Rattusin, an Intestinal α-Defensin-Related Peptide in Rats with a Unique Cysteine Spacing Pattern and Salt-Insensitive Antibacterial Activities

Abstract

c Cationic antimicrobial peptides are essential components of the innate immune system. As a major family of mammalian antimicrobial peptides, defensins are expressed mainly by mucosal epithelial cells and promyelocytes. Despite the capacity to kill a broad spectrum of bacteria through physical disruption of membranes, most defensins show substantially reduced antibacterial activities in the presence of monovalent and divalent cations, thereby limiting their therapeutic potential, particularly for the treatment of systemic infections. Genome-wide computational screening of the rat genome led to the identification of the gene for a novel ␣-defensin-related peptide that we termed rattusin. Rattusin shares a highly conserved signal and prosequence with mammalian ␣-defensins, but instead of the canonical ␣-defensin six-cysteine motif, rattusin consists of five cysteines with a distinctive spacing pattern. Furthermore, rattusin is preferentially expressed in Paneth cells of the distal small intestine with potent antibacterial activity against a broad range of Gram-negative and Gram-positive bacteria, including antibiotic-resistant strains. The MICs were mostly in the range of 2 to 4 M, with no appreciable toxicity to mammalian cells at up to 100 M. In contrast to classical ␣-and ␤-defensins, rattusin retained its activity in the presence of physiological concentrations of NaCl and Mg 2؉ , making it an attractive antimicrobial candidate for both topical and systemic applications.

Rattusin structure reveals a novel defensin scaffold formed by intermolecular disulfide exchanges

“…In a previous study, Patil et al . also found that synthetic RTSN refolded into a dimeric form that showed efficient antibacterial activity31 similar to the characteristics of r -RTSN observed in this study. They also proposed that RTSN forms a homodimer and/or oligomer through intra- and/or intermolecular SS bonds, and that the SS bond pairings may be random.…”

“…The determined MIC was 8 μM, which is slightly greater than previously reported data (~4 μM against E. coli O157:H7)31. This small difference may result from differences in the bacterial species and/or experimental conditions.…”

“…The hydrophobic region of peptides inserts into the hydrophobic interior formed by membrane lipid tails, which then changes the membrane structure. RTSN effectively exhibits antibacterial activity against both Gram-negative and Gram-positive bacteria even in the presence of NaCl and Mg 2+  31. The highly basic r -RTSN may efficiently interact with negatively charged phospholipid membranes.…”

“…The α-defensin-related peptide rattusin (RTSN), previously identified by genome-wide computational analysis of the rat genome30, has been recently studied at the protein level31. RTSN is preferentially expressed in Paneth cells of the distal small intestine in rats.…”

“…1), exhibits highly potent antimicrobial activity against Gram-negative and Gram-positive bacteria, including antibiotic-resistant bacteria. Moreover, RTSN exhibits low cytotoxicity to mammalian cells, but retains its antimicrobial activity in the presence of physiological salt concentrations, suggesting that RTSN can be a valuable antimicrobial agent31. Furthermore, although RTSN shares a highly conserved signal sequence and prosequence with mammalian α-defensins, RTSN consists of five unusual cysteines with a distinctive spacing pattern instead of the six-cysteine motif typically found in the canonical mammalian defensins, indicating that RTSN can adopt a unique structural scaffold that has not been observed previously.…”