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1.
Curr Genet ; 62(2): 343-6, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26762634

RESUMO

The exocyst is an octameric complex that orchestrates the docking and tethering of vesicles to the plasma membrane during exocytosis and is fundamental for key biological processes including growth and establishment of cell polarity. Although components of the exocyst are well conserved among fungi, the specific functions of each component of the exocyst complex unique to Candida albicans biology and pathogenesis are not fully understood. This commentary describes recent findings regarding the role of exocyst subunits Sec6 and Sec15 in C. albicans filamentation and virulence.

2.
Eukaryot Cell ; 14(12): 1228-39, 2015 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-26453654

RESUMO

In prior studies of exocyst-mediated late secretion in Candida albicans, we have determined that Sec6 contributes to cell wall integrity, secretion, and filamentation. A conditional mutant lacking SEC6 expression exhibits markedly reduced lateral hyphal branching. In addition, lack of the related t-SNAREs Sso2 and Sec9 also leads to defects in secretion and filamentation. To further understand the role of the exocyst in the fundamental processes of polarized secretion and filamentation in C. albicans, we studied the exocyst subunit Sec15. Since Saccharomyces cerevisiae SEC15 is essential for viability, we generated a C. albicans conditional mutant strain in which SEC15 was placed under the control of a tetracycline-regulated promoter. In the repressed state, cell death occurred after 5 h in the tetR-SEC15 strain. Prior to this time point, the tetR-SEC15 mutant was markedly defective in Sap and lipase secretion and demonstrated increased sensitivity to Zymolyase and chitinase. Notably, tetR-SEC15 mutant hyphae were characterized by a hyperbranching phenotype, in direct contrast to strain tetR-SEC6, which had minimal lateral branching. We further studied the localization of the Spitzenkörper, polarisomes, and exocysts in the tetR-SEC15 and tetR-SEC6 mutants during filamentation. Mlc1-GFP (marking the Spitzenkörper), Spa2-GFP (the polarisome), and Exo70-GFP (exocyst) localizations were normal in the tetR-SEC6 mutant, whereas these structures were mislocalized in the tetR-SEC15 mutant. Following alleviation of gene repression by removing doxycycline, first Spitzenkörper, then polarisome, and finally exocyst localizations were recovered sequentially. These results indicate that the exocyst subunits Sec15 and Sec6 have distinct roles in mediating polarized secretion and filamentation in C. albicans.


Assuntos
Candida albicans/metabolismo , Proteínas Fúngicas/metabolismo , Subunidades Proteicas/metabolismo , Adesividade/efeitos dos fármacos , Ácido Aspártico Proteases/metabolismo , Candida albicans/efeitos dos fármacos , Candida albicans/crescimento & desenvolvimento , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Quitinases/metabolismo , Doxiciclina/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Hidrolases/metabolismo , Hifas/efeitos dos fármacos , Hifas/metabolismo , Lipase/metabolismo , Viabilidade Microbiana/efeitos dos fármacos , Mutação/genética , Fenótipo , Tetraciclina/farmacologia
3.
Eukaryot Cell ; 14(7): 684-97, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26002719

RESUMO

The yeast exocyst is a multiprotein complex comprised of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) which orchestrates trafficking of exocytic vesicles to specific docking sites on the plasma membrane during polarized secretion. To study SEC6 function in Candida albicans, we generated a conditional mutant strain in which SEC6 was placed under the control of a tetracycline-regulated promoter. In the repressed state, the tetR-SEC6 mutant strain (denoted tSEC6) was viable for up to 27 h; thus, all phenotypic analyses were performed at 24 h or earlier. Strain tSEC6 under repressing conditions had readily apparent defects in cytokinesis and endocytosis and accumulated both post-Golgi apparatus secretory vesicles and structures suggestive of late endosomes. Strain tSEC6 was markedly defective in secretion of aspartyl proteases and lipases as well as filamentation under repressing conditions. Lack of SEC6 expression resulted in markedly reduced lateral hyphal branching, which requires the establishment of a new axis of polarized secretion. Aberrant localization of chitin at the septum and increased resistance to zymolyase activity were observed, suggesting that C. albicans Sec6 plays an important role in mediating trafficking and delivery of cell wall components. The tSEC6 mutant was also markedly defective in macrophage killing, indicating a role of SEC6 in C. albicans virulence. Taken together, these studies indicate that the late secretory protein Sec6 is required for polarized secretion, hyphal morphogenesis, and the pathogenesis of C. albicans.


