EchAMP, the tenth most abundant transcript expressed in the mammary gland of echidna, has in vitro broad-spectrum antibacterial effects. However, the effects of EchAMP on mastitis, a condition where inflammation is triggered following mammary gland infection, has not been investigated. To investigate the impact of EchAMP against mastitis, EchAMP transgenic mice were generated. In antibacterial assays, the whey fractions of milk from transgenic mice significantly reduced growth of Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Pseudomonas aeruginosa compared with whey fractions from wildtype mice. Furthermore, a mastitis model created by infecting mammary gland with these four bacterial strains displayed a significant reduction in bacterial load in transgenic mice injected with S. aureus and B. subtilis. On further confirmation, histomorphologic analysis showed absence of necrosis and cell infiltration in the mammary glands of transgenic mice. To understand the role of EchAMP against inflammation, we employed an LPS-injected mastitis mouse model. LPS is known to induce phopshorylation of NF-κB and MAPK pathways, which in turn activate downstream proinflammatory signaling mediators, to promote inflammation. In LPS-treated EchAMP transgenic mice, phosphorylation levels of NF-κB, p38 and ERK1/2 were significantly downregulated. Furthermore, in mammary gland of transgenic mice, there was a significant downregulation of mRNA levels of proinflammatory cytokines, namely TNF-α, IL-6 and IL-1β. Taken together, these data suggest that EchAMP has an antiinflammatory response and is effective against S. aureus and B. subtilis. We suggest that EchAMP may be a potential prophylactic protein against mastitis in dairy animals by expressing this gene in their mammary gland.
Echidna Antimicrobial protein EchAMP Transgenic mice Mastitis
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Present study is supported by a grant from the Department of Biotechnology, Ministry of Science and Technology, India (Grant No. BT/PR6698/AAQ/1/518/2012).
Compliance with ethical standards
Conflict of interest
The authors declare that they have no competing interests.
The present study complies with institutional ethical standards.
Supplementary fig. 1 Codon optimization of native EchAMP cDNA sequence according to murine codon usage. EchAMP gene sequence was optimized according to murine codon usage using graphical codon usage analyzer software where (a) represents the native EchAMP sequence and (b) represents the optimized EchAMP sequence (TIFF 44960 kb)
Supplementary fig. 2 Analysis of EchAMP antibody reactivity. (a) Western blotting analysis was performed to check the cross-reactivity between rabbit pre-sera and EchAMP. (b) Different dilutions of EchAMP protein were loaded onto SDS-PAGE gel to identify the reactivity of immune sera to the EchAMP protein. Decreasing concentrations of EchAMP were run on SDS-PAGE, and a western blot experiment was performed. The immune sera (1:3000) identified EchAMP protein even at 15 ng concentration. (c) SDS gel electrophoresis of EchAMP protein, Echidna milk, EchAMP transgenic (Tg1 and Tg2) and wildtype mouse milk samples. (d) Total peptide sequence of EchAMP protein. Bold indicates matched peptides. (e) Electrospray ionization mass spectroscopy was performed on the EchAMP transgenic milk, and the resultant four peptides belong to EchAMP protein (TIFF 16121 kb)
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