Stella M. Bernardo

In vitro analyses of the combination of high-dose doxycycline and antifungal agents against Candida albicans biofilms.

The potential of antifungal agents used as antimicrobial lock therapy (ALT) for the conservative management of catheter-related candidemia has not been fully defined. We sought to determine the antifungal effect of high-dose doxycycline (DOX), alone or in combination with standard concentrations of amphotericin B (AMB), caspofungin (CAS) or fluconazole (FLC), against biofilms formed by Candida albicans in vitro. DOX alone (at 2048 microg/mL and 1024 microg/mL) demonstrated up to an 85% reduction of the metabolic activity of the C. albicans biofilm. Regardless of the concentration tested, FLC alone showed minimal activity (mean 22.9% reduction) against the C. albicans biofilm. When DOX 2048 microg/mL was used in combination with FLC, antifungal activity also increased up to 85%, suggesting an additive effect. DOX 128 microg/mL in combination with FLC demonstrated synergy (mean 58.3% reduction). The combination of DOX 2048 microg/mL or 512 microg/mL and AMB was superior to AMB alone at low concentrations (0.25-0.03125 microg/mL). However, DOX 128 microg/mL was antagonistic in combination with low concentrations of AMB. Maximal efficacy against the biofilm was observed with CAS at 8-0.25 microg/mL compared with FLC and AMB alone. A paradoxical effect (PE) occurred with CAS at 16 microg/mL, which showed a marked reduction in antifungal activity compared with lower concentrations of CAS. CAS at 16 microg/mL in combination with either DOX 2048 microg/mL or 512 microg/mL resulted in attenuation of the PE. These findings suggest that a high-dose DOX-based ALT strategy in combination with traditional antifungal agents may be useful for the treatment of C. albicans biofilms.

Candida albicans VPS1 contributes to protease secretion, filamentation, and biofilm formation

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.

The interaction of induction, repression and starvation in the regulation of extracellular proteases in Aspergillus nidulans: evidence for a role for CreA in the response to carbon starvation

In Aspergillus nidulans, production of extracellular proteases in response to carbon starvation and to a lesser extent nitrogen starvation is controlled by XprG, a putative transcriptional activator. In this study the role of genes involved in carbon catabolite repression and the role of protein as an inducer of extracellular protease gene expression were examined. The addition of exogenous protein to the growth medium did not increase extracellular protease activity whether or not additional carbon or nitrogen sources were present indicating that induction does not play a major role in the regulation of extracellular proteases. Northern blot analysis confirmed that protein is not an inducer of the major A. nidulans protease, PrtA. Mutations in the creA, creB and creC genes increased extracellular protease levels in medium lacking a carbon source suggesting that they may have a role in the response to carbon starvation as well as carbon catabolite repression. Analysis of glkA4 frA2 and creAΔ4 mutants showed that the loss of glucose signalling or the DNA-binding protein which mediates carbon catabolite repression did not abolish glucose repression but did increase extracellular protease activity. This increase was XprG-dependent indicating that the effect of these genes may be through modulation of XprG activity.

Candida albicans SUR7 contributes to secretion, biofilm formation, and macrophage killing

BackgroundCandida 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 sur7 Δ null mutant and examined its contribution to key virulence attributes.ResultsStructurally, the C. albicans sur7 Δ 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°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 sur7 Δ 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 sur7 Δ mutant demonstrated increased adhesion to polystyrene and of note, was markedly defective in biofilm formation. In an in vitro macrophage model of virulence, the sur7 Δ mutant was impaired in macrophage killing.ConclusionsPlasma 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 sur7 Δ mutant.

Cranberry-derived proanthocyanidins prevent formation of Candida albicans biofilms in artificial urine through biofilm- and adherence-specific mechanisms

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.

Deletion of Vacuolar Proton-translocating ATPase Voa Isoforms Clarifies the Role of Vacuolar pH as a Determinant of Virulence-associated Traits in Candida albicans

Background: V-ATPase regulates pH, and Candida albicans virulence is pH-dependent. Results: Deletion of V-ATPase Voa subunit Vph1p, but not Stv1p, alkalinizes vacuoles; several virulence-related traits remain unaffected. Conclusion: Vacuolar acidification is not essential for in vitro filamentation, biofilm formation, and macrophage killing in C. albicans. Significance: Stv1p in non-vacuolar organelles may play important roles in C. albicans infectivity, particularly if Vph1p is not functional. 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.

Candida albicans VMA3 is necessary for V-ATPase assembly and function and contributes to secretion and filamentation.

ABSTRACT 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.

In vitro analyses of ethanol activity against Candida albicans biofilms.

ABSTRACT 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.

Candida krusei sepsis secondary to oral colonization in a hemopoietic stem cell transplant recipient

Yeasts other than Candida albicans have emerged as important causes of fungal infection in hemopoietic stem cell transplant (HSCT) patients, particularly those receiving fluconazole prophylaxis. We report on an autologous hemopoietic stem cell transplant recipient who developed Candida krusei sepsis from pre-existing oral colonization.

In vitro analyses of the effects of heparin and parabens on Candida albicans biofilms and planktonic cells

ABSTRACT 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.