Iranian Journal of Medical Microbiology

Materials and Methods

Bioinformatics Analysis

Selection of Effective Antimicrobial Peptide Derived from Lactoferrin
Camel milk lactoferrin is a glycoprotein that contains 708 amino acids and possesses two domains and two iron-binding sites (34). The strategy used for AMP synthesis from camel milk lactoferrin was in acco-rdance with bioinformatics approaches of a study conducted by Dziuba et al. (32). Based on the cleavage sites identified for various proteolytic enzymes, in-silico studies indicated that pepsin is capable of releasing numerous active peptides from lactoferrin (32, 35, 36). The initial structure of camel milk lactoferrin was obtained from the Uniport database with an ID of Q9TUM0. Next, pepsin was used to hydrolyze the protein, as this enzyme (pH>2) has proteolytic activity and is commonly applied to obtain bioactive peptides (37-39). Four online software tools, namely BIOPEP database, Collection of Antimicrobial Peptides Server, Antimicrobial Peptide Calculator and Predictor and Peptide cutter database were utilized to evaluate the antimicrobial properties of cleaved peptides derived from lactoferrin (40-43). The online server BIOPEP-UWM (www.uwm.edu.pl/biochemia) was utilized since it contains a library of bioactive peptides and comprises of the two essential coord-inated parts, namely grouping databases and appara-tuses for the assessment of proteins as the anteced-ents of bioactive peptides, which includes the proteol-ytic procedure plans. Another online database, CAMP (http://www.camp.bicnirrh. res.in), contains inform-ation about the conserved sequences, representing some examples and Hidden Markov Models (HMMs) of 1386 AMPs that are categorized into 45 families. On the other hand, the online database APD2 (http:-//aps.unmc.edu/AP/expectation/forecastmain.php) contains 2684 antimicrobial peptides from six kingdoms (266 from microbes, 4 from archaea, 8 from protists, 13 from parasites, 329 from plants, and 2018 from creatures) (44).

Experimental Procedures

 Material
S. aureus (1074, ATCC: 6538), P. aeruginosa (1060, ATTCC: 27853), and A. baumannii (1003, ATCC: BAA-747) were purchased from the Pasteur Institute of Iran.

Peptide Synthesis
The chosen peptide was named Pepsin-Camel-Lac1 (PCL1) synthesized by MIMOTOPES Pty Ltd, Australia. In order to prepare the designed peptide, 1 mg of the designed peptide was dissolved in 1 mL of sterile water and used for the experiments.
Toxicity Assay
The Methyl thiazolyl diphenyl-tetrazolium bromide (MTT) assay was utilized to assess the toxicity of PCL1 against a human cell line. The assay is based on the decrease of the tetrazolium salt into blue formazan crystals in viable cells (45, 46). The cell line was cultured in the RPMI 1640 medium supplemented with 10% Fetal Bovine Serum (FBS), 100 ml penicillin, and 100 mg/mL streptomycin. During the cell culture period, cells were incubated at 37°C in a 95% atmosphere and 5% CO₂. When the cells reached 80-90% confluence, the viability of cells was examined and passaged. In this experiment, 100 µL of the cell suspension was added to each well of a 96-well plate, each containing 100 mL of the culture medium. The cells were treated with the pre-defined concen-trations of PCL-1 and then incubated for 24 h. After dilution of PCL-1 with sterile water, cells were treated with PCL-1, and then 20 µL of the MTT solution (5 mg/mL) was added to the wells and incubated for 24 hours. After the incubation period, 50 µL of dimethyl sulfoxide (DMSO) was added to the wells to dissolve the purple formazan crystals. Afterward, according to the obtained results, the inhibitory concentration (IC50) of PCL-1 was calculated, in which 50% of the cells underwent cell death. The experiments have been carried out in triplicates, and the values were represented as the percentage of cell viability in comparison with the control cells.

Preparation of Microbial Suspension Solution
The lyophilized batches containing bacteria mentioned earlier were transferred to the liquid Mueller Hinton Broth (MHB) and incubated at 37°C for 24 h. In order to determine the antimicrobial effects, a fresh 24-hour culture medium was prepared, then 1 ml of the 24 h microbial suspension was transferred into a tube containing the MHB medium, and the turbidity of the prepared microbial suspension was assessed by McFarland standards using a spectr-ophotometer at a wavelength of 625 nm. The final suspension was used for the evaluation of drug susceptibility testing, MIC, and cell culture (47).

Determination of MIC and MBC Values by Micro-Dilution Method
The MHB medium was used to evaluate the antimicrobial effects of PCL-1 by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) methods. In general, a sterile flat 96-well plate was used after determining the half-McFarland turbidity. In this method, 50mL of MHB medium was added to well 2 to well 11. Then, 50µL of the PCL-1 peptide was added to well 2 to well 9. Next, 50 µL of the microbial suspension was added to well 1 well 10. Afterward, the plate was incubated for 24 h. Well 1 that contains bacteria were considered as the negative control, while and well 11 containing the MHB medium was considered the positive controls. In order to assess the MBC values, the contents of the wells were cultured on the Mueller Hinton Agar medium. Lack of bacterial growth is indicative of MBC determination (47, 48).

