Microbicidal Properties and Cytocidal Selectivity of Rhesus Macaque Theta Defensins

Abstract

Rhesus macaque -defensins (RTDs) are unique macrocyclic antimicrobial peptides. The three RTDs (RTD 1-3), isolated from macaque leukocytes, have broad-spectrum antimicrobial activities in vitro and share certain structural features with acyclic porcine protegrins, which are microbicidal peptides of the cathelicidin family. To understand the structural features that confer the respective cytocidal properties to -defensins and protegrins, we determined and compared the biological properties of RTD 1-3 and protegrin 1 (PG-1) in assays for antimicrobial activity, bacterial membrane permeabilization, and toxicity to human cells. RTD 1-3 and PG-1 had similar microbicidal potencies against Escherichia coli, Staphylococcus aureus, and Candida albicans in low-ionic-strength (10 mM) buffers at pH 7.4. The inclusion of physiologic sodium chloride partially inhibited the microbicidal activities of the RTDs, and the degree of inhibition depended on the buffer used in the assay. Similarly, the inclusion of 10% normal human serum partially antagonized the bactericidal activities of all four peptides. In contrast, the microbicidal activities of PG-1 and RTD 1-3 against E. coli were unaffected by physiologic concentrations of calcium chloride and magnesium chloride. Treatment of E. coli ML35 cells with RTD 1-3 or PG-1 rapidly rendered the bacteria permeable to -nitrophenyl-␤-D-galactopyranoside, and this was accompanied by the rapid entry of the RTDs. Finally, although PG-1 was toxic to human fibroblasts and caused a marked lysis of erythrocytes, the RTDs were not cytotoxic or hemolytic. Thus, compared to PG-1, RTD 1-3 possess substantially greater cytocidal selectivity against microbes. Surprisingly, the low cytotoxicity of the RTDs did not depend on the peptides' cyclic conformation.

Electropositive Charge in α-Defensin Bactericidal Activity: Functional Effects of Lys-for-Arg Substitutions Vary with the Peptide Primary Structure

“…Because electrostatic conditions influence ␣-defensin bactericidal activity (17,30,31,39), bactericidal peptide assays were performed in the presence of varied NaCl concentrations to determine whether Lys-for-Arg substitutions modulate sensitivity to inhibition by NaCl. To test this notion, bactericidal peptide assays were performed with E. coli ML35 and L. monocytogenes in the presence of RMAD-4 (3 M), (R/K)-RMAD-4 (3 M), Crp4 (6 M), or (R/K)-Crp4 (6 M) in solutions of increasing NaCl concentrations (Fig.…”

Comparative genomic identification and expression profiling of a novel β-defensin gene cluster in the equine reproductive tract

“…The three disulfide bonds 60 positioned in a predictable manner forms the defensin motif, which is conserved across 61 peptides and species (Semple et al, 2003). θ-defensins are rare peptides discovered in the 62 rhesus macaque and are present only in primates, the single θ-defensin gene in humans is 63 truncated by a stop codon creating a pseudogene (Tran et al, 2008). 64 β-defensin genes have been found in most vertebrate genomes and encode small, cationic 65 proteins produced by phagocytic cells, lymphocytes, and the epithelial cell lining of the 66 gastrointestinal and genitourinary tracts, the tracheobronchial tree, and keratinocytes 67 (Schneider et al, 2005).…”

Isolation, Synthesis, and Antimicrobial Activities of Naturally Occurring θ-Defensin Isoforms from Baboon Leukocytes

“…A synthetic -defensin designed based on the sequence of a human -defensin pseudogene was shown to possess antibacterial and antiviral activities (3,26). In addition, -defensins are reported to bind and inactivate lethal toxin from Bacillus anthracis (25).-Defensins are microbicidal in the presence of physiological concentrations of salt, divalent cations, and serum (21,22). In contrast, the antimicrobial activities of ␣-and ␤-defensins are markedly reduced in the presence of salt and divalent cations (2,11,19).…”

“…-Defensins are microbicidal in the presence of physiological concentrations of salt, divalent cations, and serum (21,22). In contrast, the antimicrobial activities of ␣-and ␤-defensins are markedly reduced in the presence of salt and divalent cations (2,11,19).…”