Introduction of cell‐selectivity in bovine cathelicidin BMAP‐28 by exchanging heptadic isoleucine with the adjacent proline at a non‐heptadic position

Abstract

Bovine cathelicidin, BMAP-28 exhibits potent antimicrobial activity without any cellselectivity. Considering the role of its C-terminus amino acid residues in its cytotoxic activities, we envisioned to rearrange the amino acids at its C-terminus without altering its composition for designing new cell-selective BMAP-28-analog. Thus an isoleucine residue at 20th position, also an "a" position of its previously identified isoleucine zipper sequence, was exchanged with a proline residue at 19th position. As a result, a remarkable reduction in the hemolytic activity of BMAP-28 toward human red blood cells (hRBCs) was observed without hampering its antibacterial activity in liquid media, and in presence of salts and serum. BMAP-28-analog, BMAP-28M depolarized S. aureus membrane and bacterial membrane-mimetic, PE/PG lipid vesicles similar to BMAP-28 but showed weaker efficacy to depolarize hRBC membrane and mammalian membrane-mimetic, PC/Chol vesicles. Differences in the localization of tryptophan residues of BMAP-28 and BMAP-28M were observed only in PC/Chol vesicles. BMAP-28 induced pores of~3.4 nm diameter onto hRBCs whereas BMAP-28M showed minor perturbation onto these cells. BMAP-28 and BMAP-28M induced the entry of calcein (size,~1 nm) and 4.4 kDa FITC-dextran (size,~2.8 nm) into B. megaterium but not of higher sized fluorescent-dextrans indicating toroidal pore formation onto these bacterial membranes. K E Y W O R D S antimicrobial and hemolytic activity, BMAP-28, cathelicidin antimicrobial peptides, dissipation of diffusion potential across membranes, estimation of peptide-induced pore sizes onto bacteria, mode of action of antimicrobial peptides, osmotic protection assay | INTRODUCTIONThere are mainly two antimicrobial peptide families in the mammalian system viz. Defensins and Cathelicidins. [1,2] Structures of Defensins are generally based on beta-sheet core, stabilized by three disulfide bonds [1][2][3] but cathelicidins are quite diverse. [1,4] By and large, the AMPs of the cathelicidin family are linear in nature comprised of 20 to 40 amino acid (a.a.) residues that adopt an alpha-helical conformation Abbreviations: AMP, antimicrobial peptides; CFU, colony-forming unit; Chol, cholesterol; HPLC, high-performance liquid chromatography; hRBCs, human red blood cells; MALDI-TOF, matrix-assisted laser desorption ionization time of flight; MICs, minimum inhibitory concentrations; PBS, phosphate-buffered saline; PC, phosphatidylcholine; PE, egg phosphatidylethanolamine; PG, egg phosphatidylglycerol.