|Year : 2015 | Volume
| Issue : 1 | Page : 110-116
Novel synthetic anti-fungal tripeptide effective against Candida krusei
K Gill1, S Kumar1, I Xess2, S Dey1
1 Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India
2 Department of Microbiology, All India Institute of Medical Sciences, Ansari Nagar, New Delhi, India
|Date of Submission||04-Nov-2013|
|Date of Acceptance||31-Jan-2014|
|Date of Web Publication||5-Jan-2015|
Department of Biophysics, All India Institute of Medical Sciences, Ansari Nagar, New Delhi
Source of Support: None, Conflict of Interest: None
Introduction: Candida species are the major fungal pathogens of humans. Among them, Candida krusei have emerged as a notable pathogen with a spectrum of clinical manifestations and is known to develop resistance against azoles mainly fluconazole. Anti-microbial peptides play important roles in the early mucosal defence against infection and are potent anti-fungal agents since they fight against fungal infection as well as have ability to regulate host immune defence system. The aim of the study was to synthesize a small anti fungal peptide. Materials and Methods: The series of tripeptides were synthesized and screened for antifungal activity against Candida strains according to CLSI guidelines. Toxicity effect of peptide was tested with human erythrocytes. The mode of action of peptide on fungus was resolved by scanning electron microscopy (SEM) studies Results: The tripeptide FAR showed a prominent anti fungal activity among the series. The minimum inhibitory concentration and minimum fungicidal concentration of tripeptide FAR was found to be 171.25 μg/ml and 685 μg/ml, respectively against Candida krusei . The therapeutic index was 2.9. The haemolytic experiment revealed that this peptide is non - toxic to human cells. The SEM studies showed disruption of cell wall and bleb-like surface changes and irregular cell surface. Conclusion: The peptide showed a significant antifungal activity against C. krusei. Thus, it can set a platform for the design of new effective therapeutic agents against C. krusei.
Keywords: Anti-fungal, Candida krusei, tripeptide electron microscopy
|How to cite this article:|
Gill K, Kumar S, Xess I, Dey S. Novel synthetic anti-fungal tripeptide effective against Candida krusei. Indian J Med Microbiol 2015;33:110-6
|How to cite this URL:|
Gill K, Kumar S, Xess I, Dey S. Novel synthetic anti-fungal tripeptide effective against Candida krusei. Indian J Med Microbiol [serial online] 2015 [cited 2019 Nov 13];33:110-6. Available from: http://www.ijmm.org/text.asp?2015/33/1/110/148404
| ~ Introduction|| |
The emergence of fungal pathogens resistant to current therapy is a serious concern among the scientists. The immunocompromised patients are always at risk for invasive fungal infections and demands for the effective anti-fungal agents. The fungi mainly belonging to Candida genus are progressively more important nosocomial pathogens. Of the various invasive candidiasis cases in the intensive care units, 60% are candidemia.  In case of candidemia, Candida albicans acted as a major pathogen but recently C. krusei has emerged as one of the most frequent pathogen causing candidemia, especially among patients having acute leukaemia. C. krusei has found to be innately resistant to fluconazole, but many infections caused by it have been associated with the therapeutic use of this anti-fungal agent. It is urgently required to develop novel anti-fungal agents which are not toxic to mammalian cells.
Antimicrobial peptides (AMPs) are one of the most important elements of the innate immune system, they are ideal for the fast and efficient defene against the microbes. In past decades, many natural and synthetic anti-fungal peptides have been found, only few of them have entered into clinical trials. Among them, most of the peptides also showed anti-bacterial activity. The AMPs make use of the fundamental differences between microbial and mammalian cells and includes membrane composition, architecture, transmembrane potential, polariation and structural features. The presence of ergosterol in the fungal cell membrane differentiates them from the mammalian cells and implicating it as a potential target for AMPs.  It was reported that Candidal surface contains specific proteins important for the binding of AMP. 
Anti-fungal peptides are classified by their mode of action and depend on a number of parameters like amino acid sequence and their charecteristic features. One group of peptide acts by disrupting the membrane structure by binding to the membrane surface without traversing the membrane and those peptides are positively charged and hydrophobic in nature. Another class of peptides aggregate in a selective manner forming aqueous pores to allow the passage of ions and other solutes.
