Characterization of Antibacterial and Hemolytic Activity of Synthetic Pandinin 2 Variants and Their Inhibition against Mycobacterium tuberculosis

Abstract

The contention and treatment of Mycobacterium tuberculosis and other bacteria that cause infectious diseases require the use of new type of antibiotics. Pandinin 2 (Pin2) is a scorpion venom antimicrobial peptide highly hemolytic that has a central proline residue. This residue forms a structural “kink” linked to its pore-forming activity towards human erythrocytes. In this work, the residue Pro14 of Pin2 was both substituted and flanked using glycine residues (P14G and P14GPG) based on the low hemolytic activities of antimicrobial peptides with structural motifs Gly and GlyProGly such as magainin 2 and ponericin G1, respectively. The two Pin2 variants showed antimicrobial activity against E. coli, S. aureus, and M. tuberculosis. However, Pin2 [GPG] was less hemolytic (30%) than that of Pin2 [G] variant. In addition, based on the primary structure of Pin2 [G] and Pin2 [GPG], two short peptide variants were designed and chemically synthesized keeping attention to their physicochemical properties such as hydrophobicity and propensity to adopt alpha-helical conformations. The aim to design these two short antimicrobial peptides was to avoid the drawback cost associated to the synthesis of peptides with large sequences. The short Pin2 variants named Pin2 [14] and Pin2 [17] showed antibiotic activity against E. coli and M. tuberculosis. Besides, Pin2 [14] presented only 25% of hemolysis toward human erythrocytes at concentrations as high as 100 µM, while the peptide Pin2 [17] did not show any hemolytic effect at the same concentration. Furthermore, these short antimicrobial peptides had better activity at molar concentrations against multidrug resistance M. tuberculosis than that of the conventional antibiotics ethambutol, isoniazid and rifampicin. Therefore, Pin2 [14] and Pin2 [17] have the potential to be used as an alternative antibiotics and anti-tuberculosis agents with reduced hemolytic effects.

Antimicrobial activity and structure of a consensus human β‐defensin and its comparison to a novel putative hBD10

“…Ethambutol and rifampicin were used as positive controls. The M tuberculosis inoculum was prepared from a 14‐day logarithmic phase culture and adjusted to 1 on the Mcfarland scale (0.76 OD, 600 nm) with 7H9‐OADC culture medium, and then diluted to 1:20 . Then, 100 μL of mycobacterium culture was added to each well to a final volume of 200 μL.…”

“…The optical density (OD) of the culture medium was measured at 600 nm; based on the relation that 0.2 OD is equivalent to 5 × 10 7 CFU/mL, a volume containing 1 × 10 6 CFU, was thoroughly mixed with 10 mL of warm sterilized culture medium 1 containing 10 mM PBS, 0.03% w/v of powder TSB, 1% w/v of low electroendosmosis‐type agarose (IBI Scientific, Peosta, Iowa) and 0.02% v/v Tween 20 (Sigma, St. Louis, Missouri). The culture medium was poured into Petri dishes (15 × 100 mm) on a perfectly level platform, to shape a uniform 1 mm deep layer.…”

“…with 7H9-OADC culture medium, and then diluted to 1:20. 25,26 Then, 100 μL of mycobacterium culture was added to each well to a final volume of 200 μL. The plates were covered with lids and incubated at 37 C. After 5 days of incubation, 20 μL of the resazurin solution was added to each control well only, and incubated again for 24 hours at 37 C for color development.…”

“…The optical density (OD) of the culture medium was measured at 600 nm; based on the relation that 0.2 OD is equivalent to 5 × 10 7 CFU/mL, 26 The cell pellet was resuspended in PBS, and the hRB cells were counted and adjusted to 7.7 × 10 6 ± 0.3 × 10 6 hRB cells/mL in a final volume of 200 μL, then incubated at room temperature for 1 hour in the presence of either 100% of deionized water (positive control) or serial dilutions of hBD peptides (from 1.6 to 100 μM). Pandinin-2, a hemolytic antimicrobial peptide, 28 was used also as positive control.…”

Enzymatic, Antioxidant, Antimicrobial, and Insecticidal Activities of Pleurotus pulmonarius and Pycnoporus cinnabarinus Grown Separately in an Airlift Reactor

“…The antimicrobial activity of CE from cultures of Pleurotus pulmonarius and Pycnoporus cinnabarinus was evaluated by the broth microdilution assay (Rodríguez et al 2014) with minor modifications. A total of 100 µL of fungal crude extract was added to 100 µL of the bacterial suspension (1 × 10 5 CFU/mL) in Müeller Hinton Broth.…”