Part:BBa_K2338003
MutantLatarcin
It is an antimicrobial peptide. This part is derived from another basic part, Native Latarcin. In the native latarcin peptide sequence, replacing phenyl alanine (F) with lysine (K) results in this mutant peptide.Through this mutation, the hemolytic activity of the native sequence has drastically reduced.
We carried out insilico analysis for this peptide using ExPAsy ProtParam tool and the results are as follows:
Molecular weight: 2883.57
Theoretical pI: 11.36
Amino acid composition:
Ala (A) 3 11.5% Arg (R) 2 7.7% Asn (N) 0 0.0% Asp (D) 0 0.0% Cys (C) 0 0.0% Gln (Q) 0 0.0% Glu (E) 0 0.0% Gly (G) 4 15.4% His (H) 1 3.8% Ile (I) 2 7.7% Leu (L) 2 7.7% Lys (K) 8 30.8% Met (M) 0 0.0% Phe (F) 1 3.8% Pro (P) 0 0.0% Ser (S) 1 3.8% Thr (T) 0 0.0% Trp (W) 0 0.0% Tyr (Y) 1 3.8% Val (V) 1 3.8% Pyl (O) 0 0.0% Sec (U) 0 0.0% (B) 0 0.0% (Z) 0 0.0% (X) 0 0.0%
Total number of negatively charged residues (Asp + Glu): 0
Total number of positively charged residues (Arg + Lys): 10
Atomic composition:
Carbon C 133 Hydrogen H 232 Nitrogen N 42 Oxygen O 29 Sulfur S 0
Formula: C133H232N42O29
Total number of atoms: 436
Extinction coefficients:
This protein does not contain any Trp residues. Experience shows that this could result in more than 10% error in the computed extinction coefficient.
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 1490
Abs 0.1% (=1 g/l) 0.517
Estimated half-life:
The N-terminal of the sequence considered is G (Gly).
The estimated half-life is: 30 hours (mammalian reticulocytes, in vitro).
>20 hours (yeast, in vivo).
>10 hours (Escherichia coli, in vivo).
Instability index:
The instability index (II) is computed to be -1.98
This classifies the protein as stable.
Aliphatic index: 82.69
Grand average of hydropathicity (GRAVY): -0.696
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
CHARACTERIZATION
We, the 2019 team of REC-CHENNAI characterised Latarcin-2a (BBa_K2338001) Latarcin-2a_F10K (BBa_K2338003) to confirm whether the mutant had retained the antimicrobial activity of the wild type while displaying decreased haemolytic and cytotoxic activity. For this, Latarcin-2a and Latarcin-2a_F10K were synthesized, purified and quantified.
The characterisation assays were carried out in E. coli DH5α, B. subtilis, and S. cerevisiae, whose growth kinetics were determined.
ANTIMICROBIAL ACTIVITY
MINIMUM INHIBITORY CONCENTRATION (MIC) ASSAY
OBJECTIVE:
Minimum Inhibitory Concentration (MIC) of Latarcin is defined as the lowest concentration of the peptide that cause 100% inhibition of cell growth. In this assay, we determine the MIC of Latarcin-2a native (Wt) and Latarcin-2a F10K (Mt) in bacterial strains E.coli DH5α, Bacillus subtilis and yeast Saccharomyces cerevisiae.
EXPERIMENT:
Bacterial strains (Bacillus subtilis and Escherichia coli DH5α) were cultured in low salt LB broth, whereas yeast cells (Saccharomyces cerevisiae) were cultured in Nutrient Broth. Prior to MIC assay, growth kinetics for all the three strains was determined from which the mid log phase was estimated as 6 hours from the addition of inoculum (Fig 1). Minimal Inhibitory Concentration (MIC) for native (Wt) and mutant (Mt) Latarcin-2a (Ltc-2a) was determined by adding the peptides to the media after 6 hours (to the mid log phase culture). The inhibition of cell growth was determined by measuring the absorbance at 630nm for every 30 minutes interval. The experiment was carried out in triplicate.
RESULT:
It has been observed that a higher concentration of the peptide inhibits the growth of the organisms immediately after the addition of the peptide whereas a lower concentration of the peptide takes comparatively more time to achieve 100% inhibition of cell growth. Both Ltc-2a Wt and Ltc-2a Mt were found to be potential inhibitors of E. coli DH5α whereas B. subtilis showed some resistance to both the native and the mutant peptides. For S. cerevisiae, Ltc-2a Wt was a better inhibitor of growth when compared to Ltc-2a Mt (Fig 2). The MIC values of Ltc-2a Wt and Ltc-2a Mt in the three microbial strains are given in Table 1. Ltc-2a Mt efficiently inhibited the growth of E. coli but both Ltc-2a Wt and Ltc-2a Mt peptides showed lesser activity on B. subtilis (Fig 3).
Table 1: MIC of native and mutant latarcin-2a for E.coli, B.subtilis and S.cerevisiae.
The growth curve of the three strains were determined by measuring absorbance at 630nm for every 30 minutes interval. The plot represents the mean of the replicates (n=3) ± s.e.m. (For data clearance, the error bars are ignored).
