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1.
Molecules ; 26(15)2021 Jul 27.
Artigo em Inglês | MEDLINE | ID: mdl-34361691

RESUMO

Lactobacillus crispatus is the dominant species in the vagina of many women. With the potential for strains of this species to be used as a probiotic to help prevent and treat dysbiosis, we investigated isolates from vaginal swabs with Lactobacillus-dominated and a dysbiotic microbiota. A comparative genome analysis led to the identification of metabolic pathways for synthesis and degradation of three major biogenic amines in most strains. However, targeted metabolomic analysis of the production and degradation of biogenic amines showed that certain strains have either the ability to produce or to degrade these compounds. Notably, six strains produced cadaverine, one produced putrescine, and two produced tyramine. These biogenic amines are known to raise vaginal pH, cause malodour, and make the environment more favourable to vaginal pathogens. In vitro experiments confirmed that strains isolated from women with a dysbiotic vaginal microbiota have higher antimicrobial effects against the common urogenital pathogens Escherichia coli and Enterococcus faecium. The results indicate that not all L. crispatus vaginal strains appear suitable for probiotic application and the basis for selection should not be only the overall composition of the vaginal microbiota of the host from which they came, but specific biochemical and genetic traits.


Assuntos
Anti-Infecciosos/metabolismo , Aminas Biogênicas/metabolismo , Doenças Urogenitais Femininas/metabolismo , Doenças Urogenitais Femininas/microbiologia , Lactobacillus crispatus/metabolismo , Microbiota , Vagina/microbiologia , Candida albicans/metabolismo , Disbiose/metabolismo , Disbiose/microbiologia , Enterococcus faecium/metabolismo , Escherichia coli/metabolismo , Feminino , Genômica/métodos , Humanos , Lactobacillus crispatus/classificação , Lactobacillus crispatus/genética , Metaboloma , Metabolômica/métodos , Filogenia , Prevotella/metabolismo , Probióticos/metabolismo
2.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069257

RESUMO

Candida albicans is an opportunistic pathogen that induces vulvovaginal candidiasis (VVC), among other diseases. In the vaginal environment, the source of carbon for C. albicans can be either lactic acid or its dissociated form, lactate. It has been shown that lactate, similar to the popular antifungal drug fluconazole (FLC), reduces the expression of the ERG11 gene and hence the amount of ergosterol in the plasma membrane. The Cdr1 transporter that effluxes xenobiotics from C. albicans cells, including FLC, is delocalized from the plasma membrane to a vacuole under the influence of lactate. Despite the overexpression of the CDR1 gene and the increased activity of Cdr1p, C. albicans is fourfold more sensitive to FLC in the presence of lactate than when glucose is the source of carbon. We propose synergistic effects of lactate and FLC in that they block Cdr1 activity by delocalization due to changes in the ergosterol content of the plasma membrane.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Fluconazol/farmacologia , Ácido Láctico/farmacologia , Candida albicans/genética , Candida albicans/metabolismo , Membrana Celular/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Sinergismo Farmacológico , Ergosterol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Ácido Láctico/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Transporte Proteico/efeitos dos fármacos
3.
Int J Mol Sci ; 22(9)2021 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-34068595

RESUMO

Systemic inflammation, from gut translocation of organismal molecules, might worsen uremic complications in acute kidney injury (AKI). The monitoring of gut permeability integrity and/or organismal molecules in AKI might be clinically beneficial. Due to the less prominence of Candida albicans in human intestine compared with mouse gut, C. albicans were orally administered in bilateral nephrectomy (BiN) mice. Gut dysbiosis, using microbiome analysis, and gut permeability defect (gut leakage), which was determined by fluorescein isothiocyanate-dextran and intestinal tight-junction immunofluorescent staining, in mice with BiN-Candida was more severe than BiN without Candida. Additionally, profound gut leakage in BiN-Candida also resulted in gut translocation of lipopolysaccharide (LPS) and (1→3)-ß-D-glucan (BG), the organismal components from gut contents, that induced more severe systemic inflammation than BiN without Candida. The co-presentation of LPS and BG in mouse serum enhanced inflammatory responses. As such, LPS with Whole Glucan Particle (WGP, a representative BG) induced more severe macrophage responses than LPS alone as determined by supernatant cytokines and gene expression of downstream signals (NFκB, Malt-1 and Syk). Meanwhile, WGP alone did not induced the responses. In parallel, WGP (with or without LPS), but not LPS alone, accelerated macrophage ATP production (extracellular flux analysis) through the upregulation of genes in mitochondria and glycolysis pathway (using RNA sequencing analysis), without the induction of cell activities. These data indicated a WGP pre-conditioning effect on cell energy augmentation. In conclusion, Candida in BiN mice accelerated gut translocation of BG that augmented cell energy status and enhanced pro-inflammatory macrophage responses. Hence, gut fungi and BG were associated with the enhanced systemic inflammation in acute uremia.