Assuntos
Candida albicans/crescimento & desenvolvimento , Candidíase/microbiologia , Parede Celular/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Hifas/crescimento & desenvolvimento , Macrófagos/patologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Candida albicans/genética , Candida albicans/metabolismo , Candidíase/genética , Candidíase/metabolismo , Membrana Celular/metabolismo , Sobrevivência Celular , Exocitose/fisiologia , Proteínas Fúngicas/genética , Hifas/genética , Hifas/metabolismo , Macrófagos/microbiologia , Camundongos , Mutação/genética , Transporte Proteico , Vesículas Secretórias/metabolismo , Proteínas de Transporte Vesicular/genética , Virulência
4.
Eukaryot Cell ; 13(9): 1207-21, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-25038082

RESUMO

Candida albicans vacuoles are central to many critical biological processes, including filamentation and in vivo virulence. The V-ATPase proton pump is a multisubunit complex responsible for organellar acidification and is essential for vacuolar biogenesis and function. To study the function of the V1B subunit of C. albicans V-ATPase, we constructed a tetracycline-regulatable VMA2 mutant, tetR-VMA2. Inhibition of VMA2 expression resulted in the inability to grow at alkaline pH and altered resistance to calcium, cold temperature, antifungal drugs, and growth on nonfermentable carbon sources. Furthermore, V-ATPase was unable to fully assemble at the vacuolar membrane and was impaired in proton transport and ATPase-specific activity. VMA2 repression led to vacuolar alkalinization in addition to abnormal vacuolar morphology and biogenesis. Key virulence-related traits, including filamentation and secretion of degradative enzymes, were markedly inhibited. These results are consistent with previous studies of C. albicans V-ATPase; however, differential contributions of the V-ATPase Vo and V1 subunits to filamentation and secretion are observed. We also make the novel observation that inhibition of C. albicans V-ATPase results in increased susceptibility to osmotic stress. Notably, V-ATPase inhibition under conditions of nitrogen starvation results in defects in autophagy. Lastly, we show the first evidence that V-ATPase contributes to virulence in an acidic in vivo system by demonstrating that the tetR-VMA2 mutant is avirulent in a Caenorhabditis elegans infection model. This study illustrates the fundamental requirement of V-ATPase for numerous key virulence-related traits in C. albicans and demonstrates that the contribution of V-ATPase to virulence is independent of host pH.


Assuntos
Autofagia/genética , Candida albicans/enzimologia , Subunidades Proteicas/genética , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Caenorhabditis elegans/genética , Caenorhabditis elegans/microbiologia , Candida albicans/genética , Candida albicans/patogenicidade , Concentração de Íons de Hidrogênio , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Estresse Fisiológico/genética , ATPases Vacuolares Próton-Translocadoras/química , ATPases Vacuolares Próton-Translocadoras/metabolismo , Vacúolos/enzimologia , Vacúolos/genética , Virulência/genética
5.
J Biol Chem ; 288(9): 6190-201, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23316054

