillus/isolamento & purificação , Lactobacillus/metabolismo , Interações Microbianas , Microbiota/fisiologia , Recidiva , Vaginite por Trichomonas/parasitologia , Vaginite por Trichomonas/patologia , Vaginite por Trichomonas/transmissão , Trichomonas vaginalis/isolamento & purificação , Trichomonas vaginalis/metabolismo , Vagina/parasitologia , Vaginose Bacteriana/microbiologia , Vaginose Bacteriana/patologia , Vaginose Bacteriana/transmissão , Fatores de Virulência/metabolismo
9.
BMC Genomics ; 21(1): 57, 2020 Jan 17.
Artigo em Inglês | MEDLINE | ID: mdl-31952470

RESUMO

BACKGROUND: Candida albicans is an opportunistic pathogenic yeast, which could become pathogenic in various stressful environmental factors including the spaceflight environment. In this study, we aim to explore the phenotypic changes and possible mechanisms of C. albicans after exposure to spaceflight conditions. RESULTS: The effect of C. albicans after carried on the "SJ-10" satellite for 12 days was evaluated by proliferation, morphology, environmental resistance and virulence experiment. The result showed that the proliferation rate, biofilm formation, antioxidant capacity, cytotoxicity and filamentous morphology of C. albicans were increased in the spaceflight group compared to the control group. Proteomics and metabolomics technologies were used to analyze the profiles of proteins and metabolites in C. albicans under spaceflight conditions. Proteomic analysis identified 548 up-regulated proteins involved in the ribosome, DNA replication, base excision repair and sulfur metabolism in the spaceflight group. Moreover, 332 down-regulated proteins related to metabolic processes were observed. The metabolomic analysis found five differentially expressed metabolites. The combined analysis of proteomic and metabolomic revealed the accumulation of cysteine and methionine in C. albicans after spaceflight. CONCLUSIONS: Mechanisms that could explain the results in the phenotypic experiment of C. albicans were found through proteomic and metabolomic analysis. And our data provide an important basis for the assessment of the risk that C. albicans could cause under spaceflight environment.


Assuntos
Candida albicans/metabolismo , Metaboloma , Proteoma , Voo Espacial , Candida albicans/citologia , Candida albicans/patogenicidade , Candida albicans/fisiologia , Metabolômica , Proteômica , Virulência
10.
Biochim Biophys Acta Proteins Proteom ; 1868(3): 140135, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-31964485

RESUMO

Heat shock proteins are best known for their role as chaperonins involved in general proteostasis, but they can also participate in specific cellular regulatory pathways, e.g. via their post-translational modification. Hsp70/Ssa1 is a central cytoplasmic chaperonin in eukaryotes, which also participates in cell cycle regulation via its phosphorylation at a specific residue. Here we analyze the role of Ssa1 phosphorylation in the morphogenesis of the fungus Candida albicans, a common human opportunistic pathogen. C. albicans can assume alternative yeast and hyphal (mold) morphologies, an ability that contributes to its virulence. We identified 11 phosphorylation sites on C. albicans Ssa1, of which 8 were only detected in the hyphal cells. Genetic analysis of these sites revealed allele-specific effects on growth or hyphae formation at 42 °C. Colony morphology, which is normally wrinkled or crenellated at 37 °C, reverted to smooth in several mutants, but this colony morphology phenotype was unrelated to cellular morphology. Two mutants exhibited a mild increase in sensitivity to the cell wall-active compounds caspofungin and calcofluor white. We suggest that this analysis could help direct screens for Ssa1-specific drugs to combat C. albicans virulence. The pleiotropic effects of many Ssa1 mutations are consistent with the large number of Ssa1 client proteins, whereas the lack of concordance between the phenotypes of the different alleles suggests that different sites on Ssa1 can affect interaction with specific classes of client proteins, and that modification of these sites can play cellular regulatory roles, consistent with the "chaperone code" hypothesis.


