Advertisement

Transgenic Research

, Volume 28, Issue 5–6, pp 573–587 | Cite as

Functional evaluation of a monotreme-specific antimicrobial protein, EchAMP, against experimentally induced mastitis in transgenic mice

  • Manjusha Neerukonda
  • Sivapriya Pavuluri
  • Isha Sharma
  • Alok Kumar
  • Purnima Sailasree
  • Jyothi  B Lakshmi
  • Julie A. Sharp
  • Satish KumarEmail author
Original Paper

Abstract

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-. 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.

Keywords

Echidna Antimicrobial protein EchAMP Transgenic mice Mastitis 

Notes

Acknowledgements

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.

Ethical approval

The present study complies with institutional ethical standards.

Supplementary material

11248_2019_174_MOESM1_ESM.tif (43.9 mb)
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)
11248_2019_174_MOESM2_ESM.tif (15.7 mb)
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)
11248_2019_174_MOESM3_ESM.tif (13.7 mb)
Supplementary fig. 3 Full-length blots of the image indicated in Fig. 6 (TIFF 13979 kb)
11248_2019_174_MOESM4_ESM.docx (49 kb)
Supplementary material 4 (DOCX 48 kb)

References

  1. Acosta AC et al (2018) Frequency of Staphylococcus aureus virulence genes in milk of cows and goats with mastitis. Pes Vet Bras 38:2029–2036CrossRefGoogle Scholar
  2. Albada B, Metzler-Nolte N (2017) Highly potent antibacterial organometallic peptide conjugates. Acc Chem Re 50:2510–2518.  https://doi.org/10.1021/acs.accounts.7b00282 CrossRefGoogle Scholar
  3. Alluwaimi AM (2004) The cytokines of bovine mammary gland: prospects for diagnosis and therapy. Res Vet Sci 77:211–222.  https://doi.org/10.1016/j.rvsc.2004.04.006 CrossRefPubMedGoogle Scholar
  4. Al-Qumber M, Tagg JR (2006) Commensal bacilli inhibitory to mastitis pathogens isolated from the udder microbiota of healthy cows. J Appl Microbiol 101:1152–1160.  https://doi.org/10.1111/j.1365-2672.2006.03004.x CrossRefPubMedGoogle Scholar
  5. Banerjee S, Batabyal K, Joardar SN, Isore DP, Dey S, Samanta I, Samanta TK, Murmu S (2017) Detection and characterization of pathogenic Pseudomonas aeruginosa from bovine subclinical mastitis in West Bengal. India. Vet World 10:5.  https://doi.org/10.14202/vetworld.2017.738-742 CrossRefGoogle Scholar
  6. Barlow J (2011) Mastitis therapy and antimicrobial susceptibility: a multispecies review with a focus on antibiotic treatment of mastitis in dairy cattle. J Mammary Gland Biol Neoplasia 16:383–407.  https://doi.org/10.1007/s10911-011-9235-z CrossRefPubMedGoogle Scholar
  7. Biasibetti E, Amedeo S, Brugiapaglia A, Destefanis G, Di Stasio L, Valenza F, Capucchio MT (2012) Lipomatous muscular ‘dystrophy’ of Piedmontese cattle. Anim Int J Anim Biosci 6:1839–1847.  https://doi.org/10.1017/s175173111200081x CrossRefGoogle Scholar
  8. Bisana S, Kumar S, Rismiller P, Nicol SC, Lefevre C, Nicholas KR, Sharp JA (2013) Identification and functional characterization of a novel monotreme-specific antibacterial protein expressed during lactation. PLoS ONE 8:e53686.  https://doi.org/10.1371/journal.pone.0053686 CrossRefPubMedPubMedCentralGoogle Scholar
  9. Bortolanza M, Cavalcanti-Kiwiatkoski R, Padovan-Neto FE, da-Silva CA, Mitkovski M, Raisman-Vozari R, Del-Bel E (2015) Glial activation is associated with l-DOPA induced dyskinesia and blocked by a nitric oxide synthase inhibitor in a rat model of Parkinson’s disease. Neurobiol Dis 73:377–387.  https://doi.org/10.1016/j.nbd.2014.10.017 CrossRefPubMedGoogle Scholar
