Inhibition of Candida species via Proteosome Inhibitor MG-262 (ZL3B)

Backround: Candida species take the fourth place among the microorganisms which cause hospital infections. New therapeutic agents are needed because of rapidly increasing resistance rates. Boric acid preparations are preferred in local treatment especially in resistant cases while azole formulations are the first choice in oral treatment. The only boric acid analog that was approved by FDA (Food and Drug Administration) for systemic use is Bortezomib (Velcade). Bortezomib is a proteasome inhibitor and proteasome inhibitors cause apoptosis of eukaryotic cells. Although the yeast also has a well preserved 20S proteasome region, the studies on the effects of proteasome inhibitors on the yeast are limited. In this study, the effects of the one of the most potent proteasome inhibitor, MG-262, on Candida spp. were investigated. Methods: C. albicans ATCC 90028, C. krusei ATCC 6258 and C. glabrata (clinical strain) strains were used in our study. Antifungal susceptibilities of the strains against MG-262 and effects of MG-262 on oxidative metabolism, proteasome activity and apoptosis as well as virulence factors such as hyphal transformation, pseudohypha, germ tube and biofilm formation were investigated. CLSI (Clinical and Laboratory Standards Institute) guidelines were used for antifungal susceptibility tests. The effect on hypha and pseudohypha formation and germ tube were determined with conventional tests. Biofilm formation was detected according to the microplate method. Oxidative metabolism experiments, proteosome activity and apoptosis tests were performed with Alamarblue dye, fluorogenic peptides and DNA ladder assays, respectively. Findings: MG-262 exhibited minimum inhibitory activity at 25 μg/ml concentration for C. albicans ATCC 90028 and at 50 μg/ml concentration for C. krusei ATCC 6258 and C. glabrata. Minimum fungicidal concentrations for all species were 50 μg/ml. Hypha, pseudohypha, germ tube and biofilm formation, proteosome activity and oxidative metabolism were all inhibited at 50 μg/ml and higher concentrations. The apoptosis was detected at 200 μg/ml. Conclusion: MG-262 caused apoptosis by inhibiting oxidative metabolism and showed fungicidal activity. It also inhibited the virulence factors such as hypha, pseudohypha germ tube and biofilm formation. Inhibition of proteasome is promising in terms of discovery of a new generation antifungal agent.


Introduction
Candida species are a leading causes of the hospital related bloodstream infections with high morbidity and mortality. They cause serious systemic diseases especially when the normal bacterial flora is supressed by widespread use of broad spectrum antimicrobials. Increasing number of solid organ and bone marrow transplantations, invasive procedures and implantable biomaterials are also other predisposing factors [1][2][3].
Oral azole drugs are the first choice in the treatment of This article is available from: www.acmicrob.com

ARCHIVES OF CLINICAL MICROBIOLOGY ISSN 1989-8436
candidiasis [4]. Boric acid is one of the local treatment alternatives in resistant cases [5]. Fungicidal effect of boric acid is attributed to the blockage in the oxidative metabolism according to some authors [6]. However, there also are some boric acid analogs which effect as proteasome inhibitor and they can be local treatment alternatives. Nevertheless, the data about the use of proteasome inhibitors for therapeutic purposes on Candida species is limited. Bortezomib (Velcade), originally codenamed PS-341 (pyrazy carbonyl-Phe-Leu-boronate), is the only boric acid analogue proteasome inhibitor, which has received Food and Drug Administration (FDA) approval and is currently used parenterally [7].
Proteasomes are the major non-lysosomal protease structures found in eukaryotic cells. It provides degradation of the misconfigured and ubiquitin labelled proteins. Ubiquitinproteasome enzymatic pathway is located in the cytoplasm and is responsible for the degradation of the abnormal proteins [8].
Ubiquitin mediated degradation of regulatory proteins plays an important role in the control of metabolic processes such as cell cycle progression, signal transmission, transcriptional regulation and apoptosis. Studies on ubiquitin-proteasome system of mammalian cells and yeast species are currently ongoing to solve the complex relationship of this system with the apoptosis pathway [9].
MG-262, originally codenamed (ZL 3 B) Z-Leu-Leu-Leu-B(OH) 2 , is one of the most potent compound among boric acid analog proteasome inhibitors. It selectively and reversibly inhibits the chymotryptic activity of the proteasome which has a critical role in cell viability and proliferation [10].
In this study, the susceptibility of the active ingredient MG-262 on Candida species was detected and then possible antifungal mechanisms (oxidative metabolism inhibition, apoptosis) and the effects on some virulence factors (hyphal growth, inhibition of cell viability in biofilm condition) were analysed in vitro.