Assuntos
Candida albicans/citologia , Candida albicans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Candida albicans/genética , Candida albicans/crescimento & desenvolvimento , Parede Celular/efeitos dos fármacos , Proteínas Fúngicas/química , Proteínas de Choque Térmico HSP70/química , Hifas/crescimento & desenvolvimento , Hifas/metabolismo , Morfogênese , Fosforilação
11.
Infect Immun ; 88(3)2020 02 20.
Artigo em Inglês | MEDLINE | ID: mdl-31792076

RESUMO

Candida albicans is a leading cause of systemic bloodstream infections, and synthesis of the phospholipid phosphatidylethanolamine (PE) is required for virulence. The psd1Δ/Δ psd2Δ/Δ mutant, which cannot synthesize PE by the cytidine diphosphate diacylglycerol (CDP-DAG) pathway, is avirulent in the mouse model of systemic candidiasis. Similarly, an ept1Δ/Δ mutant, which cannot produce PE by the Kennedy pathway, exhibits decreased kidney fungal burden in systemically infected mice. Conversely, overexpression of EPT1 results in a hypervirulent phenotype in this model. Thus, mutations that increase PE synthesis increase virulence, and mutations that decrease PE synthesis decrease virulence. However, the mechanism by which virulence is regulated by PE synthesis is only partially understood. RNA sequencing was performed on strains with deficient or excessive PE biosynthesis to elucidate the mechanism. Decreased PE synthesis from loss of EPT1 or PSD1 and PSD2 leads to downregulation of genes that impact mitochondrial function. Losses of PSD1 and PSD2, but not EPT1, cause significant increases in transcription of glycosylation genes, which may reflect the substantial cell wall defects in the psd1Δ/Δ psd2Δ/Δ mutant. These accumulated defects could contribute to the decreased virulence observed for mutants with deficient PE synthesis. In contrast to mutants with decreased PE synthesis, there were no transcriptional differences between the EPT1 overexpression strain and the wild type, indicating that the hypervirulent phenotype is a consequence of posttranscriptional changes. It was found that overexpression of EPT1 causes increased chitin content and increased hyphal length. These phenotypes may help to explain the previously observed hypervirulence in the EPT1 overexpressor.


Assuntos
Candida albicans/patogenicidade , Parede Celular/química , Hifas/citologia , Fosfatidiletanolaminas/metabolismo , Candida albicans/metabolismo , Candidíase/microbiologia , Parede Celular/metabolismo , Quitina/metabolismo , Transcrição Genética
12.
Biochim Biophys Acta Biomembr ; 1862(2): 183131, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-31734312

RESUMO

ATP-binding cassette (ABC) transporters couple ATP binding and hydrolysis to the translocation of allocrites across membranes. Two shared nucleotide-binding sites (NBS) participate in this cycle. In asymmetric ABC pumps, only one of them hydrolyzes ATP, and the functional role of the other remains unclear. Using a drug-based selection strategy on the transport-deficient mutant L529A in the transmembrane domain of the Candida albicans pump Cdr1p; we identified a spontaneous secondary mutation restoring drug-translocation. The compensatory mutation Q1005H was mapped 60 Å away, precisely in the ABC signature sequence of the non-hydrolytic NBS. The same was observed in the homolog Cdr2p. Both the mutant and suppressor proteins remained ATPase active, but remarkably, the single Q1005H mutant displayed a two-fold reduced ATPase activity and a two-fold increased drug-resistance as compared to the wild-type protein, pointing at a direct control of the non-hydrolytic NBS in substrate-translocation through ATP binding in asymmetric ABC pumps.


Assuntos
Transportadores de Cassetes de Ligação de ATP/química , Trifosfato de Adenosina/metabolismo , Antifúngicos/farmacologia , Proteínas Fúngicas/química , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sítios de Ligação , Candida albicans/efeitos dos fármacos , Candida albicans/enzimologia , Candida albicans/metabolismo , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Mutação , Ligação Proteica
13.
PLoS Pathog ; 15(12): e1007823, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31809527

RESUMO

Inside the human host, the pathogenic yeast Candida albicans colonizes predominantly oxygen-poor niches such as the gastrointestinal and vaginal tracts, but also oxygen-rich environments such as cutaneous epithelial cells and oral mucosa. This suppleness requires an effective mechanism to reversibly reprogram the primary metabolism in response to oxygen variation. Here, we have uncovered that Snf5, a subunit of SWI/SNF chromatin remodeling complex, is a major transcriptional regulator that links oxygen status to the metabolic capacity of C. albicans. Snf5 and other subunits of SWI/SNF complex were required to activate genes of carbon utilization and other carbohydrates related process specifically under hypoxia. snf5 mutant exhibited an altered metabolome reflecting that SWI/SNF plays an essential role in maintaining metabolic homeostasis and carbon flux in C. albicans under hypoxia. Snf5 was necessary to activate the transcriptional program linked to both commensal and invasive growth. Accordingly, snf5 was unable to maintain its growth in the stomach, the cecum and the colon of mice. snf5 was also avirulent as it was unable to invade Galleria larvae or to cause damage to human enterocytes and murine macrophages. Among candidates of signaling pathways in which Snf5 might operate, phenotypic analysis revealed that mutants of Ras1-cAMP-PKA pathway, as well as mutants of Yak1 and Yck2 kinases exhibited a similar carbon flexibility phenotype as did snf5 under hypoxia. Genetic interaction analysis indicated that the adenylate cyclase Cyr1, a key component of the Ras1-cAMP pathway interacted genetically with Snf5. Our study yielded new insight into the oxygen-sensitive regulatory circuit that control metabolic flexibility, stress, commensalism and virulence in C. albicans.


