Effect of phenotypic switching on expression of virulence factors by Candida albicans causing candidiasis in diabetic patients

A total of 110 strains belonging to seven species of Candida were isolated from various forms of candidiasis in diabetic patients. They were Candida albicans 53 (47%), Candida tropicalis 36 (33%), Candida glabrata 9 (8%), Candida parapsilosis 4 (4%), Candida guilliermondii 2 (2%), Candida krusei 5 (5%) and Candida kefyr 1 (1%). All 53 strains of C. albicans isolated were observed to express virulence factors such as cell surface hydrophobicity (CSH), adherence to human buccal epithelial cell (BEC) and proteinase activity (100%), while phospholipase activity was observed in 52 (98%). Phenotypic switching and its influence on the pathogenicity of C. albicans were studied. Two C. albicans strains isolated from oral and vaginal thrush, respectively, in diabetic individuals, and the control strain C. albicans NCPF 3153A were induced to undergo phenotypic switching by exposure to UV light and the degree of expression of virulence factors by the different morphological forms was determined. Three different morphological forms of C. albicans were obtained, namely Star (S), Wrinkled (W) and Ring (R) types from the original Smooth (O) variety. It was found that proteinase activity was greatest with the W type followed by the R type then the O type. The S type produced the least proteinase. The phospholipase activity was greatest with O type followed by R type. The W and S types produced the least phospholipase. Expression of CSH and adherence was greatest in the O type followed by the R and then the W type and finally the S type. Differential expression of virulence factors occurs with different phenotypic forms of C. albicans and this may provide a particular morphological type with a distinct advantage over other types in causing candidiasis.     

Adhesion of Candida albicans to epithelial cells effect of polyoxin D

Data from our previous studies suggested that the fungal cell wall component, chitin, is involved in the adhesion of Candida albicans to mucosal surfaces. In the present study, we investigated the effect of polyoxin D, an inhibitor of chitin synthase, on the interaction of the fungus with epithelial cells. The effect of polyoxin D on Candida was evaluated in in vitro assays for its capacity to adhere to buccal epithelial cells (BEC), and by fluorescent-microscopy photometry and flow cytometry using cells stained with cellufluor (CF), a fluorochrome with affinity for chitin. C. albicans grown with and without polyoxin D was stained with CF and examined in a fluorescent microscope equipped with a photometer. Measurements of fluorescence revealed a wide range of intensity among C. albicans cells and a decreased intensity in polyoxin D treated cultures. Flow cytometry analyses of yeasts revealed 2 peaks of fluorescence intensity, and pointed to differences between polyoxin D treated and non-treated microorganisms. C. albicans stained with CF were separated into 2 subpopulations by flow cytometry according to fluorescence intensity. In vitro adhesion of each subpopulation to BEC was similar. Polyoxin D treated fungi showed significantly reduced adherence to BEC, as evaluated by a radioactivity assay with radiolabelled yeasts and by microscopic readings. The reduction in adhesion was Polyoxin D concentration dependent. These observations support our previous findings suggesting involvement of chitin in the attachment process of C. albicans (CBS562) to epithelial cells.     

Role of cell surface hydrophobicity in Candida albicans biofilm

Overall cell surface hydrophobicity (CSH) is predicted to play an important role during biofilm formation in Candida albicans but is the result of many expressed proteins. This study compares the CSH status and CSH1 gene expression in C. albicans planktonic cells, sessile biofilm, and dispersal cells. Greater percentages of hydrophobic cells were found in non-adhered (1.5 h) and dispersal forms (24 or 48 h) (41.34±4.17% and 39.52±7.45%, respectively), compared with overnight planktonic cultures (21.69±3.60%). Results from quantitative real-time PCR confirmed greater up-regulation of the CSH1 gene in sessile biofilm compared with both planktonic culture and dispersal cells. Up-regulation was also greater in dispersal cells compared with planktonic culture. The markedly increased CSH found both in C. albicans biofilm, and in cells released during biofilm formation could provide an advantage to dispersing cells building new biofilm.     