Escherchia coli (DH5α):
A. Ltc-2a Wt B. Ltc-2a Mt
Bacillus subtilis:
A. Ltc-2a Wt B. Ltc-2a Mt
Saccharomyces cerevisiae:
A. Ltc-2a Wt B. Ltc-2a Mt
FIGURE 2: The growth inhibitory pattern of E. coli, B. subtilis and S. cerevisiae after the addition of Ltc-2a Wt (A) and Ltc-2a Mt (B) peptide. The plot represents the mean of the replicates (n=3) ± s.e.m. (For data clearance, the error bars are ignored)
FIGURE 3: Comparison of activity between Ltc-2a Wt and Ltc-2a Mt in E. coli, B. subtilis and S. cerevisiae.
To conclude, both Ltc-2a Wt and Ltc-2a Mt inhibited the growth of E. coli and S. cerevisiae but B. subtilis showed resistance towards both the peptides.
HEMOLYTIC ACTIVITY
The hemolytic activity of Latarcin-2a Wt and mutant peptides were determined using fresh bovine blood. The solution containing 1% isolated RBCs were exposed to varied concentrations of LTC-2a W and Mt peptides. After 24 h of incubation, the amount of hemoglobin released upon lysis of RBCs was determined by measuring the absorbance at 414 nm.
FIGURE 1: Plate layout of hemolytic activity assay for latarcin-2a Wt and Mt
CONCLUSION:
It was observed that Ltc-2a Wt showed hemolytic activity up to 16 µM concentration, whereas Ltc-2a Mt did not show any hemolytic activity. Our data reveals that the presence of F10K mutation in Ltc-2a Mt suppresses the hemolytic activity of the antimicrobial peptide when compared to the wild type.
CYTOTOXICITY ASSAY
OBJECTIVE:
To compare the cytotoxic effects of Latarcin 2a native (Ltc-2a Wt) and Latarcin 2a mutant (Ltc-2a Mt) in HeLa (immortalized human cervical cancer) and C2C12 (immortalized mouse myoblast) cells.
MTT ASSAY OF NATIVE AND MUTANT LATARCIN IN HELA CELLS (24 HRS)
FIGURE 1: MTT assay of native and mutant latarcin in HeLa cells (24 hrs)
When HeLa cells were treated with a lower concentration of the peptide (4-32 µM/ml) for 24 h, Ltc-2a Wt showed ~20% cytoxicity, whereas Ltc-2a Mt showed ~10% cytotoxicity. There was a twofold decrease in the cytotoxicity of Ltc-2a Mt when compared to Ltc-2a Wt at lower concentrations of the peptide.
When HeLa cells were treated with a higher concentration of the peptide (64,120 µM/ml) for 24 h, both Ltc-2a Wt and Ltc-2a Mt showed comparable levels of cytotoxicity of ~22%. This is attributed to the high concentration of the peptides.
MTT ASSAY OF NATIVE AND MUTANT LATARCIN IN HELA CELLS (48 HRS)
FIGURE 1: MTT assay of native and mutant latarcin in HeLa cells (48 hrs)
When HeLa cells were treated with a lower concentration of the peptide (4-32 µM/ml) for 48 h, Ltc-2a Wt showed ~65% cytoxicity, whereas Ltc-2a Mt showed ~30% cytotoxicity. There was a twofold decrease in the cytotoxicity of Ltc-2a Mt when compared to Ltc-2a Wt at lower concentrations of the peptide.
When HeLa cells were treated with higher concentration of the peptide (64,120 µM/ml) for 48 h, Ltc-2a Wt showed ~95% Cytotoxicity, whereas Ltc-2a Mt showed ~70% Cytotoxicity. The mutant peptide showed only a slightly lower cytotoxic activity when compared to the native peptide owing to the higher concentration of the peptide added.
MTT ASSAY OF NATIVE AND MUTANT LATARCIN IN C2CL2 CELLS (24 HRS)
FIGURE 3: MTT assay of native and mutant latarcin in C2C12 cells (24 hrs)
When C2C12 cells were treated with a lower concentration of the peptide (4-32 µM/ml) for 24 h, both Ltc-2a Wt and Ltc-2a Mt showed ~24% cytotoxicity. Ltc-2a Wt and Ltc-2a Mt showed the same level of toxicity to C2C12 cells at lower concentrations of the peptide for 24 h.
When C2C12 cells were treated with a higher concentration of the peptide (64,120 µM/ml) for 24 h, Ltc-2a Wt showed ~75% cytotoxicity, whereas Ltc-2a Mt showed ~55% cytotoxicity. There was a slight reduction in cytotoxicity of Ltc-2a Mt when compared to Ltc-2a Wt at higher peptide concentrations in C2C12 cells.
MTT ASSAY OF NATIVE AND MUTANT LATARCIN IN C2CL2 CELLS (24 HRS)
FIGURE 4: MTT assay of native and mutant latarcin in C2Cl2 cells (48 hrs)
When the C2C12 cells were treated with a lower concentration of the peptide (4-32 µM/ml) for 48 h, both Ltc-2a Wt and Ltc-2a Mt showed ~65% cytotoxicity. Both Native and Ltc-2a Mt showed same toxicity to C2C12 cells when treated at lower concentrations of the peptide for 48 h. When the C2C12 cells were treated with a higher concentration of the peptide (64,120 µM/ml) for 48 h, both Ltc-2a Wt and Ltc-2a Mt showed ~95% cytotoxicity. Ltc-2a Wt and Ltc-2a Mt showed the same level of toxicity to C2C12 cells when treated at higher concentrations of the peptide for 48 h.
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