Assuntos
Candida albicans/metabolismo , Candida albicans/patogenicidade , Regulação Fúngica da Expressão Gênica/fisiologia , Virulência/fisiologia , Animais , Candida albicans/genética , Proteínas Fúngicas/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Humanos , Fatores de Transcrição/metabolismo
14.
Genes (Basel) ; 10(12)2019 12 05.
Artigo em Inglês | MEDLINE | ID: mdl-31817479

RESUMO

Mad2 is a central component of the spindle assembly checkpoint required for accurate chromosome segregation. Additionally, in some organisms, Mad2 has roles in preventing mutations and recombination through the DNA damage response. In the fungal pathogen Candida albicans, CaMad2 has previously been shown to be required for accurate chromosome segregation, survival in high levels of hydrogen peroxide, and virulence in a mouse model of infection. In this work, we showed that CaMad2 promotes genome stability through its well-characterized role in promoting accurate chromosome segregation and through reducing smaller scale chromosome changes due to recombination and DNA damage repair. Deletion of MAD2 decreased cell growth, increased marker loss rates, increased sensitivity to microtubule-destabilizing drugs, and increased sensitivity to DNA damage inducing treatments. CaMad2-GFP localized to dots, consistent with a role in kinetochore binding, and to the nuclear periphery, consistent with an additional role in DNA damage. Furthermore, deletion of MAD2 increases growth on fluconazole, and fluconazole treatment elevates whole chromosome loss rates in the mad2∆/∆ strain, suggesting that CaMad2 may be important for preventing fluconazole resistance via aneuploidy.


Assuntos
Candida albicans/metabolismo , Segregação de Cromossomos , Cromossomos Fúngicos/metabolismo , Genoma Fúngico , Instabilidade Genômica , Proteínas Mad2/metabolismo , Candida albicans/genética , Cromossomos Fúngicos/genética , Fluconazol/farmacologia , Cinetocoros/metabolismo , Proteínas Mad2/genética
15.
Int J Mol Sci ; 20(23)2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31757076

RESUMO

In yeast, we reported the critical role of K+-efflux for the progress of the regulated cell death (RCD) induced by human lactoferrin (hLf), an antimicrobial protein of the innate immune system that blocks Pma1p H+-ATPase. In the present study, the K+ channel Tok1p was identified as the K+ channel-mediating K+-efflux, as indicated by the protective effect of extracellular K+ (30 mM), K+-channel blockers, and the greater hLf-resistance of TOK1-disrupted strains. K+-depletion was necessary but not sufficient to induce RCD as inferred from the effects of valinomycin, NH4Cl or nigericin which released a percentage of K+ similar to that released by lactoferrin without affecting cell viability. Cytosolic pH of hLf-treated cells decreased transiently (0.3 pH units) and its inhibition prevented the RCD process, indicating that cytosolic acidification was a necessary and sufficient triggering signal. The blocking effect of lactoferrin on Pma1p H+-ATPase caused a transitory decrease of cytosolic pH, and the subsequent membrane depolarization activated the voltage-gated K+ channel, Tok1p, allowing an electrogenic K+-efflux. These ionic events, cytosolic accumulation of H+ followed by K+-efflux, constituted the initiating signals of this mitochondria-mediated cell death. These findings suggest, for the first time, the existence of an ionic signaling pathway in RCD.