Agar Well Diffusion Assay
The antimicrobial activity test was performed using the Agar well diffusion assay (49). This technique is broadly used to assess antimicrobial activity. In this method, the microbial suspension is distributed on the plate surface. Then, a hole is created with a diameter of 6-8 mm in sterile conditions, and then specific concentrations of the PCL-1 peptide were added to the hole and left to release into the pores of Agar. Different concentrations of PCL-1 were prepared as follows; PCL-1 was transferred into 5 sterile mi-crotubes, then 100 mL of sterile water was added into microtube 2 to 5. Next, 100 mL of PCL-1 was added to microtube 1 and was serially transferred into other microtubes until the last microtube. After that, 100µL of the contents of each microtube was isolated and added to the plate agar. After incubating the plates at 37°C for 24h, the results were examined based on the diffused area around the created hole (47).

Results

Discussion

In an in-silico proteolysis conducted by Dziuba et al., 11 new peptides with potential antimicrobial activity were picked from all the digested peptides in the milk protein (32). Jahani et al. demonstrated that lact-oferrin to be effective as an antibacterial against Gram-positive (Staphylococcus epidermidis, Bacillus cereus) and Gram-negative (Campylobacter jejuni, Salmonella spp.) bacteria. However, it was more effective on the Gram-positive instead of the Gram-negative bacteria (62). In line with the investigation of peptides derived from milk lactoferrin through the milk fermentation and assessment of their anti-bacterial effects, Korheonan et al. reported that milk proteins contain a rich source of peptides that could be potentially released and activated. For example, when these peptides are activated during gastric-intestinal digestion or milk fermentation, they could be involved in modulatory processes in living systems (33). Bushra Niaz et al. reported that due to the association of lactoferrin with enterobacterial lipopolysaccharide (LPS), it acts as a permeabilizing agent of Gram-negative bacteria and destabilizes the bacterial membrane and thus enhance bacterial permeability.  Lactoferrin  is not an effective anti-bacterial agent of its own, but allows those peptides that have been derived from it, whose hydrophobicity impose a limit on their effectiveness, to enter the bacterial membrane as an antibacterial agent.(63). As a result of direct interaction between protein or lactoferrin-derived peptides, Farnaud and Evans identified that it possesses bactericidal activities (25). Cameleers believe that camel milk is more resistant to spoilage than the milk of other animals, and it has a longer shelf life. The effect of lactoperoxidase can be one of the significant factors leading to camel milk resistance to spoilage. In this work, antimicrobial peptides were identified from camel milk lactoferrin using bioinformatics; then, the PCL-1 peptide was selected, and the activity of this peptide was evaluated in-vitro. In the first step, the hydrolysis of lactoferrin was performed using the pepsin enzyme using the BIOPEP database by which 50 peptides were produced containing 5–21 amino acids. The physicochemical properties of the synthesized peptides were investigated using online tools, such as the APD2 and Peptide cutter databases, to select appropriate AMPs. The general properties of the generated peptides, including molecular weight, charge, and PI values, were analyzed to select the appropriate antimicrobial peptides (Table S1). Among the created peptides, two peptides met the initial criteria, but since the presence of lysine and cysteine amino acids in AMPs is an advantage, the peptide with an ID of 30 (PCL-1 peptide) was selected for further analyses (Table 1). Four tools of the CAMP database (ANN, DA, RF, SVM) were employed to determine the antibacterial activity of the created peptides in this study. The PCL-1 peptide was classified as AMPs since it had parameters with scores of higher than 0.45, including RF: 0.5805 and DA: 0.949 (Table 3). After synthesizing the PCL-1 peptide, the viability of bacteria in response to PCL-1, MBC, MIC, agar well diffusion was assessed in order to determine the antimicrobial activity, as well as toxicity against the human cell line. The MTT assay showed that this peptide had no toxicity against the human cell line. Our findings also showed the MIC and MBC values for S. aureus, P. aeruginosa, and A. baumannii were 31.25/500, 21.25/125, and 62.5/500, respectively. The agar well diffusion assay showed that the analyzed bacteria were resistant to the PCL-1 peptide. The experimental results of this study showed that the PCL-1 peptide had no antimicrobial properties.

Conclusion

There are naturally occurring bioactive peptides in fermented dairy products, such as yogurt, butter, milk, and cheese, which are inactivated in primary sources, and they could be activated by digestion of milk, milk fermentation, exposure to proteolytic starter media, or hydrolysis using proteolytic enzymes. Milk-derived peptides exhibit super-beneficial activity, even through oral consumption. In this study, pepsin-digested peptides in milk lactoferrin were identified by bioinformatics tools. After the synthesis of PCL-1, the MTT assay was applied to assess the toxicity of the selected peptide against the human cell line. The results showed that the chosen peptide had no toxicity against the human cell line or bacterial strains.

Abbreviations

Antimicrobial peptides: AMPs, Collection of Antimicrobial Peptides: CAMP, Antimicrobial Peptide Calculator and Predictor: APD2, Pepsin-Camel-Lac1: PCL1, Methyl thiazolyl diphenyl-tetrazolium bromide: MTT, Mueller Hinton Broth: MHB, Minimum inhibitory concentration: MIC, Minimum bactericidal concen-tration: MBC, Agar well diffusion assay: AWDA, Random Forest: RF, Support Vector Machines: SVM, Artificial Neural Network: ANN, Discriminant Analysis: DA.

 

Acknowledgements

I want to thank all those who have supported me in this work. It is worth mentioning that this essay is a thesis by Ms. Elnaz 's study at Islamic Azad University, branch of Sabzevar, Iran.

Ethics Approval and Consent to Participate

This article does not contain any studies with human participants or animals performed by any of the authors.

 

Consent for Publication

All authors gave their consent for publication.
 
 

Conflicts of Interest

The authors declared no conflict of interest.