Phenazine Methosulphate Modulating the Expression of Genes Involved in Yeast to Hyphal Form Signal Transduction in Candida albicans

Candida albicans has ability to switch from yeast to hyphal form which is an important virulence factor. The objective of the research is to study the effect of Phenazine Methosulphate (PMS) on virulence factors and to study expression profile in yeast to hyphal form transition in C. albicans. Phenazine Methosulphate (PMS) acted as an inhibitor of yeast to hyphal form transition, adhesion and biofilm formation in C. albicans. RTPCR study demonstrated that PMS Modulate the expression of genes involved in Ras1-cAMP-Efg1 and Cek1-MAPK signal transduction pathways. Cell cycle of C. albicans was arrested at S phase on treatment of PMS. Hyphal suppressor genes like Tup1, Mig1 and Nrg1 were upregulated by PMS. Based on our data on expression of genes during yeast to hyphal form transition in presence and absence of PMS, we hypothesize that inhibition of hyphal formation may be due to the overexpression of negative regulators of hyphal growth. Targeting of hyphal specific genes involved in these pathways may be a promising strategy for anti-candida drug development.


Introduction
Yeast to Hyphal (Y-H) form morphogenesis is one of the major virulence factors in C. albicans and this may facilitate penetration of epithelial tissues and escape from host immune response [1] [2].Serum induced Ras mediated signaling pathways involve Ras1-cAMP-PKA and Cek1-MAPK pathway responsible for Y-H form transition [3].Y-H transition is negatively regulated by the hyphal suppressor genes, Nrg1, Mig1, Tup1 and Rfg1 [4].Upregulation of hyphal specific genes are reported during yeast to hyphal form transformation [5].Targeting of hyphal specific genes involved in these pathways may be a promising strategy for anti-Candida drug development [6].In this study the effect of Phenazine methosulphate (PMS) on the expression of genes involved in Yeast to hyphal transition is explored.
Phenazine methosulphate (PMS), an analog of 5-methylphenazine-1-carboxylic acid (5 MPCA) is reported to exhibit antifungal activity against hyphal growth and biofilm formation of C. albicans in Candida albicans [7] [8].PMS generate reactive oxygen species (ROS) which is responsible for killing or inhibition of growth in C. albicans [7].PMS and 5MPCA generally bind with cellular amines covalently and accumulate in the cell leads to killing of C. albicans [7] [9].
In this study we have identified the potential targets of PMS in C. albicans.
The repositioning of PMS as an antifungal agent could be a promising strategy for the treatment of fungal infections.

Medium and Culture Conditions
The standard strain of Candida albicans ATCC 90028 provided by Institute of Microbial Technology, Chandigarh, India.Yeast extract Peptone Dextrose (YPD) agar medium was used to maintain culture and the culture was stored at 4˚C.PMS and all the media components purchased from HiMedia, India.A single isolated colony of C. albicans from YPD agar plate was used to inoculate 50 ml YPD broth in a 250 ml conical flask.The flasks were incubated for 24 h in an orbital shaking incubator (100 rpm) at 30˚C [10].

Minimum Inhibitory Concentration
The effect of PMS on the growth of C. albicans in planktonic form was studied by using the standard broth microdilution methodology based on the Clinical Laboratory Standards Institute guidelines (Pfaller et al., 1994).Various concentrations of PMS ranging from 0.062 to 2 mg/ml were prepared in RPMI-1640 medium in 96 well plates (Costar, Corning Inc. USA).Cell suspension (1 × 10 3 cells ml −1 ) was added to 100 μl of RPMI-1640 medium in each well.The plates were incubated at 35˚C for 48 h and growth was analyzed by measuring absorbance at 620 nm using a microplate reader (Multiskan EX, Thermo Electron Corp. USA).The lowest concentration of PMS which caused a 50% reduction in the absorbance compared to the control was considered as the minimum inhibitory concentration (MIC) for the growth of C. albicans [10].

Minimum Fungicidal Concentration (MFC)
To determine the MFC, Candida cells from the wells containing MIC and above the MIC were used.Aliquots of 10 μl from these wells were spread on YPD agar.