Assuntos
Apoptose , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Canais de Potássio/metabolismo , Bombas de Próton/metabolismo , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Lactoferrina/farmacologia , Transdução de Sinais
16.
Nat Commun ; 10(1): 5315, 2019 11 22.
Artigo em Inglês | MEDLINE | ID: mdl-31757950

RESUMO

To colonise their host, pathogens must counter local environmental and immunological challenges. Here, we reveal that the fungal pathogen Candida albicans exploits diverse host-associated signals to promote immune evasion by masking of a major pathogen-associated molecular pattern (PAMP), ß-glucan. Certain nutrients, stresses and antifungal drugs trigger ß-glucan masking, whereas other inputs, such as nitrogen sources and quorum sensing molecules, exert limited effects on this PAMP. In particular, iron limitation triggers substantial changes in the cell wall that reduce ß-glucan exposure. This correlates with reduced phagocytosis by macrophages and attenuated cytokine responses by peripheral blood mononuclear cells. Iron limitation-induced ß-glucan masking depends on parallel signalling via the iron transceptor Ftr1 and the iron-responsive transcription factor Sef1, and the protein kinase A pathway. Our data reveal that C. albicans exploits a diverse range of specific host signals to trigger protective anticipatory responses against impending phagocytic attack and promote host colonisation.


Assuntos
Candida albicans/metabolismo , Citocinas/imunologia , Evasão da Resposta Imune/fisiologia , Ferro/metabolismo , Macrófagos/imunologia , Fagocitose/imunologia , beta-Glucanas/metabolismo , Animais , Candida albicans/imunologia , Parede Celular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Fúngicas/metabolismo , Humanos , Evasão da Resposta Imune/imunologia , Imunidade Inata/imunologia , Leucócitos Mononucleares/imunologia , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Padrões Moleculares Associados a Patógenos/imunologia , Padrões Moleculares Associados a Patógenos/metabolismo , Transdução de Sinais , beta-Glucanas/imunologia
17.
Mol Med Rep ; 20(5): 4723-4730, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31702024

RESUMO

The present study aimed to evaluate the antimicrobial activity and the possible mechanisms of activity of polypeptide­enriched Gastrodia elata extracts (GEP) against the gram­negative bacteria Escherichia coli and Pseudomonas aeruginosa, the gram­positive bacterium Staphylococcus aureus and the fungus Candida albicans. The antimicrobial activity of GEP was first confirmed by determining the minimum inhibitory concentration by growth curve analysis. GEP was found to damage the cell wall and membrane of the microorganisms tested, as revealed by the morphological changes visible through scanning electron microscopy, and by the observed leakage of alkaline phosphatase and ß­galactosidase from cells. GEP was demonstrated to perturb the metabolism of the microorganisms, especially the tricarboxylic acid cycle, as indicated by the reduced intracellular activity of succinate dehydrogenase, malate dehydrogenase and ATPases, including the Na+/K+­ATPase and the Ca2+­ATPase. In addition, GEP caused the leakage of the genetic material of the bacteria and the fungus, as indicated by the increased OD260. The results of the present study indicated that GEP may exert its antimicrobial activity by damaging cell walls and membranes, causing the leakage of genetic material, and by perturbing cellular metabolism.


Assuntos
Anti-Infecciosos , Peptídeos Catiônicos Antimicrobianos , Bactérias/metabolismo , Candida albicans/metabolismo , Gastrodia/química , Proteínas de Plantas , Anti-Infecciosos/química , Anti-Infecciosos/metabolismo , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/metabolismo , Peptídeos Catiônicos Antimicrobianos/farmacologia , Proteínas de Bactérias/metabolismo , Membrana Celular/metabolismo , Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacologia
18.
Int J Mol Sci ; 20(21)2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31671552

RESUMO

Ureases from different biological sources display non-ureolytic properties that contribute to plant defense, in addition to their classical enzymatic urea hydrolysis. Antifungal and entomotoxic effects were demonstrated for Jaburetox, an intrinsically disordered polypeptide derived from jack bean (Canavalia ensiformis) urease. Here we describe the properties of Soyuretox, a polypeptide derived from soybean (Glycine max) ubiquitous urease. Soyuretox was fungitoxic to Candida albicans, leading to the production of reactive oxygen species. Soyuretox further induced aggregation of Rhodnius prolixus hemocytes, indicating an interference on the insect immune response. No relevant toxicity of Soyuretox to zebrafish larvae was observed. These data suggest the presence of antifungal and entomotoxic portions of the amino acid sequences encompassing both Soyuretox and Jaburetox, despite their small sequence identity. Nuclear Magnetic Resonance (NMR) and circular dichroism (CD) spectroscopic data revealed that Soyuretox, in analogy with Jaburetox, possesses an intrinsic and largely disordered nature. Some folding is observed upon interaction of Soyuretox with sodium dodecyl sulfate (SDS) micelles, taken here as models for membranes. This observation suggests the possibility for this protein to modify its secondary structure upon interaction with the cells of the affected organisms, leading to alterations of membrane integrity. Altogether, Soyuretox can be considered a promising biopesticide for use in plant protection.