Every year, emergence of new fungal pathogens causes morbidity and life-threatening infections in the immune-compromised host.  Some of the available anti-fungal agents have many side effects as they are ineffective against the new emergent fungi or develop resistance mainly due to their broad usage. Approximately 17% of Candida isolates exhibit resistance for azoles and the most probable reason is the wide use of fluconazole. C. krusei is one of the species exhibiting intrinsic resistance to fluconazole (inhibitor of ergosterol biosynthesis in yeast) and lower susceptibility for amphotericin B.  The mechanism of fluconazole resistance in C. krusei is due to reduction in inhibition of 14α-demethylase enzyme in the organism.  Due to relative resistance to many anti-fungal agents and capability of nosocomial transmission makes C. krusei a major nosocomial pathogen in hospitals. Therefore, the search for new and more effective anti-fungal agents is vital. AMPs are evolutionary ancient weapons of animals and plants. A diverse variety of AMPs are known till date. They are mostly derived from large precursors like signal sequences or proteolysis from larger proteins. 
Because the clinical use of AMPs is hindered by cost issues, short AMPs with high cell selectivity have been recently preferred  This work presents the development of a novel anti-fungal peptide. Here, we designed and synthesized several peptides and all are characterised for anti-fungal activity against C. krusei. This anti-fungal peptide FAR was non-toxic to human red blood cells (RBCs).
| ~ Materials and Methods|| |
Strain used and storage
The yeast strain of C krusei (ATCC 6258) and C. parapsilosis (ATCC 22019) were used in this study as a quality control as per Clinical and Laboratory Standard Institutes (CLSI) guidelines and C krusei (ATCC 6258) was used for the experiment obtained from the Mycology laboratory of All India Institute of Medical Sciences, New Delhi, India. The strains were maintained at 4°C. For the performance of the experiment the stored strain was streaked for single colonies on MuellerHinton agar (MHA) (BD Becton, Dickinson and Company, India) plates and were incubated at 37°C.
Synthesis of peptide
Five tripeptide were synthesized by introducing cationic and hydrophobic residues like Arg and Trp which facilitate to bind fungal anionic components of the cell wall. The tripeptide with amino acid sequences FWY (Phe-Trp-Tyr), FAY (Phe-Ala-Tyr), FAR (Phe-Ala-Arg), FWC (Phe-Trp-Cys) and FVY (Phe-Val-Tyr) were synthesied by solid phase peptide synthesis using Fmoc and Wang resin chemistry  in PS3 peptide synthesier (Protein Technologies, Rainin Instruments Inc. Co.). Briefly, for the synthesis of the peptide FWY the starting amino acid Fmoc-Tyr (tBu)-Wang resin was used and the active incoming acetic ester form of Fmoc-Trp-OH, formed by interaction with uronium salt 2-(1H-benzotriazole-1-yl)-1, 1, 3, 3-tetramethyluroniumhexafluorophosphates (HBTU) in the presence of base 0.4 M N-methyl morpholine (NMM) in dimethylformamide (DMF) was then, coupled to the Wang resin. Similarly, active Fmoc-Phe-OH was coupled in the same way and finally Fmoc was deprotected from the synthesied peptide using 20% piperidine in DMF. The crude peptide was cleaved from the resin using trifluoro acetic acid (TFA) and repeatedly washed with diethyl ether. Similarly, all other mentioned tripeptide were synthesied and all were lyophilized by freeze dryer.
RP-HPLC analysis of peptides
The purity of synthesied peptides was verified by analytical reversed phase-high performance liquid (RP-HPLC) using C18 reversed phase column (1.6 × 10 cm; Shimadzu). A total of 1 mg/ml of peptide was loaded onto the reversed phase chromatography (RPC) column. The linear gradients were formed by passing two different solvents, where solvent A was 0.05% aqueous TFA (pH 2) and solvent B was 0.05% TFA in acetonitrile. The flow rate was 0.5 ml/min at room temperature. The synthesied peptides purified using C-18 reverse phase column HPLC confirmed their purity by a single peak.