Assuntos
Injúria Renal Aguda/metabolismo , Candida albicans/metabolismo , Inflamação/sangue , Proteoglicanas/sangue , Injúria Renal Aguda/genética , Injúria Renal Aguda/microbiologia , Animais , Candida/metabolismo , Candida albicans/patogenicidade , Disbiose/sangue , Metabolismo Energético , Humanos , Inflamação/microbiologia , Inflamação/patologia , Inflamação/cirurgia , Lipopolissacarídeos/sangue , Macrófagos/metabolismo , Macrófagos/microbiologia , Macrófagos/patologia , Camundongos , Microbiota/genética , Nefrectomia/efeitos adversos
4.
Nat Commun ; 12(1): 3899, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162849

RESUMO

The ability of the fungal pathogen Candida albicans to undergo a yeast-to-hypha transition is believed to be a key virulence factor, as filaments mediate tissue damage. Here, we show that virulence is not necessarily reduced in filament-deficient strains, and the results depend on the infection model used. We generate a filament-deficient strain by deletion or repression of EED1 (known to be required for maintenance of hyphal growth). Consistent with previous studies, the strain is attenuated in damaging epithelial cells and macrophages in vitro and in a mouse model of intraperitoneal infection. However, in a mouse model of systemic infection, the strain is as virulent as the wild type when mice are challenged with intermediate infectious doses, and even more virulent when using low infectious doses. Retained virulence is associated with rapid yeast proliferation, likely the result of metabolic adaptation and improved fitness, leading to high organ fungal loads. Analyses of cytokine responses in vitro and in vivo, as well as systemic infections in immunosuppressed mice, suggest that differences in immunopathology contribute to some extent to retained virulence of the filament-deficient mutant. Our findings challenge the long-standing hypothesis that hyphae are essential for pathogenesis of systemic candidiasis by C. albicans.


Assuntos
Candida albicans/metabolismo , Candidíase/metabolismo , Proteínas Fúngicas/metabolismo , Hifas/metabolismo , Animais , Candida albicans/genética , Candida albicans/patogenicidade , Candidíase/microbiologia , Divisão Celular/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Humanos , Hifas/genética , Hifas/crescimento & desenvolvimento , Macrófagos/metabolismo , Camundongos Endogâmicos BALB C , Mutação , Neutrófilos/metabolismo , Virulência/genética
5.
Biochem Biophys Res Commun ; 561: 106-112, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34022710

RESUMO

Candida albicans is an important opportunistic fungal pathogen of immunocompromised individuals. The ability to switch between yeast and hyphal growth forms is critical for its pathogenesis. Hyphal development in C. albicans requires two temporally linked regulations for initiation and maintenance. Here, we performed transcriptome sequencing (RNA-Seq) to analyze the transcriptional consequences for the two different phases of hyphal development. Genome-wide transcription profiling reveals that the sets associated with hyphal initiation were significantly enriched in genes for hyphal cell wall, biofilm matrix and actin polarization. In addition to hypha-specific genes, numerous genes involved in iron acquisition, such as FTR1 and SEF1, are highly induced specifically during sustained hyphal development even when additional free iron is supplied in the medium. Therefore, iron uptake genes are induced by signals that can support prolonged hyphal development in an iron-independent manner. The induction of iron acquisition genes during hyphal elongation was further confirmed by quantitative reverse transcription-PCR under various hypha-inducing conditions. Remarkably, preventing C. albicans from acquiring iron blocks BRG1 activation, leading to impaired hyphal maintenance, and ectopically expressed BRG1 can sustain hyphal development bypassing the requirement of iron. Our study elucidates an underlying mechanism of how multiple virulence factors are interconnected and are induced simultaneously during infection.