Changes in Adhesion of Candida tropicalis Clinical Isolates Exhibiting Switch Phenotypes to Polystyrene and HeLa Cells

Background

Candida tropicalis is an important human pathogen that can undergo multiple forms of phenotypic switching.

Aim

We aimed to evaluate the effect of phenotypic switching on the adhesion ability of C. tropicalis.

Methods

C. tropicalis morphotypes included parental phenotypes (clinical isolates) and switch phenotypes (crepe, revertant of crepe—CR, rough, revertant of rough—RR, irregular center and revertant of irregular center—ICR). Adhesion to polystyrene and HeLa cells was determined by crystal violet assay. The percentage of HeLa cells with adhered yeasts and the number of adhered yeasts per HeLa cell were determined by light microscopy. Filamentation among adhered cells was assessed by direct counting.

Results

On polystyrene, 60% of the switch strains showed difference (p?<?0.05) on adhesion ability compared to their parental counterpart strains, and altered thickness of adhered cells layers. Filamentation was increased among adhered cells of the switched strains compared to parental strains. A positive correlation was observed between adhesion on polystyrene and filamentation for morphotypes of the system 49.07. The majority of the switched strains showed higher adhesion capability to HeLa cells in comparison to the adherence of the clinical strains. All revertant strains showed a higher number of yeast cells per HeLa cell compared to their variant counterparts (p?<?0.05), with exception of the ICR.

Conclusions

Our findings indicate that switching events in C. tropicalis affect adhesion and filamentation of adhered cells on polystyrene and HeLa cells. The rise of switch strains with increased adhesion ability may contribute to the success of infection associated with C. tropicalis.

     

Cell surface hydrophobicity of Candida albicans isolated from elder patients undergoing denture‐related candidosis

Background: The virulence potential of Candida albicans strains enrolled in denture‐related candidosis still remains uncertain. Candida albicans cells with higher cell surface hydrophobicity (CSH) rates, so‐called hydrophobic, present higher adhesion success in different host tissues than cells with lower rates, or even hydrophilic. Objective: The proposition of this study was to evaluate the differences in the CSH of strains isolated from denture users with and without denture‐related candidosis. Material and methods: The strains were obtained from two paired groups of patients living a same retirement house. Fungal cells were submitted to CSH evaluation by the hydrocarbon partition test using xylene. Results: The measures revealed that the yeasts from patients with candidosis had CSH values ranging from 4.52% to 12.24%, with an average of 8.22 ± 2.92%. In the countergroup, the CSH ranged from 3.86% to 14.36%, with an average of 8.38 ± 3.76%. The difference between the groups were considered not relevant (p = 0.997). Conclusion: The results let to the inference that natural populations of C. albicans from patients with and without clinical manifestation denture‐related candidosis do not differ one from the other regarding to CSH.     

Capric Acid Secreted by S. boulardii Inhibits C. albicans Filamentous Growth,Adhesion and Biofilm Formation

Candidiasis are life-threatening systemic fungal diseases, especially of gastro intestinal track, skin and mucous membranes lining various body cavities like the nostrils, the mouth, the lips, the eyelids, the ears or the genital area. Due to increasing resistance of candidiasis to existing drugs, it is very important to look for new strategies helping the treatment of such fungal diseases. One promising strategy is the use of the probiotic microorganisms, which when administered in adequate amounts confer a health benefit. Such a probiotic microorganism is yeast Saccharomyces boulardii, a close relative of baker yeast. Saccharomyces boulardii cells and their extract affect the virulence factors of the important human fungal pathogen C. albicans, its hyphae formation, adhesion and biofilm development. Extract prepared from S. boulardii culture filtrate was fractionated and GC-MS analysis showed that the active fraction contained, apart from 2-phenylethanol, caproic, caprylic and capric acid whose presence was confirmed by ESI-MS analysis. Biological activity was tested on C. albicans using extract and pure identified compounds. Our study demonstrated that this probiotic yeast secretes into the medium active compounds reducing candidal virulence factors. The chief compound inhibiting filamentous C. albicans growth comparably to S. boulardii extract was capric acid, which is thus responsible for inhibition of hyphae formation. It also reduced candidal adhesion and biofilm formation, though three times less than the extract, which thus contains other factors suppressing C. albicans adherence. The expression profile of selected genes associated with C. albicans virulence by real-time PCR showed a reduced expression of HWP1, INO1 and CSH1 genes in C. albicans cells treated with capric acid and S. boulardii extract. Hence capric acid secreted by S. boulardii is responsible for inhibition of C. albicans filamentation and partially also adhesion and biofilm formation.     