Antifungal activity screening
The anti-fungal activity of the synthesized tripeptide FWY, FAY, FAR, FWC and FVY were assessed against C. krusei using agar diffusion method.  Briefly, the Candida species were grown for overnight in MullerHilton Broth (MHB) (BD Becton, Dickinson and Company, USA) at 37°C and 0.2 ml of 24 h old culture in nutrient broth was spread onto fresh sterile MullerHilton agar (MHA) plates. Two wells of about 6 mm in diameter were bored with a borer in which 100 μl each of the test peptides (1 mg/ml) with negative control was added, respectively. Fluconazole disc (Himedia Laboratories Pvt. Ltd., India) at a concentration of 25 mcg/disc was used as a positive control. The plates were incubated at 37°C for 24 h to allow the fungus to grow and after incubation the zone of inhibition was measured. The vulnerability of each Candida species was established by the diameter of the inhibition zone of the growth surrounding the wells and the disc. The activity analysis was carried out thrice to certify the reproducibility of the result.
Determination of minimum inhibitory concentration
The minimum inhibitory concentration (MIC) values of tripeptide showing activity against C krusei were determined using the microtitre broth dilution assay according to the document M27-A3 guidelines.  The assay was done in triplicate in 96 well microtitre plates. Briefly, the fungus cells were inoculated into normal saline to achieve a concentration of 5 × 10 6 colony forming units (cfu)/ml according to 0.5 McFarland standards. A total of 100 μl of RPMI-1640 media adjusted to pH 7.4 with 0.3 M MOPS buffer (Himedia Laboratories Pvt. Ltd., India) was added to each well of 96 wells microtitre plate. Thereafter the sample peptides were twofold serially diluted from 1370 to 2.67 μg/ml in RPMI-1640 media. A total of 100 μl of C. krusei cell suspension was added to each well. Fluconazole (Himedia Laboratories Pvt. Ltd., India) (0.125-64 μg/ml) was used as positive control. Untreated cells were taken as growth control. The plates were incubated at 37°C for 48 h. The turbidity in the microtitre plate after incubation was interpreted by the visible growth of the Candida under simple microscope. The Fluconazole MIC is defined as the lowest concentration of drug displaying 50% inhibition as compared with the growth control and peptide MICs were determined as the same.
Determination of minimum fungicidal concentration
The minimum fungicidal concentration (MFC) of the sample peptides were calculated utiliing the plate used for MIC determination. After the incubation of the MIC plate, 50 μl of each of the well that indicated no Candida growth was sub-cultured on the fresh MHA plate and incubated for 24 to 48 h at 37°C until any visible growth was seen. The MFC value was determined as the concentration at which approximately 99% killing was observed, no growth or fewer than three colonies were observed.  The same experiment was done in triplicate.
Time kill assay
To illustrate the in vitro pharmacodynamics of the tripeptide, time kill assay was performed as against C. krusei. The fungal suspension as inoculum was prepared by inoculating two colonies of C. krusei in RPMI-1640 media and was incubated at 37°C for 24 h. The cells were inoculated in fresh RPMI-1640 media and were grown to achieve a logarithmic-phase growth and finally turbidity was measured with 0.5 Mc Farland standards. Exponentially growing fungus were re-suspended in fresh RPMI-1640 media at approximately 2 × 10 6 CFU/ml cells, which was further diluted to the concentration of 2 × 10 3 CFU/ml cells and exposed to the peptide at two times of MIC value for 0, 15, 30, 60, 90 and 120 min at 37°C. After each observation, samples were serially diluted and plated onto Sabouraud Dextrose Agar (SDA) plates to obtain viable colonies. The test was done in triplicates.