Assuntos
Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Hifas/crescimento & desenvolvimento , Ferro/metabolismo , Candida albicans/genética , Proteínas Fúngicas/genética , Hifas/genética , Hifas/metabolismo , Virulência
6.
Forensic Sci Int ; 324: 110809, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33993011

RESUMO

In previous research, we modeled the ethanol production by certain bacteria under controlled experimental conditions in an attempt to quantify the production of microbial postmortem ethanol in cases where other alcohols were co-detected. This contribution on the modeling of postmortem ethanol production by Candida albicans is complementary to these previous studies. Τhis work aimed to study ethanol, higher alcohols (1-propanol, isobutanol, 2-methyl-1-butanol and 3-methyl-1-butanol), and 1-butanol production by Candida albicans: (i) in different culture media (Brain Heart Infusion, BHI and, Sabouraud Dextrose Broth, SDB), (ii) under mixed aerobic/anaerobic or strict anaerobic conditions, and (iii) at different temperatures (37 °C, 25 °C and, 4 °C), and develop simple mathematical models, resulted from fungal cultures at 25 °C, to predict the microbially produced ethanol in correlation with the other alcohols. The applicability of the models was tested in the C. albicans cultures in BHI and SDB media at 37 °C, in denatured human blood at 25 °C, acidic and neutral with different concentrations of additional glucose, in acidic denatured blood diluted with dextrose solution and in blood from autopsy cases. The received results indicated that the C. albicans models could apply in cases where yeasts have been activated in blood with elevated glucose levels. Overall, the in vitro ethanol production by C. albicans in blood depended on temperature, time, glucose (or carbohydrate) content, pH of the medium and endogenous changes in the medium composition through time. Our results showed that methyl-butanol is the most significant indicator of fungal ethanol production, followed by the equally important isobutanol and 1-propanol in qualitative and quantitative terms.


Assuntos
Candida albicans/metabolismo , Etanol/metabolismo , Modelos Teóricos , Mudanças Depois da Morte , 1-Butanol/metabolismo , 1-Propanol/metabolismo , Glicemia , Butanóis/metabolismo , Técnicas de Cultura , Humanos , Pentanóis/metabolismo , Manejo de Espécimes , Temperatura
7.
J Biol Chem ; 297(1): 100727, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33933457

RESUMO

The human fungal pathogen Candida albicans responds to iron deprivation by a global transcriptome reconfiguration known to be controlled by the transcriptional regulators Hap43 (also known as Cap2), Sef1, and the trimeric Hap2-Hap3-Hap5 complex. However, the relative roles of these regulators are not known. To dissect this system, we focused on the FRP1 and ACO1 genes, which are induced and repressed, respectively, under iron deprivation conditions. Chromatin immunoprecipitation assays showed that the trimeric HAP complex and Sef1 are recruited to both FRP1 and ACO1 promoters. While the HAP complex occupancy at the FRP1 promoter was Sef1-dependent, occupancy of Sef1 was not dependent on the HAP complex. Furthermore, iron deprivation elicited histone H3-Lys9 hyperacetylation and Pol II recruitment mediated by the trimeric HAP complex and Sef1 at the FRP1 promoter. In contrast, at the ACO1 promoter, the HAP trimeric complex and Hap43 promoted histone deacetylation and also limited Pol II recruitment under iron deprivation conditions. Mutational analysis showed that the SAGA subunits Gcn5, Spt7, and Spt20 are required for C. albicans growth in iron-deficient medium and for H3-K9 acetylation and transcription from the FRP1 promoter. Thus, the trimeric HAP complex promotes FRP1 transcription by stimulating H3K9Ac and Pol II recruitment and, along with Hap43, functions as a repressor of ACO1 by maintaining a deacetylated promoter under iron-deficient conditions. Thus, a regulatory network involving iron-responsive transcriptional regulators and the SAGA histone modifying complex functions as a molecular switch to fine-tune tight control of iron homeostasis gene expression in C. albicans.


