Table 1.
Peptides’ list and sequences.
Table 2.
Antibacterial effect of different peptides and lauric acid on E. coli (ATCC 25922).
Table 3.
Percentage of viable remaining colonies of E. coli at different concentrations of ampicillin as positive control antibiotic.
Fig 1.
Effect of (A) Brevinin 2R, (B) L- Brevinin 2R, (C) CLIP, (D) L-CLIP, (E) AmB and (F) Lauric acid on L. major promastigote growth.
Fig 2.
Effect of (A) Brevinin 2R, (B) L-Brevinin 2R, (C) CLIP, (D) L-CLIP, (E) Lauric acid, and (F) AmB on L. major amastigote growth and their toxicity effect on THP1 cells. Internalized promastigotes were transformed to amastigote in THP1 cells. Imaging system captured different views of the cells exposed to different concentrations of peptides. Eventually the ratio of infected to total THP1 cells (INF ratio), number of parasites presented inside the cells and number of THP1 cells were calculated.
Fig 3.
Percentage of hemolysis on human RBCs in the presence of different concentrations of peptides and lauric acid.
Fig 4.
L. major promastigote permeability assay with Sytox green. Arrow shows the time of reagent addition. Increase in fluorescence intensity was calculated from the ratio of each sample to promastigote alone plus Sytox green.
Fig 5.
Cell membrane potential changes in L. major promastigotes exposed to (A) Brevinin 2R, (B) L- Brevinin 2R (C) CLIP and (D) L-CLIP. Arrow shows the time of peptides addition.
Fig 6.
Flow cytometry results of L. major promastigotes exposed to different peptides. Effective peptides affected L. major through necrosis. No signs of apoptosis event were detected. (A) Untreated parasite stained with Annexin/PI, (B) Parasite treated with 0.5% Triton x-100 as positive control for PI. (C) Brevinin 2R, (D) L- Brevinin 2R, (E) CLIP and (F) L- CLIP treated parasite. In each pair of graphs, the left represents SSC vs FSC and the right graph indicates PI vs Annexin value.
Fig 7.
(A) Active caspase detection in L. major promastigotes exposed to peptides. Changes in the fluorescence intensity in the presence or absence of caspase inhibitors were studied. Inh: inhibitor. (B) Ratio of red to green fluorescence zone for mitochondrial electrical potential detection. Stars indicates the significant difference in compare to valinomycin. *, ** and *** = P value < 0.05, < 0.01 and < 0.001 respectively.
Fig 8.
SEM view of promastigotes exposed to the IC50 concentration of L- Brevinin 2R (A), L- CLIP (B) and normal promastigotes (C). Pictures were captured with 7000X magnification.
Table 4.
Treatment protocol in experimental animal model.
Fig 9.
(A) Footpad swelling in weeks after challenge until the end of treatment. Swelling was calculated according to thickness and width of the footpad. Infected foot size was subtracted from intact footpad. (B) Footpad swelling in experimental groups at 5th week after challenge. (C) Real-time PCR assay for parasite burden determination in the lymph node of experimental animals. Peptide (L- Brevinin 2R) with or without CpG motif was able to control the parasite load in the lymph nodes adjacent to the infected footpad. (D) Normalized data provided from GAPDH house-keeping gene Real- time PCR assay in parallel with parasite burden in lymph node samples. *, ** and *** = P value < 0.05, < 0.01 and < 0.001 respectively.
Fig 10.
Cytokine production profile (A) IFN-γ, (B) IL-4, (C) IL-6, (D) IL-10, (E) IFN-γ/IL-10 ratio and (F) IFN-γ/IL-4 ratio in splenocytes exposed to freezed-thawed antigen of L. major. *, ** and *** = P value < 0.05, < 0.01 and < 0.001 respectively.