RESUMO

Vacuolar proton-translocating ATPase (V-ATPase) is a central regulator of cellular pH homeostasis, and inactivation of all V-ATPase function has been shown to prevent infectivity in Candida albicans. V-ATPase subunit a of the Vo domain (Voa) is present as two fungal isoforms: Stv1p (Golgi) and Vph1p (vacuole). To delineate the individual contribution of Stv1p and Vph1p to C. albicans physiology, we created stv1Δ/Δ and vph1Δ/Δ mutants and compared them to the corresponding reintegrant strains (stv1Δ/ΔR and vph1Δ/ΔR). V-ATPase activity, vacuolar physiology, and in vitro virulence-related phenotypes were unaffected in the stv1Δ/Δ mutant. The vph1Δ/Δ mutant exhibited defective V1Vo assembly and a 90% reduction in concanamycin A-sensitive ATPase activity and proton transport in purified vacuolar membranes, suggesting that the Vph1p isoform is essential for vacuolar V-ATPase activity in C. albicans. The vph1Δ/Δ cells also had abnormal endocytosis and vacuolar morphology and an alkalinized vacuolar lumen (pHvph1Δ/Δ = 6.8 versus pHvph1Δ/ΔR = 5.8) in both yeast cells and hyphae. Secreted protease and lipase activities were significantly reduced, and M199-induced filamentation was impaired in the vph1Δ/Δ mutant. However, the vph1Δ/Δ cells remained competent for filamentation induced by Spider media and YPD, 10% FCS, and biofilm formation and macrophage killing were unaffected in vitro. These studies suggest that different virulence mechanisms differentially rely on acidified vacuoles and that the loss of both vacuolar (Vph1p) and non-vacuolar (Stv1p) V-ATPase activity is necessary to affect in vitro virulence-related phenotypes. As a determinant of C. albicans pathogenesis, vacuolar pH alone may prove less critical than originally assumed.


Assuntos
Biofilmes , Candida albicans/fisiologia , Candida albicans/patogenicidade , Locos de Características Quantitativas , ATPases Vacuolares Próton-Translocadoras/metabolismo , Fatores de Virulência/metabolismo , Domínio Catalítico , Deleção de Genes , Concentração de Íons de Hidrogênio , Transporte de Íons/fisiologia , Prótons , ATPases Vacuolares Próton-Translocadoras/genética , Vacúolos/enzimologia , Vacúolos/genética , Fatores de Virulência/genética
6.
Antimicrob Agents Chemother ; 58(12): 7501-9, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25288082

RESUMO

Candida albicans is a common cause of catheter-related bloodstream infections (CR-BSI), in part due to its strong propensity to form biofilms. Drug repurposing is an approach that might identify agents that are able to overcome antifungal drug resistance within biofilms. Quinacrine (QNC) is clinically active against the eukaryotic protozoan parasites Plasmodium and Giardia. We sought to investigate the antifungal activity of QNC against C. albicans biofilms. C. albicans biofilms were incubated with QNC at serially increasing concentrations (4 to 2,048 µg/ml) and assessed using a 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) assay in a static microplate model. Combinations of QNC and standard antifungals were assayed using biofilm checkerboard analyses. To define a mechanism of action, QNC was assessed for the inhibition of filamentation, effects on endocytosis, and pH-dependent activity. High-dose QNC was effective for the prevention and treatment of C. albicans biofilms in vitro. QNC with fluconazole had no interaction, while the combination of QNC and either caspofungin or amphotericin B demonstrated synergy. QNC was most active against planktonic growth at alkaline pH. QNC dramatically inhibited filamentation. QNC accumulated within vacuoles as expected and caused defects in endocytosis. A tetracycline-regulated VMA3 mutant lacking vacuolar ATPase (V-ATPase) function demonstrated increased susceptibility to QNC. These experiments indicate that QNC is active against C. albicans growth in a pH-dependent manner. Although QNC activity is not biofilm specific, QNC is effective in the prevention and treatment of biofilms. QNC antibiofilm activity likely occurs via several independent mechanisms: vacuolar alkalinization, inhibition of endocytosis, and impaired filamentation. Further investigation of QNC for the treatment and prevention of biofilm-related Candida CR-BSI is warranted.