Adhesion Assay
The effect of Capric and Caprylic acid on the adherence of C. albicans to a solid surface (i.e.polystyrene) was studied using a microplate-based assay.Different series of concentrations were prepared in Phosphate buffered saline (PBS) and of 50 μl of aliquots were added in to wells of microtiter plates.The final volume of the assay system in each well was kept at 100 μl.The plate is incubated at 37˚C for 90 min at 100 rpm in an orbital shaking incubator to allow adherence of cells.
The adherence of cells were observed in each well by relative metabolic activity (RMA) using the XTT-assay [10].

Biofilm Development Assay
Biofilm formation was done on tissue culture treated 96-well polystyrene plates.
100 μl of Cell suspensions of 1 × 10 7 cells ml −1 were added into each well and allowed to adhere on solid surface at 37˚C for 90 min at 100 rpm.After adhesion wells were washed with PBS two times.RPMI-1640 medium (200 μl) along with various concentrations of PMS were incubated for 48 h at 37˚C.Developed biofilms were observed under an inverted light microscope (Metzer, India).XTT metabolic assay was used Quantitation of Biofilm growth.Photographs were taken with a Labomed microphotography system (Labomed, India) at 200 X magnification [12].

Mature Biofilm Assay
To determine the activity of the PMS against mature C. albicans biofilms, 24 h old biofilm was used.Wells were washed with PBS to remove nonadhered planktonic cells.Various concentrations of PMS prepared in RPMI 1640 were added to wells.Plates were incubated for 48 h and then washed with PBS.Wells were observed for the presence or absence of biofilm using an inverted light microscope (Metzer, India).XTT metabolic assay was used to analyze the biofilm growth [12].

XTT Metabolic Assay
Biofilm growth was quantified using XTT metabolic assay.XTT solution was prepared by mixing 1 mg/ml of XTT salt in PBS and stored at 20˚C.Menadione (Sigma Aldrich, India) solution was prepared in acetone (4 μM) and added to the XTT salt solution.The wells containing biofilms were washed with PBS to remove non-adhered cells and incubated with 100 μl of XTT-menadione solution in dark, at 37˚C for 5 h.Color formation by the water soluble formazan product was measured at 450 nm using a microplate reader (Multiskan EX, Advances in Microbiology Thermo Electron Corp. USA) [12].

Yeast to Hyphal (Y-H) Form Morphogenesis Assay
Serum-induced Y-H transition in C. albicans was studied in a microplate-based assay.A series of concentrations of PMS was prepared in deionized distilled water with 20% serum. 1 × 10 6 cells ml −1 Cells were inoculated in test and control wells.The final volume of the assay was 200 μl.After incubation for 2 h at 37˚C in an orbital shaker at 200 rpm, formation of germ tubes were observed microscopically.Numbers of yeast cells and hyphae were counted by using an inverted microscope (Metzer, India).Photographs were taken with a Labomed microphotography system at 200 X magnification.Inhibition of 50% hyphae formation was compared with control and considered as MIC for morphogenesis.[10].

Scanning Electron Microscopy
Adhesion of Candida albicans cells on polystyrene discs (1 × 10 7 cells/ml) were carried out in 12-well plates at 37˚C at 50 rpm for 90 min.Samples were fixed in 2.5% of glutaraldehyde in 0.1 mol phosphate buffer (pH 7.2) for 24 h at 4˚C.
Samples were post fixed in 2% aqueous solution of osmium tetraoxide for 4 h, and then dehydrated in a series of graded alcohols.The samples were mounted over stubs and gold coating was performed using an automated gold coater.Images were obtained by JOEL 6360 (Japan) scanning electron microscope [12].

Hemolytic Activity
Hemolytic activity was determined by using human red blood cells [13].Suspension of RBC was diluted 1:10 in PBS.Aliquotes of 100 μL from this suspension was added in to 100 μL of a different concentration of test molecules in the same buffer in Eppendorff tubes.1% Triton X 100 was used for total hemolysis.After

Gene Expression Assay
The expression of hyphal genes during serum induced morphogenesis was measured by using Real Time PCR. C. albicans yeast phase cells (1 × 10 6 cells/ml) were inoculated and incubated for 90 min in 20% serum containing PMS (0.062 mg/ml).Total RNA was isolated by using RNeasy ® Mini Kit (QIAGEN, USA) and converted to cDNA by using SuperScript ® III for first strand synthesis (Invitrogen, Life technologies, USA).PCR reactions were conducted by using KAPA SYBR ® Fast qPCR Kit Master mix (2x) (BIOSYSTEMS, South Africa) in 96 well PCR plates with preliminary denaturation for 3 min at 95˚C.This was followed by 32 amplifications cycles of denaturation at 95˚C for 30 s, annealing at 60˚C for 20 s and primer extension at 72˚C for 30 s (CFX 96 Real time System, Bio-Rad, USA).Primers were purchased from Eurofins Genomics India Pvt. Ltd. (Table 1).Actin was used as an internal control and the transcript levels of selected genes were calculated using the formula 2 -ΔΔCT [15].