Assuntos
Candida albicans/metabolismo , Cromatina/metabolismo , Proteínas Fúngicas/metabolismo , Homeostase , Ferro/metabolismo , Complexos Multiproteicos/metabolismo , Transcrição Genética , Acetilação , Sequência de Bases , Sítios de Ligação , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Histonas/metabolismo , Lisina/metabolismo , Modelos Genéticos , Regiões Promotoras Genéticas , Subunidades Proteicas/metabolismo , RNA Polimerase II/metabolismo , Fatores de Transcrição/metabolismo
8.
Microb Pathog ; 157: 104988, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34044051

RESUMO

This study was aimed to explore the immunomodulatory and anti-Candida mechanisms of Bacillus subtilis (B. subtilis) R0179 in macrophages. RAW 264.7 cells were first challenged with B. subtilis R0179. B. subtilis R0179 was found to down-regulate the signals of Dectin-1, Card9, P-Iκ-Bα, Iκ-Bα, and NF-κB. Meanwhile, it reduced the levels of cytokines interleukin (IL)-1ß, IL-6, IL-12, and tumor necrosis factor (TNF)-α, but increased the level of cytokine IL-10. Then RAW 264.7 cells were pretreated with B. subtilis R0179 before challenged with Candida albicans (C. albicans) or RAW 264.7 cells were co-treated with B. subtilis R0179 and C. albicans. In the presence of C. albicans, B. subtilis R0179 also showed the similar immunomodulatory effects on RAW 264.7 cells. Hence, this study provides the first insight into the immunomodulatory mechanisms of B. subtilis R0179 on the Dectin-1-related downstream signaling pathways in macrophages, which may prevent tissue damage caused by excessive pro-inflammatory response during the infection of C. albicans.


Assuntos
Bacillus subtilis , Candida albicans , Animais , Bacillus subtilis/metabolismo , Proteínas Adaptadoras de Sinalização CARD , Candida albicans/metabolismo , Imunidade , Camundongos , NF-kappa B/metabolismo , Células RAW 264.7 , Fator de Necrose Tumoral alfa
10.
Biomolecules ; 11(4)2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923411

RESUMO

Candidiasis is the wide-spread fungal infection caused by numerous strains of yeast, with the prevalence of Candida albicans. The current treatment of candidiasis is becoming rather ineffective and costly owing to the emergence of resistant strains; hence, the exploration of new possible drug targets is necessary. The most promising route is the development of novel antibiotics targeting this pathogen. In this review, we summarize such candidates found in C. albicans and those involved in the transport of (metal) cations, as the latter are essential for numerous processes within the cell; hence, disruption of their fluxes can be fatal for C. albicans.


Assuntos
Antifúngicos/farmacologia , Candidíase/tratamento farmacológico , Proteínas de Transporte de Cátions/metabolismo , Proteínas Fúngicas/metabolismo , Animais , Antifúngicos/uso terapêutico , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Proteínas de Transporte de Cátions/antagonistas & inibidores , Proteínas de Transporte de Cátions/química , Proteínas Fúngicas/antagonistas & inibidores , Proteínas Fúngicas/química , Humanos
11.
Chem Asian J ; 16(11): 1417-1429, 2021 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-33829660

RESUMO

Substantial morbidity and mortality of fungal infections have aroused concerns all over the world, and common Candida spp. currently bring about severe systemic infections. A series of pyrimidinetrione-imidazole conjugates as potentially antifungal agents were developed. Bioassays manifested that 4-fluobenzyl pyrimidinetrione imidazole 5 f exerted favorable inhibition towards C. albicans (MIC=0.002 mM), being 6.5 folds more active than clinical antifungal drug fluconazole (MIC=0.013 mM). Preliminary mechanism research indicated that compound 5 f could not only depolarize membrane potential but also permeabilize the membrane of C. albicans. Molecular docking was operated to simulate the interaction mode between molecule 5 f and CYP51. In addition, hybrid 5 f might form 5 f-DNA supramolecular complex via intercalating into DNA. The interference of membrane and DNA might contribute to its fungicidal capacity with no obvious tendency to induce the resistance against C. albicans. Conjugate 5 f endowed good blood compatibility as well as low cytotoxicity towards HeLa and HEK-293T cells.