Adherence Patterns of <Emphasis Type="Italic">Histoplasma capsulatum</Emphasis> Yeasts to Bat Tissue Sections

The adherence of Histoplasma capsulatum yeasts to lung, spleen, liver, gut, and trachea cryosections of Artibeus hirsutus bats and inbred BALB/c mice (control) was studied after in vitro yeast-tissue incubations. Candida albicans yeasts were used as a well-known adherent fungal model in the mice host, and latex beads were used as a negative adherence control. Adhered yeast cells were identified by using crystal violet staining and the immunoperoxidase method with specific antibodies. H. capsulatum yeasts adhered to all tissues tested, mainly in the lung. Moreover, H. capsulatum yeasts adhered preferentially to white and red spleen pulp, in contrast to the dispersed distribution of C. albicans yeasts. H. capsulatum yeasts were mostly found on the sinusoidal face of hepatocytes. In general, the gut showed a higher number of adhered H. capsulatum yeasts than the trachea in both bats and mice. H. capsulatum and C. albicans yeasts developed high selectivity for the lamina propria of the gut. In addition, H. capsulatum yeasts interacted better with the lamina propria and adventitia of the trachea. The number of H. capsulatum yeast cells that adhered to each tissue section type was always greater than the corresponding number of C. albicans yeast cells, and latex beads never adhered to the tissue sections. Controls with anti-H. capsulatum and normal rabbit sera showed a significant blockage of H. capsulatum yeast adherence to lung tissue. This is the first study describing the patterns of H. capsulatum yeast adherence to different bat and mouse tissues.     

Characterization of Candida albicans and Staphylococcus aureus polymicrobial biofilm on different surfaces

BackgroundStaphylococcus aureus and Candida albicans have been co-isolated from biofilm-associated diseases such as denture stomatitis, periodontitis, and burn wound infections, as well as from medical devices. However, the polymicrobial biofilm of both microorganisms has not been fully characterized.AimsTo characterize the polymicrobial biofilm of C. albicans and S. aureus in terms of microbial density, synergy, composition, structure, and stability against antimicrobials and chemical agents.MethodsCrystal violet assay was used to measure the biofilm formation. Scanning electron microscopy and confocal microscopy were used to analyze the structure and chemical composition of the biofilms, respectively.ResultsSupplemented media with fetal bovine serum (FBS) decreased the biofilm formation of S. aureus and the polymicrobial biofilm. For C. albicans, depending on the culture media, the addition of glucose or FBS had a positive effect in biofilm formation. FBS decreased the adhesion to polystyrene wells for both microorganisms. Supplementing the media with glucose and FBS enhanced the growth of C. albicans and S. aureus, respectively. It seems that C. albicans contributes the most to the adhesion process and to the general structure of the biofilms on all the surfaces tested, including a catheter model. Interestingly, S. aureus showed a great adhesion capacity to the surface of C. albicans in the biofilms. Proteins and β-1,6-linked polysaccharides seem to be the most important molecules in the polymicrobial biofilm.ConclusionsThe polymicrobial biofilm had a complex structure, with C. albicans serving as a scaffold where S. aureus adheres, preferentially to the hyphal form of the fungus. Detection of polymicrobial infections and characterization of biofilms will be necessary in the future to provide a better treatment.     

