Nutlin-3 induces apoptosis, disrupts viral latency and inhibits expression of angiopoietin-2 in Kaposi sarcoma tumor cells.

Kaposi sarcoma (KS) tumors often contain a wild-type p53. However, the function of this tumor suppressor in KS tumor cells is inhibited by both MDM2 and latent nuclear antigen (LANA) of Kaposi sarcoma-associated herpes virus (KSHV). Here, we report that MDM2 antagonist Nutlin-3 efficiently reactivates p53 in telomerase-immortalized human umbilical vein endothelial cells (TIVE) that had been malignantly transformed by KSHV as well as in KS tumor cells. Reactivation of p53 results in a G 1 cell cycle arrest, leading to inhibition of proliferation and apoptosis. Nutlin-3 inhibits the growth of "KS-like" tumors resulting from xenografted TIVE-KSHV cells in nude mice. In addition, Nutlin-3 strongly inhibits expression of the pro-angiogenic and pro-inflammatory cytokine angiopoietin-2 (Ang-2). It also disrupts viral latency by inducing expression of KSHV lytic genes. These results suggest that Nutlin-3 might serve as a novel therapy for KS.

Lipidomics of Candida albicans biofilms reveals phase-dependent production of phospholipid molecular classes and role for lipid rafts in biofilm formation.

Candida albicans-associated bloodstream infections are linked to the ability of this yeast to form biofilms. In this study, we used lipidomics to compare the lipid profiles of C. albicans biofilms and planktonic cells, in early and mature developmental phases. Our results showed that significant differences exist in lipid composition in both developmental phases. Biofilms contained higher levels of phospholipid and sphingolipids than planktonic cells (nmol per g biomass, P<0.05 for all comparisons). In the early phase, levels of lipid in most classes were significantly higher in biofilms compared to planktonic cells (P≤0.05). The ratio of phosphatidylcholine to phosphatidylethanolamine was lower in biofilms compared to planktonic cells in both early (1.17 vs 2.52, P≤0.001) and late (2.34 vs 3.81, P≤0.001) developmental phases. The unsaturation index of phospholipids decreased with time, with this effect being particularly strong for biofilms. Inhibition of the biosynthetic pathway for sphingolipid [mannosyl diinositolphosphoryl ceramide, M(IP)2C] by myriocin or aureobasidin A, and disruption of the gene encoding inositolphosphotransferase (Ipt1p), abrogated the ability of C. albicans to form biofilms. The differences in lipid profiles between biofilms and planktonic Candida cells may have important implications for the biology and antifungal resistance of biofilms.

Molecular typing and in vitro fluconazole susceptibility of Candida species isolated from diabetic and nondiabetic women with vulvovaginal candidiasis in India.

BACKGROUND: Candida albicans and Candida glabrata are the major causes of vulvovaginal candidiasis (VVC) in Indian women with diabetes mellitus. Little information is available regarding the genotyping of Candida species isolated from Indian diabetic women with VVC. METHODS: In this study, a total of 57 Candida species, comprising Candida albicans and Candida glabrata, isolated from Indian women with VVC, were genotyped and tested for fluconazole susceptibility. Arbitrarily primed polymerase chain reaction (AP-PCR) was used to genotype C glabrata isolates, whereas Southern blot hybridization using a Candida albicans repetitive element-2 (CARE-2) probe was used to genotype C albicans. RESULTS: Genotyping showed that all the C albicans isolates were genetically heterogenous. The pattern of DNA bands obtained after AP-PCR for C glabrata strains were predominantly conformed to genotype A. In vitro fluconazole-susceptibility testing of the isolates using the Clinical and Laboratory Standards Institute M27A2 protocol showed that more than 93% of the Candida isolates were susceptible. CONCLUSIONS: Ninety-five percent of the C albicans isolates analyzed were different and genetically unrelated. The analysis of the AP-PCR DNA banding pattern of C glabrata isolates showed that it resembled genotype "A". The Candida isolates were found to be susceptible to fluconazole, with minimum inhibitory concentrations ranging from 0.5 µg/mL to 8 µg/mL. This correlates with the use of fluconazole as a first-choice antifungal for treating VVC in India.

Disruption of sphingolipid biosynthetic gene IPT1 reduces Candida albicans adhesion and prevents activation of human gingival epithelial cell innate immune defense.