Assuntos
Antifúngicos/síntese química , Desenho de Fármacos , Imidazóis/química , Pirimidinas/química , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Sítios de Ligação , Candida albicans/efeitos dos fármacos , Candida albicans/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Resistência Microbiana a Medicamentos/efeitos dos fármacos , Hemólise/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Esterol 14-Desmetilase/química , Esterol 14-Desmetilase/metabolismo , Relação Estrutura-Atividade
12.
G3 (Bethesda) ; 11(3)2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33793759

RESUMO

A MAPK cascade consists of three kinases, (MEKK, MEK and MAPK), that are sequentially activated in response to a stimulus and serve to transmit signals. In C. albicans and in yeast, an MAPK cascade is linked to the pheromone pathway through a scaffold protein (Cst5 and Ste5, respectively). Cst5 is much shorter and lacks key domains compared to Ste5, so in C. albicans, other elements, in particular the MEKK Ste11, play key roles in controlling the associations and localizations of network components. ABSTRACT: Candida albicans opaque cells release pheromones to stimulate cells of opposite mating type to activate their pheromone response pathway. Although this fungal pathogen shares orthologous proteins involved in the process with Saccharomyces cerevisiae, the pathway in each organism has unique characteristics. We have used GFP-tagged fusion proteins to investigate the localization of the scaffold protein Cst5, as well as the MAP kinases Cek1 and Cek2, during pheromone response in C. albicans. In wild-type cells, pheromone treatment directed Cst5-GFP to surface puncta concentrated at the tips of mating projections. These puncta failed to form in cells defective in either the Gα or ß subunits. However, they still formed in response to pheromone in cells missing Ste11, but with the puncta distributed around the cell periphery in the absence of mating projections. These puncta were absent from hst7Δ/Δ cells, but could be detected in the ste11Δ/Δ hst7Δ/Δ double mutant. Cek2-GFP showed a strong nuclear localization late in the response, consistent with a role in adaptation, while Cek1-GFP showed a weaker, but early increase in nuclear localization after pheromone treatment. Activation loop phosphorylation of both Cek1 and Cek2 required the presence of Ste11. In contrast to Cek2-GFP, which showed no localization signal in ste11Δ/Δ cells, Cek1-GFP showed enhanced nuclear localization that was pheromone independent in the ste11Δ/Δ mutant. The results are consistent with CaSte11 facilitating Hst7-mediated MAP kinase phosphorylation and also playing a potentially critical role in both MAP kinase and Cst5 scaffold localization.


Assuntos
Candida albicans , Proteínas Fúngicas , Feromônios , Candida albicans/metabolismo , Proteínas Fúngicas/genética , Regulação Fúngica da Expressão Gênica , Proteínas Quinases Ativadas por Mitógeno/metabolismo
13.
Int J Mol Sci ; 22(7)2021 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-33800643

RESUMO

Candida albicans and Klebsiella pneumoniae frequently co-exist within the human host as a complex biofilm community. These pathogens are of interest because their association is also related to significantly increased morbidity and mortality in hospitalized patients. With the aim of highlighting metabolic shifts occurring in the dual-species biofilm, an untargeted GC-MS-based metabolomics approach was applied to single and mixed biofilms of C. albicans and K. pneumoniae. Metabolomic results showed that among the extracellular metabolites identified, approximately 40 compounds had significantly changed relative abundance, mainly involving central carbon, amino acid, vitamin, and secondary metabolisms, such as serine, leucine, arabitol, phosphate, vitamin B6, cyclo-(Phe-Pro), trehalose, and nicotinic acid. The results were related to the strict interactions between the two species and the different microbial composition in the early and mature biofilms.


Assuntos
Candida albicans/metabolismo , Klebsiella pneumoniae/metabolismo , Metabolômica/métodos , Algoritmos , Biofilmes , Meios de Cultura , Cromatografia Gasosa-Espectrometria de Massas , Glucose/metabolismo , Análise dos Mínimos Quadrados , Análise de Componente Principal
14.
Nucleic Acids Res ; 49(8): 4655-4667, 2021 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-33823547