Candida strains
Standard strains; C. albicans ATCC 90028, C. krusei ATCC 6258 and a clinical isolate; C. glabrata strain were included. C. glabrata was identified via the standard methods, such as chlamydospore production, germ tube assays, micro-morphology studies in cornmeal-Tween 80 agar and biochemical tests using the commercial system ID32C (bioMérieux Marcy l'Etoile, France). Antifungal susceptibility tests were performed according to the Clinical and Laboratory Standards Institute (CLSI) M27-A3 document and interpreted in accordance with the CLSI M27-S4 suplement [11,12]

Germ tube test
Germ tube formation was monitored microscopically according to the conventional germ tube test protocol [14]. C. albicans ATCC 90028 strain was placed in human serum-containing media (10% v/v) with or without chemical compounds (25 μg/ml, 50 μg/ ml, 100 μg/ml and 200 μg/ml), and incubated at 37°C for 3 hours. The samples were visuliazed with 400X magnification via an optical microscope (Nikon Eclipse Ci, Japan). MG-262 untreated cells were used as control.

Corn meal agar tests
Hyphal transformation and pseudohypha production of C. albicans ATCC 90028 strain and pseudohypha production of C. krusei ATCC 6258 strain were determined via Cornmeal Agar (Oxoid, UK) with 10% Tween 80 (Oxoid, UK). Before and after incubation with MG-262 for 3 hours, Corn Meal Agar was cut deeply in the form of horizontal furrow with a loopful of suspension colonies and a flamed sterile coverslip was placed over the line of inoculum [14].
After the incubation at 22°C the results were observed both at 24 and 48 hours and documented with 400X magnification under an optical microscope (Nikon Eclipse Ci, Japan). MG-262 untreated cells were used as control.

Oxidative metabolism experiments
In our study, Candida strains were exposed to Alamar Blue stain in order to determine the effect of MG-262 on the oxidative metabolism with the method described by Seta et al. with slight modifications [6]. The Alamar Blue system is composed of an oxidation-reduction indicator, which fluoresces and turns from blue to pink secondary to oxidative metabolism and cell growth. The active ingredient of the dye is resazurin which is cell permeable, nonfluorescent and blue in color. Upon entering cells, resazurin is reduced to resorufin, which produces very bright pink fluorescence viable cells continuously convert resazurin to resorufin [15]. In order to determine the effect of chemical compound on oxidative metabolism, 45 µl samples of each strain (1 × 10 3 CFU/ ml) were exposed to 45 µl of serial dilutions (25 µg/ ml, 50 µg/ml, 100 µg/ml and 200 µg/ml) of MG-262 and stained with 10 µl AlamarBlue (Invitrogen, USA) dye. MG-262 untreated cells with alamarblue were used as positive control. The cells were incubated with MG-62 for 1 hour and washed gently. They were also observed for 24 hour in order to detect the colour change.
After 24 hours the plate was read at 570 nm using a microtiter plate reader (Multiskan Ascent, Labsystems, Helsinki, Finland).

Biofilm assays
The effect of MG-262 on the cell viability in a biofilm condition were evaluated according to the microplate method described by Evensen and Braun with some modifications [16].

Apoptosis (DNA ladder assay)
In order to detect the apoptosis effect of MG-262 on test strains, different concentrations of the compound (25 µg/ml, 50 µg/ ml, 100 µg/ml and 200 µg/ml) were tested on 0.5 × 10 3 -2.5 × 10 3 CFU/ml of the yeast cells. After 3 hours of incubation with MG-262, DNA was extracted according to the protocol described by Suman et al. [17,18]. DNA fragmentation was visualized using a 50-10000 bp (base pair) Hi-Lo DNA marker/mass ladder (Bionexus, ABD) in the 1% agaroz gel via the gel electrophoresis method at 25 V for 10 min and at 50 V for 30 min. Untreated cells were used as positive control. The results were documented via Gel Logic 2000 gel magnification system (Kodak, USA).