Minimal haemolytic concentration
Human blood of blood group A, B and O was collected from a healthy volunteer from the laboratory under sterile conditions. The human RBCs were washed three times with phosphate buffer saline (PBS) (137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 2H 2 O and 1.76 mM KH 2 PO 4 , pH 7.4) and diluted to a concentration of 2% (v/v) with sterile PBS. The peptide with the lowest MIC was further assessed for its haemolytic activity. Fluconazole at 1000 to 0.97 μg/ml concentration was used as drug control. A total of 100 μl of test peptide, FAR, was added to the 96 well polypropylene microtitre plate and was twofold serially diluted in sterile PBS (1000 to 0.97 μg/ml). A total of 100 μl of RBC suspension was added to each well. The detergent 0.1% Triton X 100 known to lyse all blood cells was used for 100% haemolysis. All the concentrations were analyzed in triplicates and were incubated at 37°C for 1 h. The plate was centrifuged at 1000 g for 10 min and the supernatant (100 μl) was transferred to a fresh 96 well plate without disturbance. Eventually, the absorbance of the released haemoglobin was measured at 541 nm with a microplate ELISA reader (Bio-Tek Instrument). The MHC is defined as the minimal peptide concentration that produces haemolysis. For negative and positive controls, hRBCs in PBS and in 0.1% Triton X-100 were used, respectively. The haemolysis percentage was calculated by using the following equation: Percentage haemolysis = [(Abs 541 nm in the peptide solution - Abs 541 nm in a PBS)/(Abs 541 nm in 0.1% Triton (X-100) - Abs 541 nm in a PBS)] ×100.
The potential and cell selectivity of the tripeptide FAR was calculated in terms of therapeutic index (TI), which represents the specificity of anti-microbial reagents and is determined as MHC/MIC. Thus, larger the TI, greater is the anti-microbial specificity of the peptide. 
Electron Microscopy studies
The effect of peptide on the yeast strain was also studied using scanning electron microscope. The C. krusei was allowed to grow overnight and then inoculated to fresh media. It was further incubated for 2 h at 37°C till logarithmic phase and subsequently washed with PBS and centrifuged. The cells were then suspended in 10 mM sodium phosphate buffer to a final concentration of 2 × 10 6 CFU/ml. A dose of peptide FAR (MFC 685 μg/ml) was used to treat the Candida cells for 30 and 60 min at 37°C. The cells were then centrifuged and fixed with a fixation mixture (2% paraformaldehyde and 2.5% glutareldehyde in 0.05 M sodium cacodylate buffer, pH 7.2) diluted in PBS in a ratio 1:1 for 6 h at 4°C and was then washed with PBS. 
After this the cells were plated onto poly-L-lysine treated gold coated Thermanox TM coverslips and incubated for 30 min at room temperature for proper adherence to the surface. The cover slips were washed with PBS. The cells were subsequently prepared for SEM using OTOTO method. The cells were repeatedly treated with osmium tetroxide and thiocarbohydrazine. The cells on the cover slips were dehydrated with ethanol and infiltrated with hexamethyldisilazane. It was then evaporated in a fume hood. The resultant dried specimens were then mounted on aluminium SEM stubs and examined under scanning electron microscope (Leo Imaging Systems, USA).
Protease inhibitor assay
The protease inhibition activity was performed on porcine protease enzyme trypsin (Sigma Aldrich, USA) spectrophotometrically. The casein solution was prepared by dissolving 1 g of casein into 5 ml distilled water and 5 ml NaOH to which 30 ml of distilled water was added. The solution was stirred and the pH was adjusted to 7.5 with HCl. The solution was heated at 90°C for 15 min, cooled and diluted with 50 ml 100 mM Tris-HCl (pH 8.0) buffer containing 40 mM CaCl 2 . The precipitate obtained was removed by centrifugation at 13,000 rpm for 15 min and the supernatant obtained was used as casein solution.
Initially, the FAR peptide was incubated in a ratio of 1:1 with trypsin in 50 mM Tris-HCl buffer (pH 8.0) for 30 min at 25°C. The mixture was incubated for 25 min with 350 μl of casein solution. After incubation, 1 ml of 4% (w/v) trichloroacetic acid (TCA) was added and again incubated at room temperature for 30 min. The resultant was centrifuged at 15,000 rpm for 15 min. The amount of casein fragments produced in the supernatant after proteolytic action was determined spectophotometrically at 280 nm against water as a blank.
| ~ Results|| |
Characterisation of purified peptide
The synthesized cationic peptides were purified on C-18 reverse phase column using HPLC and confirmed by single peak. [Figure 1] shows the single peak of FAR.
