Preparation and Purification of ß-1,3-glucan-Linked Candida glabrata Cell Wall Proteases by Ion-Exchange Chromatography, Gel Filtration, and MDPF-Gelatin-Zymography Assay.

Candida glabrata is an opportunistic pathogen that may cause serious infections in an immunocompromised host. C. glabrata cell wall proteases directly interact with host cells and affect yeast virulence and host immune responses. This protocol describes methods to purify ß-1,3-glucan-bonded cell wall proteases from C. glabrata. These cell wall proteases are detached from the cell wall glucan network by lyticase treatment, which hydrolyzes ß-1,3-glucan bonds specifically without rupturing cells. The cell wall supernatant is further fractioned by centrifugal devices with cut-offs of 10 and 50 kDa, ion-exchange filtration (charge), and gel filtration (size exclusion). The enzymatic activity of C. glabrata proteases is verified with MDPF-gelatin zymography and the degradation of gelatin is visualized by loss of gelatin fluorescence. With this procedure, the enzymatic activities of the fractions are kept intact, differing from methods used in previous studies with trypsin digestion of the yeast cell wall. The protein bands may be eventually located from a parallel silver-stained gel and identified with LC-MS/MS spectrometry. The advantage of this methodology is that it allows further host protein degradation assays; the protocol is also suitable for studying other Candida yeast species. Key features • Uses basic materials and laboratory equipment, enabling low-cost studies. • Facilitates the selection and identification of proteases with certain molecular weights. • Enables further functional studies with host proteins, such as structural or immune response-related, or enzymes and candidate protease inhibitors (e.g., from natural substances). • This protocol has been optimized for C. glabrata but may be applied with modifications to other Candida species.

Isolation, characterization and regulation of moonlighting proteases from Candida glabrata cell wall.

Candida glabrata (C. glabrata) cell wall proteins play a role in virulence and in initial host immune recognition and responses. We isolated and characterized C. glabrata cell wall proteases from a clinical hospital C. glabrata T-1638 blood isolate and estimated the enzymatic activities and their ability to degrade gelatin and processing proMMP-8 and assess the regulation of these proteases with salt treatment, mercaptoethanol and fermented lingonberry juice from Vaccinium vitis idaea L. The cell wall proteases were enzymatically released from the cell wall and beta- 1,3- bonded proteases were fractioned into 10-50 kDa and >50 kDa fractions with anionic DEAE-sepharose ion-exchange chromatography and gel filtration. Proteins were monitored and analyzed with MDPF- zymography, and five gelatinolytic bands were cut out from a parallel silver-stained gel for the LC- MS/MS analysis. The proteases lacked a signal sequence, indicating that they are moonlighting proteases. Human proMMP-8 activation assays were performed with both fractions and verified by western-immunoblot using aMMP-8 specific antibody. Inhibition of proMMP-8 conversion to the lower molecular active enzyme species were demonstrated with fermented lingonberry juice. The results indicate that moonlighting proteases may play a role in the virulence of C. glabrata.

Antimicrobial and Anti-inflammatory Lingonberry Mouthwash - A Clinical Pilot Study in the Oral Cavity.

Fermented lingonberry juice was designed to be used as a mouthwash. Our aim was to study the antimicrobial and anti-inflammatory effects of the mouthwash in the oral cavity. A clinical study of 30 adult participants was performed. A total of 20 participants used 10 mL of the mouthwash twice daily for two weeks and 10 participants used 20 mL twice daily for one week. Streptococcus mutans, Candida and Lactobacilli were cultivated at the beginning, after the mouthwash period and after a washout period. At the same timepoints an additional oral mouthrinse was collected for chair-side/point-of-care (POC)-PerioSafe®/OraLyzer® aMMP-8 quantitative on-line evaluation, and an oral clinical investigation was performed. Mean Streptococcus mutans and Candida counts, visible plaque index (VPI) and bleeding on probing (BOP) were reduced, and Lactobacilli counts increased during the lingonberry mouthwash period. The aMMP-8 mouthrinses showed reduced values in both test groups when compared to the startpoint. The mouthrinse aMMP-8 reduction correlated with the reductions in microbial counts, VPI and BOP. Based on the results, fermented lingonberry juice seems a promising aid in oral homecare, diminishing the microbial and related proinflammatory burden by balancing the oral microbial flora and gradually lowering the inflammatory load in the oral cavity.

The Effect of Fermented Lingonberry Juice on Candida glabrata Intracellular Protein Expression.

Lingonberries have a long traditional use in treating fungal infections on mucosal membranes, but very little is known about the exact antifungal mechanisms. We tested the effects of fermented lingonberry juice on Candida glabrata intracellular protein expression. A Candida glabrata clinical strain was grown in the presence of fermented lingonberry juice (FLJ). Also the effect of lowered pH was tested. Intracellular protein expression levels were analyzed by the 2D-DIGE method. Six proteins detected with ≥1.5-fold lowered expression levels from FLJ treated cells were further characterized with LC-MS/MS. Heat shock protein 9/12 and redoxin were identified with peptide coverage/scores of 68/129 and 21/26, respectively. Heat shock protein 9/12 had an oxidized methionine at position 56. We found no differences in protein expression levels at pH 3.5 compared to pH 7.6. These results demonstrate that FLJ exerts an intracellular stress response in Candida glabrata, plausibly impairing its ability to express proteins related to oxidative stress or maintaining cell wall integrity.

A novel Candida glabrata cell wall associated serine protease.

We set out to identify the Candida glabrata cell wall attached proteases which may play a role as virulence factors in candidosis, particularly in the immunocompromized host. We studied a clinical C. glabrata strain T-1639, which was isolated from a patient from the Helsinki University Central Hospital. With non-reducing 2-D electrophoresis using parallel fluorogenic gels and mass spectrometry we identified a novel appr. 25 kDa (192 aa in length) cell wall located protease with an estimated pI of 7.6. The LC-MS/MS peptides matched with the ORF of predicted C. glabrata CBS138 cell wall protein Cwp1.2p/pI 7.7/212 aa (http://cbi.labri.fr/Génolevures/[NCBI access 49525604, UniProt access Q6FTZ7]), which is an ortholog to Saccharomyces cerevisiae cell wall protein Cwp1p (UniProt access P28319). The novel serine protease was released by ß-1,3-glucanase treatment from the cell wall. In contrast to previous predictions this protease has an enzymatic function instead of being merely a structural cell wall protein. The protease showed gelatinolytic activity and was inhibited by PMSF, a known serine protease inhibitor. Further characterization of the protease may give insight to its role in infections caused by C. glabrata and possibly aid in the development of new kinds of antifungal drugs.