RESUMO

Ribosomes of different species share an evolutionarily conserved core, exhibiting flexible shells formed partially by the addition of species-specific ribosomal RNAs (rRNAs) with largely unexplored functions. In this study, we showed that by swapping the Saccharomyces cerevisiae 25S rRNA genes with non-S. cerevisiae homologs, species-specific rRNA variations caused moderate to severe pre-rRNA processing defects. Specifically, rRNA substitution by the Candida albicans caused severe growth defects and deficient pre-rRNA processing. We observed that such defects could be attributed primarily to variations in expansion segment 7L (ES7L) and could be restored by an assembly factor Noc2p mutant (Noc2p-K384R). We showed that swapping ES7L attenuated the incorporation of Noc2p and other proteins (Erb1p, Rrp1p, Rpl6p and Rpl7p) into pre-ribosomes, and this effect could be compensated for by Noc2p-K384R. Furthermore, replacement of Noc2p with ortholog from C. albicans could also enhance the incorporation of Noc2p and the above proteins into pre-ribosomes and consequently restore normal growth. Taken together, our findings help to elucidate the roles played by the species-specific rRNA variations in ribosomal biogenesis and further provide evidence that coevolution of rRNA expansion segments and cognate assembly factors specialized the ribosome biogenesis pathway, providing further insights into the function and evolution of ribosome.


Assuntos
Candida albicans/genética , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ribossomos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Candida albicans/metabolismo , Evolução Molecular , Mutação , Filogenia , Proteínas de Ligação a RNA/genética , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
15.
Genetics ; 217(2)2021 02 09.
Artigo em Inglês | MEDLINE | ID: mdl-33724418

RESUMO

The assimilation of inorganic sulfate and the synthesis of the sulfur-containing amino acids methionine and cysteine is mediated by a multibranched biosynthetic pathway. We have investigated this circuitry in the fungal pathogen Candida albicans, which is phylogenetically intermediate between the filamentous fungi and Saccharomyces cerevisiae. In S. cerevisiae, this pathway is regulated by a collection of five transcription factors (Met4, Cbf1, Met28, and Met31/Met32), while in the filamentous fungi the pathway is controlled by a single Met4-like factor. We found that in C. albicans, the Met4 ortholog is also a core regulator of methionine biosynthesis, where it functions together with Cbf1. While C. albicans encodes this Met4 protein, a Met4 paralog designated Met28 (Orf19.7046), and a Met31 protein, deletion, and activation constructs suggest that of these proteins only Met4 is actually involved in the regulation of methionine biosynthesis. Both Met28 and Met31 are linked to other functions; Met28 appears essential, and Met32 appears implicated in the regulation of genes of central metabolism. Therefore, while S. cerevisiae and C. albicans share Cbf1 and Met4 as central elements of the methionine biosynthesis control, the other proteins that make up the circuit in S. cerevisiae are not members of the C. albicans control network, and so the S. cerevisiae circuit likely represents a recently evolved arrangement.


Assuntos
Regulação Fúngica da Expressão Gênica , Redes Reguladoras de Genes , Metionina/biossíntese , Fatores de Transcrição de Zíper de Leucina Básica/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Candida albicans/genética , Candida albicans/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Metionina/genética
16.
Commun Biol ; 4(1): 342, 2021 03 16.
Artigo em Inglês | MEDLINE | ID: mdl-33727664

RESUMO

Severe infection often causes a septic cytokine storm followed by immune exhaustion/paralysis. Not surprisingly, many pathogens are equipped with various anti-inflammatory mechanisms. Such mechanisms might be leveraged clinically to control septic cytokine storms. Here we show that N-glycan from pathogenic C. albicans ameliorates mouse sepsis through immunosuppressive cytokine IL-10. In a sepsis model using lipopolysaccharide (LPS), injection of the N-glycan upregulated serum IL-10, and suppressed pro-inflammatory IL-1ß, TNF-α and IFN-γ. The N-glycan also improved the survival of mice challenged by LPS. Analyses of structurally defined N-glycans from several yeast strains revealed that the mannose core is key to the upregulation of IL-10. Knocking out the C-type lectin Dectin-2 abrogated the N-glycan-mediated IL-10 augmentation. Furthermore, C. albicans N-glycan ameliorated immune exhaustion/immune paralysis after acute inflammation. Our results suggest a strategy where the immunosuppressive mechanism of one pathogen can be applied to attenuate a severe inflammation/cytokine storm caused by another pathogen.