Assuntos
Anfotericina B/farmacologia , Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Equinocandinas/farmacologia , Quinacrina/farmacologia , Antiprotozoários/farmacologia , Biofilmes/crescimento & desenvolvimento , Candida albicans/crescimento & desenvolvimento , Caspofungina , Combinação de Medicamentos , Reposicionamento de Medicamentos , Farmacorresistência Fúngica , Sinergismo Farmacológico , Endocitose/efeitos dos fármacos , Fluconazol/farmacologia , Concentração de Íons de Hidrogênio , Hifas/efeitos dos fármacos , Hifas/crescimento & desenvolvimento , Lipopeptídeos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Plâncton/efeitos dos fármacos , Plâncton/crescimento & desenvolvimento
7.
J Antimicrob Chemother ; 69(2): 428-36, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24114570

RESUMO

OBJECTIVES: Candida albicans is a common cause of nosocomial urinary tract infections (UTIs) and is responsible for increased morbidity and healthcare costs. Moreover, the US Centers for Medicare & Medicaid Services no longer reimburse for hospital-acquired catheter-associated UTIs. Thus, development of specific approaches for the prevention of Candida urinary infections is needed. Cranberry juice-derived proanthocyanidins (PACs) have efficacy in the prevention of bacterial UTIs, partially due to anti-adherence properties, but there are limited data on their use for the prevention and/or treatment of Candida UTIs. Therefore, we sought to systematically assess the in vitro effect of cranberry-derived PACs on C. albicans biofilm formation in artificial urine. METHODS: C. albicans biofilms in artificial urine were coincubated with cranberry PACs at serially increasing concentrations and biofilm metabolic activity was assessed using the XTT assay in static microplate and silicone disc models. RESULTS: Cranberry PAC concentrations of ≥16 mg/L significantly reduced biofilm formation in all C. albicans strains tested, with a paradoxical effect observed at high concentrations in two clinical isolates. Further, cranberry PACs were additive in combination with traditional antifungals. Cranberry PACs reduced C. albicans adherence to both polystyrene and silicone. Supplementation of the medium with iron reduced the efficacy of cranberry PACs against biofilms. CONCLUSIONS: These findings indicate that cranberry PACs have excellent in vitro activity against C. albicans biofilm formation in artificial urine. We present preliminary evidence that cranberry PAC activity against C. albicans biofilm formation is due to anti-adherence properties and/or iron chelation.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Proantocianidinas/farmacologia , Vaccinium macrocarpon , Antifúngicos/isolamento & purificação , Biofilmes/crescimento & desenvolvimento , Candida albicans/fisiologia , Humanos , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Proantocianidinas/isolamento & purificação , Urina/microbiologia
8.
FEMS Yeast Res ; 14(5): 762-75, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24911595

RESUMO

To study the role of late secretion in Candida albicans pathogenesis, we created conditional mutant C. albicans strains in which the t-SNARE-encoding genes SSO2 or SEC9 were placed under the control of a tetracycline-regulated promoter. In repressing conditions, C. albicans tetR-SSO2 and tetR-SEC9 mutant strains were defective in cytokinesis and secretion of aspartyl proteases and lipases. The mutant strains also exhibited a defect in filamentation compared with controls, and thus, we followed the fate of the C. albicans Spitzenkörper, an assembly of secretory vesicles thought to act as a vesicle supply center for the growing hyphae. In the absence of Ca Sso2p, the Spitzenkörper dissipated within 5 h and thin-section electron microscopy revealed an accumulation of secretory vesicles. Moreover, the hyphal tip developed into a globular yeast-like structure rather than maintaining a typical narrow hyphae. These studies indicate that late secretory t-SNARE proteins in C. albicans are required for fundamental cellular processes and contribute to virulence-related attributes of C. albicans pathogenesis. Moreover, these results provide direct evidence for a key role of SNARE proteins in vesicle-mediated polarized hyphal growth of C. albicans.