We demonstrated the effect of a Candida albicans sphingolipid biosynthetic gene, IPT1, on the interaction between gingival epithelial and Candida cells using monolayer cultures and engineered human oral mucosa tissue (EHOM). Disrupting the IPT1 gene greatly reduced Candida adhesion to gingival epithelial cells, compared to the wild-type and revertant strains. The yeasts adhesion to epithelial cells may activate toll-like receptors (TLRs). Cell response against Candida infection was thus investigated by evaluating TLR expression and antimicrobial peptide (AMP) production. The wild-type and revertant strains both activated TLR2, TLR4, TLR6, and TLR9 gene expression in the epithelial cells, whereas the Δipt1 mutant Candida strain had no effect on this expression. This finding was supported by an increased AMP expression (human ß-defensin HBD-2 and HBD-3) in the EHOM tissue infected with the wild-type and revertant Candida strains, and a decreased expression in the Δipt1 mutant-infected model. HBD protein secretion confirmed the absence of any effect by the Δipt1 on epithelial cell innate defense. This is the first study to demonstrate that a disruption of the IPT1 gene affects Candida-host interaction, thus preventing TLR activation and ß-defensin expression.

Characterization of biofilms formed by Candida parapsilosis, C. metapsilosis, and C. orthopsilosis.

Infections due to Candida parapsilosis have been associated with the ability of this fungus to form biofilms on indwelling medical devices. Recently, C. parapsilosis isolates were reclassified into 3 genetically non-identical classes: C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Little information is available regarding the ability of these newly reclassified species to form biofilms on biomedical substrates. In this study, we characterized biofilm formation by 10 clinical isolates each of C. parapsilosis, C. orthopsilosis, and C. metapsilosis. Biofilms were allowed to form on silicone elastomer discs to early (6h) or mature (48 h) phases and quantified by tetrazolium (XTT) and dry weight assays. Surface topography and three-dimensional architecture of the biofilms were visualized using scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM), respectively. Metabolic activity assay revealed strain-dependent biofilm forming ability of the 3 species tested, while biomass determination revealed that all 3 species formed equivalent biofilms (P>0.05 for all comparisons). SEM analyses of representative isolates of these species showed biofilms with clusters of yeast cells adherent to the catheter surface. Additionally, confocal microscopy analyses showed the presence of cells embedded in biofilms ranging in thickness between 62 and 85 microm. These results demonstrate that similar to C. parapsilosis, the 2 newly identified Candida species (C. orthopsilosis and C. metapsilosis) were able to form biofilms.

Parenteral lipid emulsion induces germination of Candida albicans and increases biofilm formation on medical catheter surfaces.

BACKGROUND: The administration of parenteral nutrition, including lipid emulsion (LE), to patients via medical catheters is an unexplained risk factor for the development of candidemia. Germination and biofilm formation are recognized virulence determinants of Candida albicans. No studies have addressed the effect of LE on candidal biofilm production. In this study, we investigated the effect of LE on candidal germination and its ability to form biofilm on medical catheter material. METHODS: C. albicans strain SC-5314 was grown in standard growth medium in the presence or absence a commercially available LE. Biofilms grown on silicone-elastomer catheter discs in these media were compared for mass by dry weight measurements. Biofilm morphology was analyzed by scanning electron microscopy and confocal laser microscopy. The effect of LE on C. albicans germination and growth was evaluated microscopically and by determination of colony-forming units, respectively. RESULTS: Addition of LE to standard growth medium increased C. albicans biofilm production and resulted in observed changes in biofilm morphology and architecture. Furthermore, LE induced germination and supported the growth of C. albicans. CONCLUSIONS: LE-inducible candidal virulence determinants, such as germination and enhanced biofilm production, may help to explain the increased risk of candidemia in patients receiving LE via medical catheters.

Iron deprivation induces EFG1-mediated hyphal development in Candida albicans without affecting biofilm formation.