Adhesion of Candida albicans mutant strains to host tissue

Adhesion of Candida albicans to host cells is believed to represent a fungal virulence factor and a significant step in the development of candidiasis. As C. albicans strains may differ in their in vitro adhesion ability we initiated a study to investigate whether mutant strains differ in this respect from their parent wild-type. We assessed the in vitro adhesion of C. albicans CBS562 and two mutants obtained by mutagenesis with N′-nitrosoguanidine: a histidine auxotroph, SAG5, derived from CBS562, and a respiratory-deficient strain (a petite mutant), SAR1, derived from SAG5. The adhesion was tested in vitro using two target cell systems: (1) exfoliated human buccal epithelial cells (BEC); and (2) human keratinocyte tissue line cells (HaCaT cells). Adhesion to BEC was evaluated microscopically and that to HaCaT cells by a direct ELISA technique. The results indicated a 54% reduction in adhesion to BEC for SAG5 and 30% for SAR1 as compared to the wild-type, and a 25% reduction in adhesion to HaCaT cells for SAG5 and 20% for SAR1. To verify whether the prototrophy restores the adhesion ability, we complemented the his-negative auxotroph by transforming the strain with the HIS4 gene. Then we assayed the adhesion to BEC of the complemented his-negative mutant in comparison to that of the wild-type, the his-negative mutant (SAG5) and the plasmid-cured transformant. The adhesion values of the complemented his-negative strain were similar to those of the wild-type, whereas the values of the plasmid-cured strain were similar to those of SAG5.     

Surface hydrophobicity of "rheumatogenic" and "nephritogenic" strains of group A streptococci and the ultrastructural surface feature of pharyngeal cells exposed to group A streptococci.

The present study was carried out to determine the surface hydrophobicity of group A streptococcal strains responsible for rheumatic fever (RF), "rheumatogenic" strains (RG strains) and strains causing glomerulonephritis, "nephritogenic" strains (NG strains) in relation to their adhesion to human pharyngeal cells. Scanning electronmicroscopic (SEM) studies were carried out to the difference, if any, in the adherence of group A streptococci (M type 5) to pharyngeal and buccal cells (PEC and BEC). By employing two techniques for hydrophobicity determination, salt aggregation titre (SAT) and n-hexadecane binding technique, it was observed that RG strains (M5, M1 and M6) were more hydrophobic than NG strain, M49. However, NG strain M12 was almost equally as hydrophobic as RG strains. The adherence of RG strains, except M1 and M24, to PEC was greater in number than that of NG strains. Although M1 strain was hydrophobic, its adherence to PEC was less. Pepsin and trypsin treatment with streptococci reduced the hydrophobicity and adherence of RG and NG strains to PEC. SEM studies revealed firmly adhered indigenous bacteria on PEC and BEC. Streptococci (M5) adhered more to PEC than to BEC. SEM studies also showed that PEC had a peculiar ultrastructural surface feature to which streptococci adhered. These findings suggest that streptococcal hydrophobicity alone does not determine their adhesion to PEC. The surface nature of PEC might be a characteristic feature of the epithelial cells that allows streptococci to adhere and colonize or it might be a consequence of streptococcal adhesion.     

Species Distribution and Virulence Factors of Candida spp. Isolated from the Oral Cavity of Kidney Transplant Recipients in Brazil

Although yeasts belonging to the genus Candida are frequently seen as commensals in the oral cavity, they possess virulence attributes that contribute for pathogenicity. The aims of the present study were to study the prevalence of Candida spp. isolated from the oral cavity of renal transplant recipients and to analyze strains virulence factors. We isolated a total of 70 Candida strains from 111 transplant recipients, and Candida albicans was the most prevalent species (82.86 %). Oral candidiasis was diagnosed in 14.4 % kidney transplant patients, while 11 isolates (15.7 %) corresponded to non-Candida albicans Candida (NCAC) species. C. albicans adhered to a higher extension than NCAC strains. Some isolates of Candida tropicalis were markedly adherent to human buccal epithelial cells and highly biofilm-forming strains. Regarding proteinase activity, Candida orthopsilosis was more proteolytic than Candida metapsilosis. Candida glabrata and Candida dubliniensis showed very low ability to form biofilm on polystyrene microtiter plates. We have demonstrated here diverse peculiarities of different Candida species regarding the ability to express virulence factors. This study will contribute for the understanding of the natural history and pathogenesis of yeasts belonging to the genus Candida in the oral cavity of patients who were submitted to kidney transplant and are under immunosuppressive therapies.     