Assuntos
Candida albicans/imunologia , Candidíase/imunologia , Parede Celular/imunologia , Citocinas/imunologia , Glicoproteínas de Membrana/imunologia , Polissacarídeos/imunologia , Sepse/imunologia , Animais , Candida albicans/metabolismo , Candidíase/metabolismo , Candidíase/microbiologia , Parede Celular/metabolismo , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Interferon gama/imunologia , Interferon gama/metabolismo , Interleucina-10/imunologia , Interleucina-10/metabolismo , Interleucina-1beta/imunologia , Interleucina-1beta/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/imunologia , Macrófagos/metabolismo , Glicoproteínas de Membrana/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polissacarídeos/metabolismo , Sepse/metabolismo , Sepse/microbiologia , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fatores de Tempo , Fator de Necrose Tumoral alfa/imunologia , Fator de Necrose Tumoral alfa/metabolismo
17.
mSphere ; 6(2)2021 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-33731469

RESUMO

Fluorescence microscopy is a standard research tool in many fields, although collecting reliable images can be difficult in systems characterized by low expression levels and/or high background fluorescence. We present the combination of a photochromic fluorescent protein and stochastic optical fluctuation imaging (SOFI) to deliver suppression of the background fluorescence. This strategy makes it possible to resolve lowly or endogenously expressed proteins, as we demonstrate for Gcn5, a histone acetyltransferase required for complete virulence, and Erg11, the target of the azole antifungal agents in the fungal pathogen Candida albicans We expect that our method can be readily used for sensitive fluorescence measurements in systems characterized by high background fluorescence.IMPORTANCE Understanding the spatial and temporal organization of proteins of interest is key to unraveling cellular processes and identifying novel possible antifungal targets. Only a few therapeutic targets have been discovered in Candida albicans, and resistance mechanisms against these therapeutic agents are rapidly acquired. Fluorescence microscopy is a valuable tool to investigate molecular processes and assess the localization of possible antifungal targets. Unfortunately, fluorescence microscopy of C. albicans suffers from extensive autofluorescence. In this work, we present the use of a photochromic fluorescent protein and stochastic optical fluctuation imaging to enable the imaging of lowly expressed proteins in C. albicans through the suppression of autofluorescence. This method can be applied in C. albicans research or adapted for other fungal systems, allowing the visualization of intricate processes.


Assuntos
Candida albicans/química , Candida albicans/genética , Proteínas Fúngicas/genética , Microscopia de Fluorescência/métodos , Imagem Óptica/métodos , Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo
18.
Eur J Immunol ; 51(4): 864-878, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33616974

RESUMO

Intestinal epithelial cells (IECs) are the first to encounter luminal antigens and play an active role in intestinal immune responses. We previously reported that ß-glucans, major fungal cell-wall glycans, induced chemokine secretion by IEC lines in a Dectin-1- and Syk-dependent manner. Here, we show that in contrast to ß-glucans, stimulation of IEC lines with Candida albicans and Saccharomyces cerevisiae did not induce secretion of any of the proinflammatory cytokines IL-8, CCL2, CXCL1, and GM-CSF. Commensal fungi and ß-glucans activated Syk and ERK in IEC lines. However, only ß-glucans activated p38, JNK, and the transcription factors NF-κB p65 and c-JUN, which were necessary for cytokine secretion. Furthermore, costimulation of IEC lines with ß-glucans and C. albicans yielded decreased cytokine secretion compared to stimulation with ß-glucans alone. Finally, ex vivo stimulation of human colonic mucosal explants with zymosan and C. albicans, leads to epithelial Syk and ERK phosphorylation, implying recognition of fungi and similar initial signaling pathways as in IEC lines. Lack of cytokine secretion in response to commensal fungi may reflect IECs' response to fungal glycans, other than ß-glucans, that contribute to mucosal tolerance. Skewed epithelial response to commensal fungi may impair homeostasis and contribute to intestinal inflammation.


Assuntos
Candida albicans/imunologia , Parede Celular/imunologia , Células Epiteliais/imunologia , Mucosa Intestinal/imunologia , beta-Glucanas/imunologia , Células CACO-2 , Candida albicans/metabolismo , Candida albicans/fisiologia , Linhagem Celular Tumoral , Parede Celular/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , MAP Quinases Reguladas por Sinal Extracelular/imunologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Células HT29 , Interações Hospedeiro-Patógeno/imunologia , Humanos , Mucosa Intestinal/microbiologia , Fosforilação/imunologia , Quinase Syk/imunologia , Quinase Syk/metabolismo , Zimosan/imunologia , Zimosan/metabolismo , beta-Glucanas/metabolismo
19.
mSphere ; 6(1)2021 02 10.
Artigo em Inglês | MEDLINE | ID: mdl-33568458