Assuntos
Agentes de Controle Biológico/farmacologia , Peptídeos/farmacologia , Soja/enzimologia , Urease/química , Animais , Agentes de Controle Biológico/química , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Dicroísmo Circular , Hemócitos/efeitos dos fármacos , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Simulação de Dinâmica Molecular , Peptídeos/química , Proteínas de Plantas/química , Dobramento de Proteína , Espécies Reativas de Oxigênio/metabolismo , Rhodnius/efeitos dos fármacos
19.
Int J Mol Sci ; 20(20)2019 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-31614738

RESUMO

The essential role of dolichyl phosphate (DolP) as a carbohydrate carrier during protein N-glycosylation is well established. The cellular pool of DolP is derived from de novo synthesis in the dolichol branch of the mevalonate pathway and from recycling of DolPP after each cycle of N-glycosylation, when the oligosaccharide is transferred from the lipid carrier to the protein and DolPP is released and then dephosphorylated. In Saccharomyces cerevisiae, the dephosphorylation of DolPP is known to be catalyzed by the Cwh8p protein. To establish the role of the Cwh8p orthologue in another distantly related yeast species, Candida albicans, we studied its mutant devoid of the CaCWH8 gene. A double Cacwh8∆/Cacwh8∆ strain was constructed by the URA-blaster method. As in S. cerevisiae, the mutant was impaired in DolPP recycling. This defect, however, was accompanied by an elevation of cis-prenyltransferase activity and higher de novo production of dolichols. Despite these compensatory changes, protein glycosylation, cell wall integrity, filamentous growth, and biofilm formation were impaired in the mutant. These results suggest that the defects are not due to the lack of DolP for the protein N-glycosylation but rather that the activity of oligosacharyltransferase could be inhibited by the excess DolPP accumulating in the mutant.


Assuntos
Candida albicans/metabolismo , Dolicóis/biossíntese , Proteínas Fúngicas/genética , Oligossacarídeos de Poli-Isoprenil Fosfato/metabolismo , Processamento de Proteína Pós-Traducional , Pirofosfatases/genética , Candida albicans/crescimento & desenvolvimento , Parede Celular/metabolismo , Dolicóis/genética , Proteínas Fúngicas/metabolismo , Glicosilação , Morfogênese , Pirofosfatases/metabolismo
20.
BMC Res Notes ; 12(1): 681, 2019 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-31640775

RESUMO

OBJECTIVE: Candida species is implicated in a wide array of clinical infections. Speciation of Candida strains is of prime importance in the epidemiological survey and laboratory diagnosis as there is an upswing of antifungal resistance and changing trends in the antifungal resistance pattern among C. albicans and non albicans Candida. Varied phenotypic methods are available for the identification of Candida species which vary in principles and cost factors. Chromogenic agar medium (HiCrome Candida differential agar) is one of the preferred phenotypic methods in limited resource laboratories. Hence, this study was aimed to assess the reliability of HiCrome Candida differential agar, M1297A (HiMedia) in the identification of Candida species compared polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Oral Candida isolates (n = 194) were inoculated onto HiCrome Candida differential agar and the potential of Candida differential Agar was compared with PCR-RFLP. RESULTS: The results were not in agreement with PCR-RFLP. Percentage of disagreement was 40.2, 50.0, 100.0 and 25.0 for Candida albicans, Candida krusei, Candida glabrata and Candida tropicalis respectively. PCR-RFLP demonstrated a very high discriminatory power in the identification of Candida species compared to agar.


Assuntos
Candida/genética , Candidíase/diagnóstico , Compostos Cromogênicos/metabolismo , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Fragmento de Restrição , Ágar , Candida/classificação , Candida/metabolismo , Candida albicans/genética , Candida albicans/isolamento & purificação , Candida albicans/metabolismo , Candida glabrata/genética , Candida glabrata/isolamento & purificação , Candida glabrata/metabolismo , Candida tropicalis/genética , Candida tropicalis/isolamento & purificação , Candida tropicalis/metabolismo , Candidíase/microbiologia , Meios de Cultura/química , Meios de Cultura/metabolismo , Humanos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da Espécie
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