Thetripeptide FWY, FAY, FAR, FWC and FVY were tested against C. krusei. They showed a significant anti-fungal potential against C. krusei by diffusion method. The zone of inhibition was observed in all tested peptides against C. krusei. The most prominent inhibition zone was seen in case of FAR peptide (Data not shown).
MIC and MFC
The MIC was evaluated for the yeast strain that was inhibited in the diffusion assay. The tripeptide FWY, FAY, FAR, FWC and FVY showed anti-fungal activity against C. krusei and were subjected to MIC determination. The MIC value of FAR for 50% inhibition of the fungal growth was 171.25 μg/ml. The MFC of the FAR was the concentration where no fungal growth was seen after plating and was found to be higher than the MIC value, 685 μg/ml. The MIC and MFC values of the peptides are summaried in [Table 1].
|Table 1: The MIC and MFC values of the tripeptide exhibiting anti - fungal activity against Candida krusei|
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Time kill assay
Time-kill studies with the tripeptide FAR at concentration twice the MIC value demonstrated nearly 98% killing of C. krusei growth at 120 min. Results are presented as percentage of cell viability change in the viable colony number. A significant decrease in cell viability of isolates was observed at each time interval. There was steep decline in the cell number after the completion of 15 min [Figure 2].
|Figure 2: Time kill plot of FAR. The 2× MIC concentration of FAR was tested against Candida krusei and was sampled after 0, 15, 30, 60, 90 and 120 min after the peptide treatment|
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The haemolytic assay for FAR was performed on 2% RBC solution prepared in PBS. As it showed a very high MIC concentration but, at this concentration, there was no visible haemolysis. The visible haemolysis was found after 250 μg/ml concentration of FAR in blood group A and O. At the concentration of 1 mg/ml of FAR the percentage haemolysis is ~3% in the case of group A and group O. This peptide was found ineffective to the blood group B. With the comparison of Flu haemolysis, FAR is non-toxic to human cells [Figure 3].
|Figure 3: Haemolytic activity of FAR and Fluconazole (Flu) with 2% RBCs solution from human blood group A, B and O: Here, FAR A, FAR B and FAR O denote FAR haemolysis on different blood group A, B, and O while Flu A, Flu B and Flu O denote fluconazole haemolysis on blood group A, B, and O|
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The TI is a widely accepted parameter of the cell selectivity of anti-microbial agents. The TI was determined to evaluate the cell selectivity of FAR peptides. The TI was calculated as the ratio of the MHC of peptide to the MIC against tested fungal strain. The MHC and MIC values were carried out by two fold serial dilution. When no haemolytic activity was found at the concentration tested (250 μg/ml), the twofold tested concentration (500 μg/ml) as MHC value was used to calculate TI. The TI obtained against C. krusei was 2.92.
Electron microscopic studies
The scanning electron microscopic studies were performed with a dose of FAR (MFC 685 μg/ml) on C. krusei to observe the morphological changes after the treatment. The cells were visualized after 30 and 60 min from the treatment. The untreated cell surface was intact [[Figure 4]a], whereas the bleb-like surface changes and irregular cell surface and cell wall disruption was seen on the surface of cells after 30 and 60 min treatment, respectively [[Figure 4]b and c].
|Figure 4: Scanning electron microscopic study of FAR on Candida krusei showing (A) Untreated cells, (B) bleb-like surface changes and irregular cell surface after 30 min treatment (C) cell wall and cell membrane disruption after 1 h treatment|
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Protease inhibitory assay
The tripeptide FAR demonstrated inhibition activity against C. krusei. The absorbance of trypsin treated casein at 280 nm was 2.8 (positive control) while the absorbance of FAR mixed trypsin treated casein was 0.2. The absorbance of casein fragments produced by trypsin was taken at 280 nm. Thus, it inhibits protease activity with 71% inhibition (Data not shown).
