Identification and characterization of anti-microbial peptides from rabbit vaginal fluid

Antimicrobial peptides (AMPs) serve as a first line of host defense and represent an important, though poorly understood components of the innate immune system. The present study was an attempt to identify and characterize the major molecules having anti-bacterial activities from the vaginal fluid of rabbit, Oryctologus cuniculus. AMPs from the rabbit vaginal fluid (RVF) were identified in the acid extracts of pooled RVF samples after RP-HPLC purification. The protein, RVFAMP was effective against gram negative (Escherichia coli and Pseudomonas aeruginosa) and gram positive (Staphylococcus aureus and Streptococcus pyogenes) bacteria. The results of acid urea-PAGE-gel overlay assay (AU-PAGE-GOA) demonstrated clear zone of growth inhibition of E. coli corresponding to 6 and 15 kDa protein bands. LC-MS data of these proteins indicated that 15 kDa protein consists of lysozyme, lipopolysaccharide binding protein (LBP), hemoglobin-α and β subunits (Hb-α/β), whereas 9 kDa protein band consists of transthyretin and calcyclin while uteroglobulin and neutrophil antibacterial peptide-5 (NAMP-5) are present in the 6 kDa protein band. Of the eight proteins, Hb-α derived protein was further characterized, as it showed the highest Probability Based Mowse Score (PBMS) of 288. A 25mer peptide, RVFHbαp was active against several clinical pathogens as demonstrated by minimum inhibitory concentration (MIC) and radial diffusion assays (RDA). The interaction of RVFHbαP with bacterial liposome membrane was assessed by calcein dye leakage assay. RVFHbαP did not show cytotoxicity against human endocervical cells (End1/E6E7) or erythrocytes. RT-PCR and immunofluorescence results revealed the expression of RVFHbαP mRNA and protein in rabbit vaginal tissue. To the best our knowledge, this is the first report describing the detection of AMPs in RVFs. In conclusion, these studies indicated that vaginal epithelial cells (VEC) derived RVFHbαP may have therapeutic potential in the management of reproductive well being of rabbits. The major reason for undertaking this study in rabbits is that, it forms an excellent in vivo model system for human's studies.