In this study, we investigated the role of cellular iron status in hyphae and biofilm formation in Candida albicans. Iron deprivation by a chelator, bathophenanthrolene disulfonic acid, promoted hyphal development even in nonhyphal-inducing media without affecting growth of C. albicans cells. Iron-acquisition defective mutants, Deltaftr1 and Deltaccc2, also showed hyphal formation, which was prevented by iron supplementation. Notably, most of the tested morphological mutants Deltacph1, Deltaefh1 and Deltatpk1 continued to form hyphae under iron-deprived conditions, except the Deltaefg1 null mutant, which showed a complete block in hyphae formation. The role of EFG1 in filamentation under iron-deprived conditions was further confirmed by Northern analysis, which showed a considerable upregulation of the EFG1 transcript. Of notable importance, all the morphological mutants including Deltaefg1 mutant possessed enhanced membrane fluidity under iron-deprived conditions; however, this did not appear to contribute to hyphal development. Interestingly, iron deprivation did not affect the ability of C. albicans to form biofilms on the catheter surface and led to no gross defects in azole resistance phenotype of these biofilms of C. albicans cells. Our study, for the first time, establishes a link between cellular iron, Efg1p and hyphal development of C. albicans cells that is independent of biofilm formation.

Characterization and partial purification of Candida albicans Secretory IL-12 Inhibitory Factor.

BACKGROUND: We have previously shown that supernatant from Candida albicans (CA) culture contains a Secretory Interleukin (IL)-12 Inhibitory Factor (CA-SIIF), which inhibits IL-12 production by human monocytes. However, the effect of CA-SIIF on secretion of other cytokines by monocytes is unknown, and detailed characterization of this factor has not been performed. RESULTS: In this study, we demonstrate that the IL-12 inhibitory activity of CA-SIIF was serum-independent, based on the reduction of IL-12 levels in monocytes stimulated under serum-independent conditions. The minimal inhibitory dose of CA-SIIF was found to be 200 mug/ml. Investigation of CA-SIIF's effect on macrophages IL-12 production in vitro and in vivo also showed that CA-SIIF inhibited IL-12 production by murine macrophages both in vitro (from 571 +/- 24 pg/ml to 387 +/- 87 pg/ml; P = 0.05) and in vivo (from 262 +/- 6 pg/ml to 144 +/- 30 pg/ml; P < 0.05). In addition to IL-12, cytokine array analysis revealed that CA-SIIF induced differential production of other cytokines also. In this regard, reduction in levels were observed for IL-8, IL-10, IL-13, monocyte chemoattractant protein (MCP)-1, MCP-2, macrophage inflammatory protein (MIP)-1, RANTES, etc. In contrast, levels of other chemokines e.g. MCP-4, MIF and MIP-3alpha (P < 0.05) were increased. We also found that CA-SIIF suppressed the maturation of human monocytes to dendritic cells (CD1a expression = 13 +/- 3% vs 36 +/- 2% of the control; P < 0.01). Next, to identify the biochemical nature of CA-SIIF, we separated this factor into a Concanavalin A (ConA)-binding glycoprotein fraction (CA-SIIF-GP) and a non-ConA-binding protein fraction (CA-SIIF-NGP) using ConA affinity chromatography. Both fractions were then tested for this inhibitory effect on human monocyte IL-12 production. CA-SIIF-GP produced a higher inhibitory effect on IL-12 production compared to CA-SIIF-NGP and CA-SIIF crude (P < 0.01), proving that CA-SIIF is a glycoprotein in nature. CONCLUSION: CA-SIIF is a glycoprotein which exhibits serum-independent inhibition of IL-12 production from monocytes in vitro and in vivo, and also modulates differentiation of monocytes into dendritic cells. These results suggest important role for CA-SIIF in interactions of C. albicans with the host immune system.

Fusarium and Candida albicans biofilms on soft contact lenses: model development, influence of lens type, and susceptibility to lens care solutions.

Fungal keratitis is commonly caused by Fusarium species and less commonly by Candida species. Recent outbreaks of Fusarium keratitis were associated with contact lens wear and with ReNu with MoistureLoc contact lens care solution, and biofilm formation on contact lens/lens cases was proposed to play a role in this outbreak. However, no in vitro model for contact lens-associated fungal biofilm has been developed. In this study, we developed and characterized in vitro models of biofilm formation on various soft contact lenses using three species of Fusarium and Candida albicans. The contact lenses tested were etafilcon A, galyfilcon A, lotrafilcon A, balafilcon A, alphafilcon A, and polymacon. Our results showed that clinical isolates of Fusarium and C. albicans formed biofilms on all types of lenses tested and that the biofilm architecture varied with the lens type. Moreover, differences in hyphal content and architecture were found between the biofilms formed by these fungi. We also found that two recently isolated keratitis-associated fusaria formed robust biofilms, while the reference ATCC 36031 strain (recommended by the International Organization for Standardization guidelines for testing of disinfectants) failed to form biofilm. Furthermore, using the developed in vitro biofilm model, we showed that phylogenetically diverse planktonic fusaria and Candida were susceptible to MoistureLoc and MultiPlus. However, Fusarium biofilms exhibited reduced susceptibility against these solutions in a species- and time-dependent manner. This in vitro model should provide a better understanding of the biology and pathogenesis of lens-related fungal keratitis.