| ~ Discussion|| |
Cationic AMPs are important candidates for anti-fungal therapeutic agents due to broad spectra of anti-microbial activity. The presence of Phe in these peptides suggests that it plays a critical role in anti-microbial activities. revious studies showed that peptides containing hydrophobic, positively charged residues like Arg, Lys are effective AMP and Phe serve as a membrane anchor in immunogenic.  Very few small (tripeptide) AMPs are known so far. One tripeptide, GHK, was reported to inhibit the growth of Escherichi coli. 3-bromocoumarins and 2-methylimidazoles analogues of di-, tri-, tetra-peptides were found to exhibit anti-microbial activity.  It has also been reported that conjugation of a palmitic acid to the N-terminus of very short cationic di- and tripeptide further provided them with potent anti-microbial activities.  The phenylalanine (Phe) in peptide has great importance for providing anti-microbial activity. An AMP named prophenin is rich in Pro and Phe and exhibits activity against the Gram-negative bacteria.  Moreover, molecular dynamics has shown that Phe plays a crucial role in the membrane permeabiliation.  In this study five tripeptide were synthesized containing Phe with different combination of cationic and aliphatic amino acids to have efficient anti-fungal properties. The cationic peptides with hydrophobic residues deeply penetrate the membrane of the microbes.  The Arg and Trp make the peptide more anti-microbial in nature. Arg endowed the peptide with cationic property and hydrogen bonding, which is very crucial for interaction with anionic component of the fungal membrane. The small peptides are non-immunogenic. Keeping these fundamental things in mind, we have designed and synthesized tripeptide with different combinations and found that FAR was showing better anti-fungal effect than other four peptides. FAR is the first tripeptide has anti-fungal activity by inhibiting the growth of C. krusei.
Preliminary screenings of peptides for their anti-fungal activities were done by agar well diffusion method. The tripeptide, FAR demonstrated the zone of inhibition against C. krusei. The MIC value confirmed that this peptide inhibited 50% of the fungal growth effectively. The peptide exhibited visible haemolysis up to 3% at the concentration of 1000 ug/ml, which showed that the peptide is nearly non-toxic to human erythrocytes.
The cytoplasmic membranes are the main targets for AMPs. The peptide accumulates in the membrane causing an increase in permeability and loss of barrier function resulting in the leakage of cytoplasmic components, which leads to cell death. The cationic peptide has two features, one is positive charge and another is amphipathic characteristic of non-polar and polar face.  The SEM studies showed that the peptide-treated fungal cells were disrupted on the cell wall as well as cytoplasmic membrane and a hole appeared on the surface. The formation of hole and cell membrane rupture may have resulted in the leakage of fungal cellular cytoplasm. The time kill assay showed the killing of fungus by FAR is very rapid. This rapid killing revealed that, there may be a lesser chance for developing resistance against it. The exposure and killing time encountered in microbes against a particular antibiotic makes them resistant as the strains get enough time to revert themselves.
The sequences and structures of AMPs are highly diverse, amphipathic in structures within membranes, have a positive net charge under physiological conditions, small size, rapid binding to membranes of the microbes and usually have the ability to kill microorganisms very fast.  The mode of action of AMPs is not fully understood, but it is believed that they mainly target the cytoplasmic membrane of the organism.  It is also believed that it is very difficult to develop resistance to AMPs as it kill microbial cells very fast mainly through their actions on the entire cytoplasmic membrane or can act through complex mechanisms.  The resistance for AMP may occur due to the change in the structure or by proteolytic cleavage. The other disadvantage of AMP is the toxicity.
According to the guidelines of Code of Federal Register 21 (section 320.33c) of Food and Drug Administration (FDA), US the TI value of FAR showed to be significant.
Our data suggests that this small synthetic tripeptide having amphipathic properties may serve as an anti-fungal agent by potentially targeting the cytoplasmic membrane through cell wall. The small size of peptide attributed to the fast mechanism of action on the fungi. It did exhibit protease inhibition activity as well as was non-toxic to human blood cells. This AMP can be the next generation of therapeutic agent for combating multi-drug resistant fungal infections.
It can be concluded that small peptides will become the drugs of choice for emerging fungal infections in future. The present work is a significant in case of C. krusei as it is developing resistance against the drugs in use. Thus, it can set a platform for the design of new effective therapeutic agents against Candida particularly C. krusei.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]