β-Defensin Antibiotic Peptides in the Innate Immunity of the Buffalo: In Vivo and In Vitro Studies

Abstract

β-Defensin antimicrobial peptides are multifunctional biomolecules, which are a major component of the oxygen-independent microbicidal system of buffalo polymorphonuclear (PMN) cells. They have great potential for use as proteomic biomarkers of host cell responses in the presence of microbial agents. On purifying these peptides by RP-HPLC, four defensin peptides were revealed. The results from testing against Escherichia coli, Staphylococcus aureus, Streptococcus pyogenes, Candida albicans, Rinderpest Virus (RPV) and Newcastle Disease Virus (NDV), showed that the peptides possessed antimicrobial and antiviral activities. Minimum inhibitory concentration (MIC) values varied according to the peptide amounts and the exposure time. Furthermore, an increase in the levels of these cationic antimicrobial peptides was apparent in milk obtained from natural cases of mastitis, as compared to the levels in normal milk. MALDI-TOF-based amino acid sequencing confirmed the expression of two β-defensins (LAP and BNBD-2) in mastitis milk. A comparison of peptide sequences revealed that buffalo LAP and BNBD-2 share 98.6% and 100% sequence identity, respectively, with those of cattle. In vitro, Bovine Viral Diarrhoea Virus (BVDV) infection was shown to induce the expression of the β-defensin gene, as evidenced by the PCR amplification of cDNA with specific primers. The determination of the enhanced expression of β-defensin peptides in mastitis milk and in PMN cells, can be considered as an advanced approach to the assessment of cellular and molecular responses to cell injury. It is hoped that in vitro studies on phagocytes such as PMN cells and other cell lines, will eventually replace the use of animals in elucidating the roles of these cytokines in response to microbe-derived cell damage. It will also be possible to use defensins as biomarkers to correlate failure in host cell defence systems with peptide heterogeneity.