Membrane fluidity and lipid composition in clinical isolates of Candida albicans isolated from AIDS/HIV patients.

In this study, we describe the membrane lipid composition of eight clinical isolates (azole resistant and sensitive strains) of Candida albicans isolated from AIDS/ HIV patients. Interestingly, fluorescence polarization measurements of the clinical isolates displayed enhanced membrane fluidity in fluconazole resistant strains as compared to the sensitive ones. The increase in fluidity was reflected in the change of membrane order, which was considerably decreased (decrease in fluorescence polarization "p" value denotes higher membrane fluidity) in the resistant strains. The ergosterol content in azole susceptible isolates was greater, almost twice as compared to the resistant isolates. However, no significant alteration was observed in phospholipid and fatty acid composition of these isolates. Labeling experiments with fluorescamine dye revealed that the percentage of phosphatidylethanolamine exposed to the membrane's outer leaflet was higher in the resistant strains as compared to the sensitive strains, indicating increased floppase activity of the two major ABC drug efflux pumps, CDR1 and CDR2 possibly due to their overexpression in resistant strains. The results of the present study suggest that changes in the status of membrane lipid phase especially the ergosterol content and increased activity of drug efflux pumps by overexpression ofABC transporters, CDR1 and CDR2 might contribute to fluconazole resistance in C. albicans isolated from AIDS/HIV patients.

Allelic variants of ABC drug transporter Cdr1p in clinical isolates of Candida albicans.

Candida drug resistance protein (Cdr1p) is a major drug efflux protein, which plays a key role in commonly encountered clinical azole resistance in Candida albicans. We have analyzed its sequence in several azole resistant clinical isolates to evaluate the allelic variation within CDR1 gene and to relate it to its functional activity. The sequence analysis revealed 53 single nucleotide polymorphisms (SNPs), out of which six were non-synonymous single nucleotide polymorphisms (NS-SNPs) implying a change in amino acid and were found in two or more than two allelic combinations in different sensitive or resistant isolates. We have identified three new NS-SNPs namely, E948P, T950S, and F1399Y, in isolates wherein F1399Y appeared to be unique and was present in one of the naturally occurring azole resistant isolates obtained from Indian diabetic patients. However, site-directed mutagenesis showed that the residue F1399 in between TMS 11 and TMS 12 does not affect the functionality of Cdr1p. Taken together, our SNPs analyses reveal that unlike human P-gp, the naturally acquired allelic variations are mostly present in non-conserved regions of the protein which do not allow Cdr1p to genetically evolve in a manner, that would allow a change in its functionality to affect substrate recognition, specificity, and drug efflux activity of C. albicans cells.

The glyoxylate cycle enzyme activities in the pathogenic isolates of Candida albicans obtained from HIV/AIDS, diabetic and burn patients.

Recently it has been found that Candida albicans harbours enzymes involved in the glyoxylate cycle (GC), which have a role in its virulence, especially the two key enzymes, isocitrate lyase (ICL) and malate synthase (MS). There are however, few studies on the GC enzyme activities isolated in the clinical isolates. Samples were collected from three groups of patients namely, HIV/AIDS, diabetic and burn patients suffering from candidiasis at different body locations. Isolation, identification and the antifungal susceptibility test of all the isolates of C. albicans were followed by the standard techniques. Measurements of all the GC enzyme activities were also carried out by the standard methods. Levels of the principal GC enzymes showed significant changes when calculated and compared taking control strains of C. albicans. The activity of the two key enzymes of the GC, ICL and MS were significantly higher in the isolates from diabetic patients. No significant relationship between the drug susceptibility and the level of enzymes of the GC was observed. As GC activity is absent in mammalian cells, a specific inhibitor for the GC could be developed and these enzymes therefore can be used as a new antifungal target.

Pattern of Candida species isolated from patients with diabetes mellitus and vulvovaginal candidiasis and their response to single dose oral fluconazole therapy.