Assuntos
Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Citocinese , Enzimas/metabolismo , Proteínas Fúngicas/metabolismo , Hifas/genética , Proteínas SNARE/metabolismo , Candida albicans/citologia , Microscopia Eletrônica
9.
Med Mycol ; 52(2): 131-139, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24576999

RESUMO

Echinocandin-resistant clinical isolates of Candida albicans have been reported, and key-hot spot mutations in the FKS1 gene, which encodes a major glucan synthase subunit, have been identified in these (caspofungin-resistant [CAS-R]) strains. Although these mutations result in phenotypic resistance to echinocandins in planktonic cells, there is little data on antifungal susceptibilities of CAS-R C. albicans strains within biofilms. Thus, we analyzed biofilms formed by 12 C. albicans CAS-R clinical strains in which we previously identified FKS1 hot-spot mutations and compared the sessile antifungal and paradoxical activity of anidulafungin (ANID), caspofungin (CAS), and micafungin (MICA). Biofilms were formed in a 96-well static microplate model and assayed using both tetrazolium-salt reduction and crystal violet assays, as well as examination by scanning electron microscopy. We first sought to assess biofilm formation and structure in these fks1 mutants and found that the biofilm mass and metabolic activities were reduced in most of the fks1 mutants as compared with reference strain SC5314. Structural analyses revealed that the fks1 mutant biofilms were generally less dense and had a clear predominance of yeast and pseudohyphae, with unusual "pit"-like cell surface structures. We also noted that sessile minimum inhibitory concentrations (MICs) to ANID, CAS, and MICA were higher than planktonic MICs of all but one strain. The majority of strains demonstrated a paradoxical effect (PE) to particular echinocandins, in either planktonic or sessile forms. Overall, biofilms formed by echinocandin-resistant clinical isolates demonstrated varied PEs to echinocandins and were structurally characterized by a preponderance of yeast, pseudohyphae, and pit-like structures.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida albicans/efeitos dos fármacos , Candida albicans/fisiologia , Equinocandinas/farmacologia , Anidulafungina , Candida albicans/isolamento & purificação , Candida albicans/ultraestrutura , Candidíase/microbiologia , Caspofungina , Glucosiltransferases/genética , Humanos , Lipopeptídeos/farmacologia , Proteínas de Membrana/genética , Micafungina , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Microscopia Eletrônica , Proteínas Mutantes/genética , Coloração e Rotulagem
10.
Eukaryot Cell ; 12(10): 1369-82, 2013 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23913543

RESUMO

The vacuolar membrane ATPase (V-ATPase) is a protein complex that utilizes ATP hydrolysis to drive protons from the cytosol into the vacuolar lumen, acidifying the vacuole and modulating several key cellular response systems in Saccharomyces cerevisiae. To study the contribution of V-ATPase to the biology and virulence attributes of the opportunistic fungal pathogen Candida albicans, we created a conditional mutant in which VMA3 was placed under the control of a tetracycline-regulated promoter (tetR-VMA3 strain). Repression of VMA3 in the tetR-VMA3 strain prevents V-ATPase assembly at the vacuolar membrane and reduces concanamycin A-sensitive ATPase-specific activity and proton transport by more than 90%. Loss of C. albicans V-ATPase activity alkalinizes the vacuolar lumen and has pleiotropic effects, including pH-dependent growth, calcium sensitivity, and cold sensitivity. The tetR-VMA3 strain also displays abnormal vacuolar morphology, indicative of defective vacuolar membrane fission. The tetR-VMA3 strain has impaired aspartyl protease and lipase secretion, as well as attenuated virulence in an in vitro macrophage killing model. Repression of VMA3 suppresses filamentation, and V-ATPase-dependent filamentation defects are not rescued by overexpression of RIM8, MDS3, EFG1, CST20, or UME6, which encode positive regulators of filamentation. Specific chemical inhibition of Vma3p function also results in defective filamentation. These findings suggest either that V-ATPase functions downstream of these transcriptional regulators or that V-ATPase function during filamentation involves independent mechanisms and alternative signaling pathways. Taken together, these data indicate that V-ATPase activity is a fundamental requirement for several key virulence-associated traits in C. albicans.