Statistical analysis
All experiments were performed three times in triplicate. Chisquare test was used to compare filamentous growth, germ tube and DNA ladder assays. Arithmetic mean of absorbance values of oxidative metabolism, biofilm and proteasome activity experiments were calculated. The absorbance values of the negative control wells (containing no cells) were subtracted from the values of the test wells in order to eliminate any background absorbance and compared with the absorbance of the MG-262 untreated cell absorbance results. In the comparison of groups that provide the normal distribution independent samples T test was used while Mann-Whitney U test was used to compare the groups in which normality assumption was not provided. Analyses were performed, using the SPSS for Windows (Version 11.3.8.0) pocket program. Descriptive statistics were shown as mean ± standart deviation. A probability value of p less than 0.05 was considered statistically significant.

Corn meal agar and germ tube tests
The serial dilutions of MG-262 at 25 µg/ml concentration, virulence factors; such as filamentous growth and germ tube formation were still continuing. However at 50 µg/ml, 100 µg/ml and 200 µg/ml concentrations, they were all inhibited. Figure 1 shows the results of germination and germ tube formation of C. albicans at 25 µg/ml and 100 µg/ml concentrations of MG-262.

Oxidative metabolism inhibition
The control group and 25 µg/ml of MG-262 treated cells developed a pink colour, which is a visual evidence of reduction of AlamarBlue. However, 50 µg/ml, 100 µg/ml and 200 µg/

ARCHIVES OF CLINICAL MICROBIOLOGY ISSN 1989-8436
ml treated cells showed no colour change during 24 hour after the addition of the dye. The statistical analysis results of the absorbance values are given both as table (Table 1) and as figure (Figure 2) in order to be more visually descriptive. When values were analyzed statistically, it was observed that MG-262 has a significant effect on the oxidative metabolism of Candida isolates at 50 µg/ml and higher concentrations. At 50 µg/ml concentrations, p values for C. albicans, C. krusei ve C. glabrata isolates were 0.000, 0.046 and 0.000, respectively. At 100 µg/ml concentration, p values for C. albicans, C. krusei ve C. glabrata isolates were 0.000, 0.043 and 0.000, respectively. Finally at 200 µg/ml concentrations, p values for C. albicans, C. krusei ve C. glabrata were found to be 0.003, 0.046 and 0.001 respectively.

Biofilm assays
The inhibitory effect of MG-262 on cell viability of Candida strains in biofilm condition was observed to be enhanced with increasing concentrations (Figure 3). The ability of MG-262 to inhibit the cells in biofilm was found to be significantly increased at 50 µg/ml and higher concentrations.

Proteasome activity
We observed a prominent decrease in the proteasome activity with increasing concentrations of MG-262. The statisticall analysis of absorbance results are given both in (Table 1 and Figure 4).  When analysed statistically, this effect was significant at 50 µg/ ml and higher concentrations. At 50 µg/ml concentrations, p values for C. albicans, C. krusei ve C. glabrata were 0.000, 0.000 and 0.046 respectively. At 100 µg/ml concentrations, p values for C. albicans, C. krusei ve C. glabrata were 0.000, 0.00 and 0.046 respectively. At 200 µg/ml concentrations, p values for C. albicans, C. krusei ve C. glabrata were 0.000, 0.000 and 0.046 respectively.

Discussion
The aim of our study was to investigate the antifungal activity of MG-262 in for Candida infections. We determined that one of the most potent proteasome inhibitors, MG-262 inhibits virulence factors, and also cause apoptosis by proteasome inhibition and blockage of oxidative metabolism.
The researches on the apoptosis of the yeast give opinion about the apoptotic process of the eukaryotic cells and are promising in competing against unicellular eukaryotic pathogens. New drug molecules are required for life threatening resistant C. albicans and non-albicans Candida isolates. Agents performing fungal-programmed cell death can be a model for future novel antifungals [18].
Chymotrypsin-like region of the 20S proteasome is a well conserved region both in human and fungus cells. MG-262 is a peptide boronic acid. It inhibits proteasome by binding to chymotrypsin-like region of the 20S proteasome in human cells.
The properties such as being a more potent agent than other proteasome inhibitors and being resistant to be easily inactivated by oxidation may make MG-262 a good candidate for a novel antifungal drug [10]. The previous in vitro sensitivity analysis have demonstrated that C. krusei and C. glabrata are more resistant than other Candida strains and MIC values of these two subspecies have shown to be higher than other species [19]. In our study, these two strains which are believed to have a Multi Drug Resistance (MDR) potential were also 2-fold more resistant to MG-262.
Unlike the fungistatic effects of the clinically used local boric acid preparation, MG-262 showed fungicidal activity [6]. The concentration for lethal effect of MG-262 was 2 times higher than for inhibitory effect only for C. albicans, for the 2 other strains it was the same concentration. Previous studies have revealed that proteosome inhibitor Bortezomib shows inhibitory effect on both P. falciparum and human cells in nM concentrations [7,20]. In our study MIC and MFC of the MG-262 were in µM concentration