Statistical Analysis
Values mentioned are the mean with standard deviations, obtained from three different observations.Values in the control and treatment groups were compared using Student's t-test.A value of p < 0.05 was considered statistically significant [10].

The Anti-Candida Efficacy of Phenazine Methosulphate (PMS)
PMS inhibited planktonic growth of Candida albicans ATCC 90028 in a concentration dependent fashion (Figure 1).More than 90% (p < 0.05) of the growth was inhibited at 0.062 mg/ml.99% of planktonic growth was inhibited by PMS after 48 hrs of exposure at 0.25 mg/ml (Table 1).PMS caused significant inhibition of developing as well as mature biofilm formation (Figure 1, Figure 2).
MIC values for developing biofilm and mature biofilm were 0.25 mg/ml and 0.5 mg/ml respectively.>70% of 48 h old mature biofilm growth was eradicated at 0.5 mg/ml (Figure 1).MIC values were established for Adhesion for PMS (  1).MIC50 for inhibition of adhesion was 16 µg /ml (Table 2).After treatment of PMS (0.031 mg/ml) on C. albicans caused cell cycle arrest in S phase (Figure 3).
Table 1.Gene specific primers used in qRT-PCR.

PMS Inhibited Serum Induced Yeast to Hyphal Form Morphogenesis
Significant (p < 0.05) inhibition of serum induced yeast to hyphal form transition was observed in presence of PMS in a concentration dependent manner.
More than fifty percentage inhibition of germ tube induction was found at 0.062 mg/ml (Figure 4).

PMS Altered the Expression of Genes Involved in Yeast to Hyphal Form transition Pathways
Since PMS has shown inhibition of serum induced hyphal induction in C. albicans, we analyzed changes in expression levels of the genes involved in Ras1- Advances in Microbiology cAMP-Efg1, Cek1-MAPK pathways with quantitative real-time PCR.It was found that the expression of Ras1 which is a master regulator was downregulated up to 1.14 fold (p < 0.05) by treatment with PMS.Cdc35 and Pde2, the upstream components of cAMP dependent PKA pathway were upregulated by 1.09 and 1.46 fold respectively.Similarly expression of its downstream components Bcy1, Efg1 and Tec1 were upregulated by 2.75, 1.12, 1.65 fold.Ece1 and Hwp1 downstream of Efg1 were significantly downregulated by 1.56 and 11.16 fold respectively (Figure 5).Expression of Hst7, Cek1 and Cph1 involved in Cek1-MAPK pathways were upregulated by 1.44, 1.43 and 2.57 fold in presence of PMS respectively (Figure 5).Negative regulators of hyphal induction, Nrg1, Mig1 and Tup1 were upregulated by 1.53, 2.96 fold and 17.95 fold respectively (Figure 5).
Serum induced yeast to hyphal transition was also inhibited by PMS at 0.062 mg/ml (Figure 4).After 3 hr of PMS treatment, it was found that cell cycle of C. albicans was arrested at the S phase (Figure 3).Adherence of Candida cells to surface of polystyrene Microtitre plates was analyzed by microscopically and XTT assay.It was found that adhesion was encodes a glycosylphosphatidylinositol-modified cell wall protein that can serve as a target for mammalian transglutaminases to form covalent attachments between C. albicans and host epithelial cells [17].
To know the effect of PMS at the transcription levels during yeast to hypha switching we performed qPCR.Ras1 gene which is a master regulator of both the pathways [18] was downreguated up to 1.14 fold by treatment with PMS.Expression its downstream components like Efg1 [4] which is a strong inducer of Hyphal suppressor gene like Nrg1, Mig1 and Rfg1 suppresses hyphal induction [4] [23] [24] and Tup1 repressor which is constitutively expressed forms a complex with MIG1, RFG1 and NRG1 negative regulators and thereby inhibit the activity of hyphal specific gene [4].Tup1 encodes a transcriptional repressor that negatively controls filamentous growth in Candida albicans [4].Expression of Nrg1, a negative regulator of hyphal induction was upregulated by 1.53 fold by treatment of PMS.Similarly Mig1 and Tup1 over expressed by 2.96 fold and 17.95 fold (Figure 5).