Biomaterial-Associated Infection with Candida albicans in Mice

Candida yeasts are frequently isolated from patients with continuous ambulatory peritoneal dialysis peritonitis or other biomaterial-associated infections. The mouse model of candidal peritonitis was used to study the interaction of Candida cells with end-point attached heparinized polyethylene (H-PE) and with polymorphonuclear leukocytes (PMNs) or macrophages (Mφ). Two Candida strains differing in cell surface hydrophobicity and in expression of fibronectin (Fn) binding were used for the study. Cells of both Candida strains adhered at higher numbers to H-PE surfaces preadsorbed with Fn or with human dialysis fluid (HDF) than to non-modified H-PE, supporting a role of Fn in mediating adhesion. C. albicans 4016 cells expressing low hydrophobicity and low binding of soluble Fn demonstrated stronger adhesion to PMNs than the more hydrophobic C. albicans 3248 yeasts, which express high binding of soluble Fn. However, C. albicans 4016 cells were more resistant to phagocytic killing and were hardly eradicated in intraperitoneally infected mice. The animals depleted in PMNs by treatment with CY were neither able to eradicate C. albicans 3248 (rapidly eliminated by normal mice) nor C. albicans 4016 yeasts (with a tendency to persist in the tissues of normal mice).     

Adhesive Properties and Hydrolytic Enzymes of Oral <Emphasis Type="Italic">Candida albicans</Emphasis> Strains

Several virulence factors in Candida albicans strains such as production of hydrolytic enzymes and biofilm formation on surfaces and cells can contribute to their pathogenicity. For this, control of this opportunistic yeast is one of the factors reducing the nosocomial infection. The aim of this study was to investigate biofilm formation on polystyrene and polymethylmethacrylate and the production of hydrolytic enzymes in Candida albicans strains isolated from the oral cavity of patients suffering from denture stomatitis. All strains were identified by macroscopic, microscopic analysis and the ID 32 C system. Our results showed that 50% of the total strains produced phospholipase. Furthermore, protease activity was detected in seven (35%) strains. All Candida albicans strains were beta haemolytic. All C. albicans strains adhered to polystyrene 96-well microtiter plate at different degrees, and the metabolic activity of C. albicans biofilm formed on polymethylmethacrylate did not differ between tested strains. The atomic force micrographs demonstrated that biofilm of Candida albicans strains was organized in small colonies with budding cells.     

Actividad de la proteinasa en cepas de Candida albicans aisladas de la cavidad oral de pacientes inmunodeprimidos,con candidiasis oral y sujetos sanos

BackgroundCandida albicans has a variety of virulence factors, including secreted aspartyl proteases, which are determinant factors in the pathogenesis of this yeast in immunocompromised patients.AimsProteinase activity was identified in C. albicans strains isolated from the oral cavity of immunocompromised patients with cancer, diabetes and HIV+, with oral candidiasis and in healthy subjects.MethodsTwo hundred and fifty C. albicans strains were analyzed, distributed in 5 different groups: patients with cancer, diabetes, HIV+, with oral candidiasis and healthy subjects.ResultsProteolytic activity was identified in 46% of the strains from cancer patients, 54% from HIV+ patients, 60% from diabetics, 70% from oral candidiasis patients, and 42% from healthy subjects. Activity was higher in strains from immunocompromised and oral candidiasis patients than in healthy subjects. Differences were observed between the candidiasis-healthy, candidiasis-HIV+, and diabetic-healthy groups. No differences were observed between the oral candidiasis, diabetes and cancer patients, between the diabetes and HIV+ patients, or between the cancer patients, HIV+ patients and healthy subjects.ConclusionsThe present results suggest that although secreted aspartyl proteases are important in the pathogenesis of C. albicans, their activity depends on host conditions.     

