eNAP-2, a novel cysteine-rich bactericidal peptide from equine leukocytes.
We purified a novel cysteine-rich antibiotic peptide, eNAP-2 (M(r), approximately 6,500), from acid extracts of equine neutrophils by sequential gel filtration and reversed-phase high-performance liquid chromatography and determined its partial N-terminal amino acid sequence. Although its cysteine motif distinguished eNAP-2 from all other currently known endogenous antibiotic peptides, including defensins and granulins, it showed substantial sequence similarity to WDNM1, a putative member of the four-disulfide-core protein family that also includes animal and human antiproteases, snake venom neurotoxins, and rat and mouse whey proteins. The antibacterial properties of eNAP-2 were tested against several equine uterine pathogens, namely, Streptococcus zooepidemicus, Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae. Killing of S. zooepidemicus was very efficient, as evidenced by a 94% decrease in numbers of CFU per milliliter after exposure to 100 micrograms of eNAP-2 per ml (approximately 15 microM) for 2 h. Exposure of E. coli and P. aeruginosa to 200 micrograms of eNAP-2 per ml for 2 h resulted in 90.2 and 77.6% reduction, respectively, in the numbers of CFU per milliliter. Bacteriostasis, without bactericidal activity, occurred after K. pneumoniae was incubated with 200 micrograms of eNAP-2 per ml. Additional studies will be required in other species and cell types to determine whether eNAP-2 is restricted to equine neutrophils or is the index member of a larger family of endogenous antibiotics.
Couto
M A
MA
Department of Reproduction, School of Veterinary Medicine, University of California, Davis 95616.
Harwig
S S
SS
Cullor
J S
JS
Hughes
J P
JP
Lehrer
R I
RI
D000595:Amino Acid Sequence; D000818:Animals; D000900:Anti-Bacterial Agents / Q000302:isolation & purification*; D001419:Bacteria / Q000187:drug effects*; D001798:Blood Proteins / Q000302:isolation & purification*; D003545:Cysteine / Q000032:analysis; D005260:Female; D006736:Horses / Q000097:blood*; D008969:Molecular Sequence Data; D009504:Neutrophils / Q000737:chemistry*; D010455:Peptides / Q000032:analysis / Q000302:isolation & purification* / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D001798:Blood Proteins; D010455:Peptides; C078461:equine neutrophil antibiotic peptide 2; D003545:Cysteine