OBJECTIVE: Patients with diabetes mellitus are at increased risk of vulvovaginal candidiasis (VVC). Besides Candida albicans, they often have infection due to non-C. albicans Candida species such as C. glabrata. Oral single dose fluconazole (150 mg) is commonly used to treat VVC in non-diabetic individuals with response rate varying from 70 to 90%. However, there is paucity of related information in diabetic women with VVC. Present study has been conducted to systematically assess the effect of fluconazole therapy among diabetic patients with clinically symptomatic VVC. METHODS: Study subjects included 85 consecutive patients with diabetes mellitus (type 2=70 and type 1=15) and 62 non-diabetic women who had clinical signs and symptoms of VVC and in whom evidence of candidiasis was documented by presence of yeast on direct microscopy followed by culture. Single dose fluconazole (150 mg) was given orally to all the subjects in a supervised manner. Subjects were reassessed on 14th day after fluconazole therapy and a repeat high vaginal swab was taken for direct microscopy and fungal culture. Total glycosylated haemoglobin (HbA1) was measured to assess glycaemic control. RESULTS: There were no significant differences in the frequency of pruritus (55.9 vs. 56.7%), vaginal discharge (63.8 vs. 69.0%), dyspareunia (25.0 vs. 20.0%), and percentage yeast positivity (67.5 vs. 54.7%) between diabetic and control groups before the start of fluconazole therapy. Following fluconazole therapy, vaginal discharge on examination and yeast positivity on direct microscopy continued to remain positive in higher percentage of subjects in the diabetic group as compared to non-diabetic subjects (52.5 vs. 36.4%; P =0.22 and 50.7 and 29.0%, respectively, P =0.07, respectively). Overall 67.1% of patients with diabetes and 47.3% of controls continued to show persistence of Candida growth on high vaginal swab culture following fluconazole treatment (P=0.042). Candida glabtara was the most common species isolated in patients with diabetes mellitus and its frequency was significantly higher in them when compared to control group (54.1 vs. 22.6%, P<0.001). C. albicans was the most common species isolated in controls. Species-specific response to fluconazole showed that 81.3% of patients in the diabetic group and 78.6% of the non-diabetic controls continued to show fungal growth when C. glabrata was the organism grown (P=0.99). However, in case of C. albicans, 45.4% of the patients in the diabetic group and only 21.5% of the controls had persistent Candida growth following fluconazole therapy (P=0.22). CONCLUSION: Overall only one third of patients with diabetes mellitus and VVC respond to single dose 150 mg of fluconozole therapy. Limited response in the clinical symptoms and culture negativity following single dose fluconazole therapy in diabetic subjects with VVC is explained by the high prevalence of C. glabrata in them. The present study involved only 85 patients and majority of them had type-2 diabetes mellitus. There is need to perform similar study in large number of diabetics subjects including patients with type-1 diabetes mellitus and assess various alternative treatment protocol which are also effective in C. glabrata infection.

Susceptibility pattern and molecular type of species-specific Candida in oropharyngeal lesions of Indian human immunodeficiency virus-positive patients.

A study of oropharyngeal candidiasis (OPC) in Indian human immunodeficiency virus (HIV)/AIDS patients was conducted over a period of 15 months. This study revealed that 75% of the HIV/AIDS patients had OPC. MIC testing revealed that 5% of the Candida isolates were fluconazole resistant. A correlation between CD4(+)-T-cell counts and development of OPC in HIV/AIDS patients was also observed. Molecular typing of C. albicans isolates showed that all were genetically unrelated.

Epidemiology and molecular typing of Candida isolates from burn patients.

This study, spread over a span of 2 years describes Candida infections in burn patients of an Indian hospital. A total of 220 burn patients were monitored and Candida could be isolated from 138 patients. A total of 228 different Candida species were obtained from various body locations of these patients. Species identification revealed that Candida albicans was the most predominant (45%) followed by Candida tropicalis (33%), Candida glabrata (13.5%), C. parapsilosis (4%), C. krusei (2.75%) and C. kefyr (1.75%). DNA fingerprinting of all C. albicans isolates was done by using CARE-2 probe. Fingerprinting analyses of all the C. albicans strains revealed that strains collected from different patients were different. It is noteworthy that patients with disseminated candidiasis had a similar, but unique strain isolated from all body locations, suggesting a possibility that commensal isolates might be turning pathogenic. Taken together, this is probably the first ever detailed survey of Candida infections in burn patients in India and is expected to lead to better clinical management of this group of patients.