ARCHIVES OF CLINICAL MICROBIOLOGY ISSN 1989-8436
range and far above nM levels. We suggest that this can be due to the mechanical strength of the cell wall which consists of β1,3glucan and chitin [21]. On the other hand, when compared with the other proteasome inhibitors, it showed efficacy at much lower concentration on the yeast probably secondary to being a more potent agent [22].
In addition to the effective concentration, the effects on virulence factors are also of great importance when an agent is being investigated as a potential novel antimicrobial. In our study, the effects of MG-262 on germ tube, hyphe, pseudohypha and cell viability in biofilm condition were examined. Candida infections generally originate from implants such as prosthetic valves, endotracheal tubes and joint prostheses. The major virulence factors are biofilm formation and adhesion to the biomaterial surface [23]. The hyphal growth and germ tube development capabilities of the fungus cells are also important in the formation of cell damage and deeply located infections [24]. MG-262 inhibited hyphal growth, germ tube and pseudohypha formation at 50 µg/ml and higher concentrations. The effects on cell viability in biofilm were more prominent with increasing MG-262 concentrations (Figure 3). The antimicrobial resistance of the biofilm-producing microorganisms can be up to 1000-fold greater than planctonic cells [25]. Therefore, the inhibition at higher concentration and the most efficacy at 200 µg/ml concentration can not to be regarded as a surprising finding.
Proteaosome inhibitors cause apoptosis in almost all cells at high concentrations [10]. Our study was in line with this literature, indicating that MG-262 provided a significant proteasome inhibition only at 50 µg/ml and higher concentration.
The breakdown of the oxidative metabolism in cells and associated oxidative stress are the most substantial signals portending the death due to apoptosis [26]. In the oxidationreduction experiments of our study, the color had changed to pink both in control strains and 25 µg/ml MG-262 treated strains in about 1 hour whereas the blue color had not changed in the following 24 hours beginning from 50 µg/ml concentration. These results showed that oxidative metabolism was blocked in increasing concentrations from 50 μg/ml. It has been reported that the effect of some protease inhibitors is reversible [26].
However, in our study, the effect of MG-262 lasts for 24 hours, suggesting that the effect of MG-262 on tested Candida species is not reversible.
The specific finding of apoptosis is fragmentation of the cellular DNA from the regions between nucleosomes leading to formation of about 180 to 200 bp or multiples sized DNA pieces [27]. DNA ladder appearance is the final stage and precise finding of apoptosis. However DNA fragments in yeast are composed of much larger base pairs. DNA fragments as large as 800 bp were demonstrated in Saccharomyces pompe in the previous studies [28]. In our study, the DNA fragments were approximately 750 bp and 1450-1500 bp in size when compared to DNA HiLo marker.
Although statistically significant proteasome inhibition was detected at 50, 100 and 200 µg/ml concentrations, DNA ladder image could only be detected at 200 µg/ml concentration. This might be because of the limitation of the DNA ladder assay since it may causes loss of DNA fragments during sample processing [17,27].
The current study demonstrates that MG-262 caused antifungal activity on Candida species via apoptosis. The possible mechanism of action was inhibition of chymotrypsin-like activity. Although the data about the dose related toxic effects and antimicrobial resistance is currently missing, this novel therapeutic approach deserves further researches since it has the potential to remediate Candida infections resistant to existing treatment modalities. We conclude that this boric acid anolog not only inhibits virulence factors, but also cause apoptosis by proteasome inhibition and blockage of oxidative metabolism on several Candida spp. However our work was a pre-study. Many different types of new proteasome inhibitors on human cells have been described, including grape, soy polyphenols and Bortezomib, a drug approved by FDA and these molecules are still untouched for yeast species. Determination of mechanisms of actions of different proteasome inhibitors and boric acid anologs may open the way to drug discovery for Candida infections.