Assuntos
Candida albicans/enzimologia , Exocitose , Proteínas Fúngicas/metabolismo , Multimerização Proteica , ATPases Vacuolares Próton-Translocadoras/metabolismo , Ácido Aspártico Proteases/metabolismo , Candida albicans/citologia , Candida albicans/metabolismo , Candida albicans/patogenicidade , Proteínas Fúngicas/genética , Lipase/metabolismo , Mutação , ATPases Vacuolares Próton-Translocadoras/genética , Vacúolos/metabolismo , Vacúolos/ultraestrutura , Virulência
11.
Antimicrob Agents Chemother ; 56(8): 4487-9, 2012 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-22615286

RESUMO

Candida albicans is a common cause of catheter-related bloodstream infections (CR-BSI). Ethanol (EtOH) lock therapy has been attempted despite limited data on optimal dose and duration. Concentrations of 35% EtOH or higher for a minimum of 4 h demonstrated a >99% reduction in mature C. albicans biofilm metabolic activity and prevented regrowth. Concentrations of 10% EtOH or higher reduced C. albicans biofilm formation by >99%. Further investigation of EtOH lock therapy for treatment and prevention of C. albicans CR-BSI is warranted.


Assuntos
Anti-Infecciosos Locais/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Etanol/farmacologia , Biofilmes/crescimento & desenvolvimento , Candida albicans/crescimento & desenvolvimento , Infecções Relacionadas a Cateter/tratamento farmacológico , Infecções Relacionadas a Cateter/microbiologia , Catéteres/microbiologia , Testes de Sensibilidade Microbiana
12.
Antimicrob Agents Chemother ; 56(1): 148-53, 2012 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21986822

RESUMO

Infections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin on Candida albicans biofilms and planktonic cells have not been previously studied. Therefore, we sought to determine the in vitro effect of a heparin sodium preparation (HP) on biofilms and planktonic cells of C. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformed C. albicans biofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P < 0.0001). Pure-H, MP, and PP each inhibited C. albicans biofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H have in vitro antifungal activity against C. albicans mature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention of C. albicans biofilms is warranted.


Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Heparina/farmacologia , Parabenos/farmacologia , Plâncton/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida albicans/crescimento & desenvolvimento , Candida albicans/ultraestrutura , Infecções Relacionadas a Cateter/prevenção & controle , Cateterismo Venoso Central , Relação Dose-Resposta a Droga , Humanos , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Plâncton/crescimento & desenvolvimento , Plâncton/ultraestrutura , Sais de Tetrazólio
16.
BMC Microbiol ; 10: 133, 2010 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-20433738

RESUMO

BACKGROUND: Candida albicans SUR7 has been shown to be required for plasma membrane organization and cell wall synthesis, but its role in virulence is not known. Using a bioinformatics strategy, we previously identified several novel putative secretion pathway proteins potentially involved in virulence, including the C. albicans homolog of the Saccharomyces cerevisiae endocytosis-related protein Sur7p. We therefore generated a C. albicans sur7Delta null mutant and examined its contribution to key virulence attributes. RESULTS: Structurally, the C. albicans sur7Delta mutant was impaired in response to filamentation-inducing conditions, and formed aberrant hyphae with extensive accumulation of plasma membrane-derived structures within the cell. Absence of SUR7 resulted in a temperature-sensitive growth defect at high temperatures (42 degrees C), which was partially rescued by addition of NaCl. We next examined the role of the SUR7 paralog C. albicans FMP45 in this temperature-sensitive phenotype. Analysis of C. albicans Fmp45p-GFP demonstrated co-localization of Fmp45p with Sur7p and increased fluorescence in the plasma membrane in the presence of high salt. We next focused on key virulence-related phenotypes. The C. albicans sur7Delta null mutant exhibited secretory defects: reduced lipase secretion, and increased levels of secreted Sap2p. The null mutant was hyper-susceptible to sub-inhibitory concentrations of caspofungin, but not amphotericin B and 5-fluorocytosine. Functionally, the sur7Delta mutant demonstrated increased adhesion to polystyrene and of note, was markedly defective in biofilm formation. In an in vitro macrophage model of virulence, the sur7Delta mutant was impaired in macrophage killing. CONCLUSIONS: Plasma membrane and cell wall organization are important for cell morphology, and alterations of these structures contributed to impairment of several key virulence-associated phenotypes in the C. albicans sur7Delta mutant.


Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/fisiologia , Morte Celular , Proteínas Fúngicas/fisiologia , Macrófagos/microbiologia , Fatores de Virulência/fisiologia , Animais , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Candida albicans/patogenicidade , Linhagem Celular , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Deleção de Genes , Temperatura Alta , Hifas/citologia , Hifas/crescimento & desenvolvimento , Proteínas de Membrana/genética , Camundongos , Proteínas de Saccharomyces cerevisiae/genética , Cloreto de Sódio/metabolismo , Virulência , Fatores de Virulência/genética
17.
Med Mycol ; 48(3): 557-60, 2010 May.
Artigo em Inglês | MEDLINE | ID: mdl-20370365

RESUMO

We report a case of fluconazole-resistant oropharyngeal colonization caused by a strain of Candida glabrata that rapidly regained susceptibility once prophylaxis with this agent was discontinued and echinocandin therapy was initiated. Isolates collected before and after discontinuation of fluconazole were confirmed to be isogenic by RAPD analysis. Transcription analysis demonstrated constitutive expression of genes encoding efflux pumps in the isolate recovered on fluconazole prophylaxis and transient expression in those isolates collected after fluconazole was discontinued.


Assuntos
Antifúngicos/uso terapêutico , Candida glabrata/efeitos dos fármacos , Quimioprevenção/métodos , Farmacorresistência Fúngica , Fluconazol/uso terapêutico , Transplante de Células-Tronco Hematopoéticas , Antifúngicos/farmacologia , Candida glabrata/classificação , Candida glabrata/genética , Candida glabrata/isolamento & purificação , Candidíase/microbiologia , Portador Sadio/microbiologia , Impressões Digitais de DNA , DNA Fúngico/genética , Equinocandinas/uso terapêutico , Fluconazol/farmacologia , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Técnicas de Tipagem Micológica , Orofaringe/microbiologia , Técnica de Amplificação ao Acaso de DNA Polimórfico
18.
Front Microbiol ; 10: 1012, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31143168

RESUMO

Candida albicans occupies diverse ecological niches within the host and must tolerate a wide range of environmental pH. The plasma membrane H+-ATPase Pma1p is the major regulator of cytosolic pH in fungi. Pma1p extrudes protons from the cytosol to maintain neutral-to-alkaline pH and is a potential drug target due to its essentiality and fungal specificity. We characterized mutants in which one allele of PMA1 has been deleted and the other truncated by 18-38 amino acids. Increasing C-terminal truncation caused corresponding decreases in plasma membrane ATPase-specific activity and cytosolic pH. Pma1p is regulated by glucose: glucose rapidly activates the ATPase, causing a sharp increase in cytosolic pH. Increasing Pma1p truncation severely impaired this glucose response. Pma1p truncation also altered cation responses, disrupted vacuolar morphology and pH, and reduced filamentation competence. Early studies of cytosolic pH and filamentation have described a rapid, transient alkalinization of the cytosol preceding germ tube formation; Pma1p has been proposed as a regulator of this process. We find Pma1p plays a role in the establishment of cell polarity, and distribution of Pma1p is non-homogenous in emerging hyphae. These findings suggest a role of PMA1 in cytosolic alkalinization and in the specialized form of polarized growth that is filamentation.

