Expression of the Bovine NK-Lysin Gene Family and Activity against Respiratory Pathogens

Abstract

Unlike the genomes of many mammals that have a single NK-lysin gene, the cattle genome contains a family of four genes, one of which is expressed preferentially in the lung. In this study, we compared the expression of the four bovine NK-lysin genes in healthy animals to animals challenged with pathogens known to be associated with bovine respiratory disease (BRD) using transcriptome sequencing (RNA-seq). The expression of several NK-lysins, especially NK2C, was elevated in challenged relative to control animals. The effects of synthetic peptides corresponding to functional region helices 2 and 3 of each gene product were tested on both model membranes and bio-membranes. Circular dichroism spectroscopy indicated that these peptides adopted a more helical secondary structure upon binding to an anionic model membrane and liposome leakage assays suggested that these peptides disrupt membranes. Bacterial killing assays further confirmed the antimicrobial effects of these peptides on BRD-associated bacteria, including both Pasteurella multocida and Mannhemia haemolytica and an ultrastructural examination of NK-lysin-treated P. multocida cells by transmission electron microscopy revealed the lysis of target membranes. These studies demonstrate that the expanded bovine NK-lysin gene family is potentially important in host defense against pathogens involved in bovine respiratory disease.

Fig 1. Expression of four bovine NK-lysin genes in bronchial lymph node (BLN, left panel) and lung (LNG, right panel) among healthy animals and animals challenged with P.multocida, M.bovis, M.haemolytica, BRSV, BVDV and IBR.

The Y axis shows the FPKM value, and each black dot represents the FPKM value of an individual. Three or four individuals were included in each control and challenged group.

Fig 2. Secondary structural changes of the four synthetic bovine NK-lysin peptides upon liposome binding.

CD spectra of NK-lysin peptides in lipid-free (A) and lipid-bound states (B) are compared. Estimated secondary structural contents, including alpha-helices, beta-sheet, beta-turn and the total secondary structure in lipid-free and lipid-bound states are shown in (C) and (D), respectively.

Fig 3. Intensities of released fluorescent dye from liposome plotted against concentration of bovine NK-lysin peptides.

Fig 4

Antimicrobial effects of bovine NK-lysin peptides on BRD-causing pathogens P. multocida strains ATCC 43019 (A) and ATCC 43137 (B), M. haemolytica strains ATCC BAA-410 (C) and ATCC 33396 (D). Surviving cell numbers after peptide treatment are shown on the Y axis. Error bars represent the standard deviations calculated from four biological replications.

Fig 5. Influence of 20 μM of bovine NK1 peptide on the cell membrane of P. multocida (ATCC 43019) examined by transmission electron microscopy.

A) Control cells. (B) and (C) Cells treated with 20 μM NK1 peptide for 30 mins. (D) Statistical analysis of the average electron intensity of control cells versus NK1-treated cells. Thirty cells from each group were used for statistical analysis.