RESUMO

Pleiotropic drug resistance (PDR) ATP-binding cassette (ABC) transporters of the ABCG family are eukaryotic membrane proteins that pump an array of compounds across organelle and cell membranes. Overexpression of the archetype fungal PDR transporter Cdr1 is a major cause of azole antifungal drug resistance in Candida albicans, a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR transporter has been solved. The objective of this project was to investigate the role of the 23 Cdr1 cysteine residues in the stability, trafficking, and function of the protein when expressed in the eukaryotic model organism, Saccharomyces cerevisiae The biochemical characterization of 18 partially cysteine-deficient Cdr1 variants revealed that the six conserved extracellular cysteines were critical for proper expression, localization, and function of Cdr1. They are predicted to form three covalent disulfide bonds that stabilize the large extracellular domains of fungal PDR transporters. Our investigations also revealed a novel nucleotide-binding domain motif, GX2[3]CPX3NPAD/E, at the peripheral cytosolic apex of ABCG transporters that possibly contributes to the unique ABCG transport cycle. With this knowledge, we engineered an "almost cysteine-less," yet fully functional, Cdr1 variant, Cdr1P-CID, that had all but the six extracellular cysteines replaced with serine, alanine, or isoleucine (C1106I of the new motif). It is now possible to perform cysteine-cross-linking studies that will enable more detailed biochemical investigations of fungal PDR transporters and confirm any future structure(s) solved for this important protein family.IMPORTANCE Overexpression of the fungal pleiotropic drug resistance (PDR) transporter Cdr1 is a major cause of antifungal drug resistance in Candida albicans, a significant fungal pathogen that can cause life-threatening invasive infections in immunocompromised individuals. To date, no structure for any PDR ABC transporter has been solved. Cdr1 contains 23 cysteines; 10 are cytosolic and 13 are predicted to be in the transmembrane or the extracellular domains. The objective of this project was to create, and biochemically characterize, CDR1 mutants to reveal which cysteines are most important for Cdr1 stability, trafficking, and function. During this process we discovered a novel motif at the cytosolic apex of PDR transporters that ensures the structural and functional integrity of the ABCG transporter family. The creation of a functional Cys-deficient Cdr1 molecule opens new avenues for cysteine-cross-linking studies that will facilitate the detailed characterization of an important ABCG transporter family member.


Assuntos
Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Candida albicans/genética , Candida albicans/metabolismo , Cisteína/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Cisteína/genética , Mutação , Dobramento de Proteína , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
20.
Fungal Genet Biol ; 149: 103529, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33596477

RESUMO

Candida albicans is a commensal organism and opportunistic pathogen that can form biofilms that colonize surfaces of medical devices, such as implants, catheters, and dentures. Compared to planktonic C. albicans cells, cells in biofilms exhibit increased resistance to treatment. Histatin 5 (Hst-5) is an antimicrobial peptide that is natively secreted by human salivary glands and has strong antifungal activity against C. albicans. However, C. albicans produces secreted aspartic proteases (Saps) that can cleave and inactivate Hst-5, limiting its antifungal properties. We previously showed that residue substitutions K11R and K17R within Hst-5 improve its antifungal activity and prevent proteolytic degradation by Saps when treating planktonic C. albicans. Here, we investigated the use of the K11R-K17R peptide as an alternative therapeutic against C. albicans biofilms by assessing its ability to reduce viability of pre-formed biofilms and to inhibit the formation of biofilms and showed that K11R-K17R had improved activity compared to Hst-5. Based on these results, we incorporated K11R-K17R and Hst-5 into polyelectrolyte multilayer (PEM) surface coatings and demonstrated that films functionalized with K11R-K17R reduced the formation of C. albicans biofilms. Our results demonstrate the therapeutic potential of the K11R-K17R Hst-5 variant in preventing and treating biofilms.


Assuntos
Candida albicans/crescimento & desenvolvimento , Histatinas/genética , Antifúngicos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/metabolismo , Biofilmes/crescimento & desenvolvimento , Candida albicans/genética , Candida albicans/metabolismo , Histatinas/metabolismo , Histatinas/fisiologia , Humanos , Proteólise
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