19.
Fungal Genet Biol ; 45(6): 861-77, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18296085

RESUMO

To investigate the pre-vacuolar secretory pathway in Candida albicans, we cloned and analyzed the C. albicans homolog of the Saccharomyces cerevisiae vacuolar protein sorting gene VPS1. C. albicans VPS1 encodes a predicted 694-aa dynamin-like GTPase that is 73.3% similar to S. cerevisiae Vps1p. Plasmids bearing C. albicans VPS1 complemented the temperature-sensitive growth, abnormal class F vacuolar morphology, and carboxypeptidase missorting of a S. cerevisiae vps1 null mutant. To study VPS1 function in C. albicans, a conditional mutant strain (tetR-VPS1) was generated by deleting the first allele of VPS1 and placing the second allele under control of a tetracycline-regulatable promoter. With doxycycline, the tetR-VPS1 mutant was hyper-susceptible to sub-inhibitory concentrations of fluconazole, but not amphotericin B, 5-fluorocytosine, or non-specific osmotic stresses. The repressed tetR-VPS1 mutant was defective in filamentation and secreted less extracellular protease activity. Biofilm production and filamentation within the biofilm were markedly reduced. These results suggest that C. albicans VPS1 has a key role in several important virulence-related phenotypes.


Assuntos
Biofilmes/crescimento & desenvolvimento , Candida albicans/enzimologia , Candida albicans/fisiologia , Proteínas de Ligação ao GTP/metabolismo , Peptídeo Hidrolases/metabolismo , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Proteínas de Ligação ao GTP/genética , Regulação Fúngica da Expressão Gênica , Teste de Complementação Genética , Pressão Osmótica , Transporte Proteico , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcrição Gênica , Vacúolos/metabolismo , Proteínas de Transporte Vesicular
20.
PLoS One ; 13(8): e0201969, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30089157

RESUMO

Candida albicans is one of the most common causes of hospital-acquired urinary tract infections (UTIs). However, azoles are poorly active against biofilms, echinocandins do not achieve clinically useful urinary concentrations, and amphotericin B exhibits severe toxicities. Thus, novel strategies are needed to prevent Candida UTIs, which are often associated with urinary catheter biofilms. We previously demonstrated that cranberry-derived proanthocyanidins (PACs) prevent C. albicans biofilm formation in an in vitro urinary model. To elucidate functional pathways unique to urinary biofilm development and PAC inhibition, we investigated the transcriptome of C. albicans in artificial urine (AU), with and without PACs. C. albicans biofilm and planktonic cells were cultivated with or without PACs. Genome-wide expression analysis was performed by RNA sequencing. Differentially expressed genes were determined using DESeq2 software; pathway analysis was performed using Cytoscape. Approximately 2,341 of 6,444 total genes were significantly expressed in biofilm relative to planktonic cells. Functional pathway analysis revealed that genes involved in filamentation, adhesion, drug response and transport were up-regulated in urinary biofilms. Genes involved in carbon and nitrogen metabolism and nutrient response were down-regulated. In PAC-treated urinary biofilms compared to untreated control biofilms, 557 of 6,444 genes had significant changes in gene expression. Genes downregulated in PAC-treated biofilms were implicated in iron starvation and adhesion pathways. Although urinary biofilms share key features with biofilms formed in other environments, many genes are uniquely expressed in urinary biofilms. Cranberry-derived PACs interfere with the expression of iron acquisition and adhesion genes within urinary biofilms.


Assuntos
Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Candida albicans/genética , Candidíase/microbiologia , Extratos Vegetais/farmacologia , Proantocianidinas/farmacologia , Infecções Urinárias/microbiologia , Vaccinium macrocarpon/química , Candida albicans/classificação , Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala , Extratos Vegetais/química , Proantocianidinas/química , Transcriptoma
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