Efecto inhibitorio in vitro de ajoeno sobre aislamientos de Candida recuperados de secreciones vaginales

AimsThe main purpose of this work was to evaluate the in vitro activity of ajoene of the Candida, obtained from vaginal discharges.MethodsFor this, 136 samples were analyzed. The yeasts were recovered and identified by conventional mycological methods. The susceptibility to ajoene (at 20, 15, 12.5, 10, 6.25 and 3.125 μg/ml) was performed according to the CLSI M27-A2 document with the EUCAST modifications. The ATCC reference strains 90028 (Candida albicans), 22019 (Candida parapsilosis), and 6258 (Candida krusei) were included in this study. The minimal inhibitory concentration (MIC) was considered as the minimal concentration of ajoena able to inhibit 80% of the fungal growth.ResultsFifty five yeasts were recovered, 36 (65.4%) of them were causing candidosis and 19 (34.5%) were colonizing. C. albicans was the most frequent (81.8%) of the six isolated species, prevailing on the patients with candidosis (54.5%). The non-albicans species were less frequently isolated (18.2%), and Candida glabrata was the prevailing agent (7.3%) followed by Candida tropicalis (3.6%), C. krusei, C. parapsilosis, Candida guilliermondii and Candida sp. (1.8% each of them). The susceptibility tests to ajoeno showed inhibition of fungal growth in 98.2% of the isolates, showing MIC values ?15 μg/ml, and in (one isolate of C. glabrata) (1.8%) this value was >20 μg/ml. The reference strains showed MIC values of 3.125 and 10 μg/ml.ConclusionsThe results here presented, obtained from a significant number of isolates, mainly C. albicans, demonstrate, once more, the potential of ajoeno as an antifungal agent.     

Cyclooxygenase inhibitors reduce biofilm formation and yeast-hypha conversion of fluconazole resistant Candida albicans

The incidence of fluconazole-resistant Candida albicans has been increasing worldwide. Both biofilm and fungal morphogenesis are main virulence factors of C. albicans cells. Extracellular fungal prostaglandins are synthesized during biofilm adhesion and development and through yeast-hypha conversion. Hence, we targeted prostaglandin synthesis with various cyclooxygenase (COX) inhibitors (aspirin, diclofenac, ketoprofen, tenoxicam, and ketorolac) and assessed their effect on fungal adhesion, biofilm formation, and yeast-hypha conversion in clinical isolates of Fluconazole resistant C. albicans. Significant reduction in fungal adhesion and detachment of mature biofilm was attained down to 1 mM concentrations of anti-inflammatory agents. Microscopical examination of fungal cells in the presence of the tested drugs showed significant reduction of germ tube formation. Therefore, COX inhibitors have a significant effect on reduction of Candida adhesion and biofilm development in correlation with fungal morphogenesis. Moreover, inhibition of C. albicans by COX inhibitors gave synergistic activity with fluconazole suggesting that combination therapeutic strategies may be fruitful for management of infection of Fluconazole resistant C. albicans.     

Enhancement of <Emphasis Type="Italic">Candida albicans</Emphasis> Virulence After Exposition to Cigarette Mainstream Smoke

The habit of cigarette smoking is associated with higher oral candidal carriage and possible predisposition to oral candidosis. The effects of exposure to smoke on the virulence properties of oral yeasts remain obscure. Hence, we showed in vitro the effect of cigarette smoke condensate (CSC) on ten clinical isolates of Candida albicans obtained from nonsmoking volunteers, as well the type-strain CBS562. CSC was generated by complete burn of five commercial cigarettes in an in-house smoking machine and used to prepare the culture broth in which the strains were grown. In 24-h intervals (T₂₄, T₄₈, and T₇₂), the cells were harvested, washed, subcultured, and the resultant growth were evaluated for possible variations for secreted aspartyl protease, phospholipase, chondroitinase, and hemolysins, adhesion to acrylic and cell surface hydrophobicity (CSH). The results indicated a temporal increase in the secretion rates of enzymes, particularly when yeast cells were exposed to CSC for 48–72 h (P < 0.05). Similarly, adhesion to acrylic and CSH increased with exposure period (P < 0.05). Based on foregoing, we concluded that CSC may promote significant enhance in the secretion of candidal histolytic enzymes and adherence to denture surfaces, thereby promoting oral yeast carriage and possible infection.     