Conclusion
Our in vitro study revealed that PMS inhibited major virulence factors in C. albicans like morphogenesis and adhesion.qPCR studies showed that PMS affects the signal transduction pathways in yeast to hyphal transition in C. albicans.Most of the genes involved in serum induced Ras1-cAMP-Efg1 and Cek1-MAPK pathway were affected differentially by the treatment with PMS.Upregulation of Hyphal suppressor gene like Tup1 may result in inhibition of yeast to hyphal induction.PMS arrested the cell cycle of C. albicans at S phase.PMS did not cause hemolysis of RBCs at the effective concentrations.PMS like molecules could be good molecules to treat candidiasis.In vivo studies need to be done to confirm the anti-Candida efficacy of PMS.

2 . 11 .
incubation of 1 h at 37˚C it was centrifuged for 10 min at 2000 rpm at 20˚C. 150 μL from supernatant was transferred to a flat-bottomed microtiter plate, and O.D. is taken at 450 nm.All the experiments were done in triplicates.The hemolysis percentage was calculated by following formula: % of hemolysis = [(A 450 of test compound treated Sample − A 450 of buffer treated sample)/(A 450 of 1% Triton X 100 treated sample − A 450 of buffer treated sample)] × 100 Cell Cycle Studies C. albicans yeast cells from the log phase were harvested, washed and starved for nutrients for 1 h.cells (10 3 cfu/ml) were inoculated to each flask containing 50 ml YPD broth and MIC of PMS.Flask without treatment of PMS was considered as Control.All the flasks were incubated at 30˚C for 3 h.Cells were centrifuged at 1000 rpm for 5 min.washed and fixed in 1 ml of 70% ethanol by incubating at room temperature on a rotary shaker (REMI, India) for 30 min.Cells were har-vested by centrifugation (Genei, Bangalore, India) at 1000 rpm for 5 min, washed twice with 1 ml of 50 mM Tris (pH 7.8).Washed cells were resuspended in 500 μl of 50 mM Tris containing 10 μg RNase A and incubated for 2 h at 37˚C.After incubation samples were subjected to protease treatment (5 mg/ml pepsin in 0.05 M HCl) for 1 h at 25˚C to reduce cellular clumps.Cells were harvested by centrifugation and resuspended in 500 μl FACS-buffer (200 mM Tris/HCl pH 7.5; 200 mM NaCl; 78 mM MgCl 2 ).15 μl of propidium iodide (1 mg/ml) was added to each tube and incubated for 30 min and stored at 4˚C.Before analysis, cells were sonicated for 10 s and analyzed using a FACS Calibur cytometer (Becton-Dickinson, San Jose, Calif.) and analyzed with Cell Quest 3.0 software[14].

Figure 3 .Figure 4 .
Figure 3.Effect of PMS on Cell cycle of C. albicans.

Figure 5 .
Figure 5. Gene expression profile of Candida albicans during serum induced yeast to hyphal form transition in presence of PMS.
hyphal development was unaffected by the treatment of PMS.The expression of Tec1 was upregulated by 1.65 fold.Ece1 and Hwp1 are downstream of Efg1 were significantly downregulated by 1.56 and 11.16 fold respectively (Figure 5).Ece1 is required for cell elongation in C. albicans and Hwp1 is associated with adhesion and biofilm formation [17] [19].In addition Cek1-MAPK pathway was affected by change in expression of Hst7 followed by Cek1 and Cph1.Their expression was slightly upregulated by 1.44, 1.43 and 2.57 fold respectively.Cph1 in C. albicans is regulated by a mitogen-activated protein MAP kinase cascade that includes Cst20, Hst7, and Cek1 [20] [21] [22].

Table 2 .
The efficacies of PMS against C. albicans ATCC 90,028, showing the MICs for growth, adhesion, morphogenesis, and biofilm formation and MFCs for planktonic growth.