Dispersion as an Important Step in the Candida albicans Biofilm Developmental Cycle

Biofilms are dynamic microbial communities in which transitions between planktonic and sessile modes of growth occur interchangeably in response to different environmental cues. In the last decade, early events associated with C. albicans biofilm formation have received considerable attention. However, very little is known about C. albicans biofilm dispersion or the mechanisms and signals that trigger it. This is important because it is precisely C. albicans cells dispersed from biofilms that are the main culprits associated with candidemia and establishment of disseminated invasive disease, two of the gravest forms of candidiasis. Using a simple flow biofilm model recently developed by our group, we have performed initial investigations into the phenomenon of C. albicans biofilm dispersion, as well as the phenotypic characteristics associated with dispersed cells. Our results indicate that C. albicans biofilm dispersion is dependent on growing conditions, including carbon source and pH of the media used for biofilm development. C. albicans dispersed cells are mostly in the yeast form and display distinct phenotypic properties compared to their planktonic counterparts, including enhanced adherence, filamentation, biofilm formation and, perhaps most importantly, increased pathogenicity in a murine model of hematogenously disseminated candidiasis, thus indicating that dispersed cells are armed with a complete arsenal of “virulence factors” important for seeding and establishing new foci of infection. In addition, utilizing genetically engineered strains of C. albicans (tetO-UME6 and tetO-PES1) we demonstrate that C. albicans biofilm dispersion can be regulated by manipulating levels of expression of these key genes, further supporting the evidence for a strong link between biofilms and morphogenetic conversions at different stages of the C. albicans biofilm developmental cycle. Overall, our results offer novel and important insight into the phenomenon of C. albicans biofilm dispersion, a key part of the biofilm developmental cycle, and provide the basis for its more detailed analysis.     

Evaluation of the antifungal photodynamic activity of Thymophylla pentachaeta extracts against Candida albicans and its virulence factors

BackgroundCandida albicans is one of the most common causative of opportunistic infections. Treatment of candidiasis is challenging considering the few antifungal drugs available and the increase in resistance. Antimicrobial photodynamic therapy (aPDT) is a recently developed therapeutic option that combines a non-toxic photosensitizer (PS) and light to kill the microbial pathogens. Targeting virulence, defined as the ability of a pathogen to cause overt disease, represents another attractive target for the development of novel antifungal agents. Thymophylla pentachaeta (DC.) Small var. belenidium (DC.) is an endemic plant from Argentina in which the presence of thiophenes, biologically active compounds whose antifungal activity is enhanced by irradiation with Ultraviolet A (UVA), have been already described.PurposeThe purpose of this study was to evaluate the photodynamic antifungal activity of hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and methanol (MeOH) extracts from T. pentachaeta var. belenidium and their inhibitory effects on C. albicans virulence factors as well as biofilm formation and eradication.Study Design/MethodsAntifungal photodynamic activity of Hex, DCM, EtOAc and MeOH extracts from different parts of the plant were assessed with the microbroth dilution, bioautography and the time-kill assays, under light and darkness conditions. The capacities of the most active extracts of inhibiting Candida virulence factors (adherence to epithelial cells, germ tube and pseudomycelium formation and hydrolytic enzyme secretion) were assessed. In addition, the activity against biofilm formation and eradication has been investigated by reaction with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) that quantifies living cells in these structures.ResultsHex and DCM extracts from T. pentachaeta roots exhibited high photodynamic antifungal activity against C. albicans [Minimal fungicide concentrations (MFCs)= 7.8 µg/ml] under UVA light irradiation. Chemical analysis of active extracts (Hex and DCM from roots) revealed the presence of photoactive thiophenes. Both extracts generate reactive oxygen species through type I and II mechanisms. These extracts, at sub-inhibitory concentrations, under light conditions decreased the adherence of C. albicans to Buccal Epithelial Cells (BEC), inhibited germ tube formation and reduced esterase production. Finally, they demonstrated activity against preformed biofilms submitted to irradiation (MFCs= 3.91 µg/ml and 15.63 µg/ml for Hex and DCM extracts, respectively).ConclusionTaking together, results demonstrated the strong photodynamic effects of T. pentachaeta root extracts under UVA irradiation, making them valuable alternatives to the already established antifungal drugs against C. albicans.