  10. Choi H, Lee DG (2012) Synergistic effect of antimicrobial peptide arenicin-1 in combination with antibiotics against pathogenic bacteria. Res Microbiol 163:479–486.  https://doi.org/10.1016/j.resmic.2012.06.001 CrossRefPubMedGoogle Scholar
  11. Cooper CA, Klobas LCG, Maga EA, Murray JD (2013) Consuming transgenic goats’ milk containing the antimicrobial protein lysozyme helps resolve diarrhea in young pigs. PLoS ONE 8:e58409.  https://doi.org/10.1371/journal.pone.0058409 CrossRefPubMedPubMedCentralGoogle Scholar
  12. Cooper CA, Maga EA, Murray JD (2015) Production of human lactoferrin and lysozyme in the milk of transgenic dairy animals: past, present, and future. Transgenic Res 24:605–614.  https://doi.org/10.1007/s11248-015-9885-5 CrossRefPubMedGoogle Scholar
  13. Das D, Panda SK, Jena B, Sahoo AK (2018) Economic impact of subclinical and clinical mastitis in Odisha, India. Int J Curr Microbiol Appl Sci 7:4.  https://doi.org/10.20546/ijcmas.2018.703.422 CrossRefGoogle Scholar
  14. De Schepper S, De Ketelaere A, Bannerman DD, Paape MJ, Peelman L, Burvenich C (2008) The toll-like receptor-4 (TLR-4) pathway and its possible role in the pathogenesis of Escherichia coli mastitis in dairy cattle. Vet Res 39:5.  https://doi.org/10.1051/vetres:2007044 CrossRefPubMedGoogle Scholar
  15. Devi M, Dutta JB (2018) Incidence of bovine subclinical mastitis in organized and unorganized farms based on somatic cell count. Int J Chem Stud 6:5Google Scholar
  16. Elhadidy M, Elsayyad A (2013) Uncommitted role of enterococcal surface protein, Esp, and origin of isolates on biofilm production by Enterococcus faecalis isolated from bovine mastitis. J Microbiol Immunol Infect Wei mian yu gan ran za zhi 46:80–84.  https://doi.org/10.1016/j.jmii.2012.02.002 CrossRefPubMedGoogle Scholar
  17. Enjapoori AK, Grant TR, Nicol SC, Lefevre CM, Nicholas KR, Sharp JA (2014) Monotreme lactation protein is highly expressed in monotreme milk and provides antimicrobial protection. Genome Biol Evol 6:2754–2773.  https://doi.org/10.1093/gbe/evu209 CrossRefPubMedPubMedCentralGoogle Scholar
  18. Fossum K, Herikstad H, Binde M, Pettersen KE (1986) Isolation of Bacillus subtilis in connection with bovine mastitis. Nord Vet 38:233–236Google Scholar
  19. Foxman B, D’Arcy H, Gillespie B, Bobo JK, Schwartz K (2002) Lactation mastitis: occurrence and medical management among 946 breastfeeding women in the United States. Am J Epidemiol 155(2):103–114CrossRefGoogle Scholar
  20. Gomes F, Henriques M (2016) Control of bovine mastitis: old and recent therapeutic approaches. Curr Microbiol 72:377–382.  https://doi.org/10.1007/s00284-015-0958-8 CrossRefPubMedGoogle Scholar
  21. Hagiwara K et al (2003) Mouse SWAM1 and SWAM2 are antibacterial proteins composed of a single whey acidic protein motif. J Immunol (Baltimore, Md: 1950) 170:1973–1979CrossRefGoogle Scholar
  22. Halasa T, Huijps K, Osteras O, Hogeveen H (2007) Economic effects of bovine mastitis and mastitis management: a review. Vet Q 29:18–31.  https://doi.org/10.1080/01652176.2007.9695224 CrossRefPubMedGoogle Scholar
  23. Kerr DE et al (2001) Lysostaphin expression in mammary glands confers protection against staphylococcal infection in transgenic mice. Nat Biotechnol 19:66–70.  https://doi.org/10.1038/83540 CrossRefPubMedGoogle Scholar
  24. Kim MO et al (2007) Ectopic expression of tethered human follicle-stimulating hormone (hFSH) gene in transgenic mice. Transgenic Res 16:65–75.  https://doi.org/10.1007/s11248-006-9031-5 CrossRefPubMedGoogle Scholar
  25. Krishnamoorthy P, Suresh KP, Saha S, Govindaraj G, Shome BR, Roy P (2017) Meta-analysis of prevalence of subclinical and clinical mastitis, major mastitis pathogens in dairy cattle in India. Int J Curr Microbiol Appl Sci 6:21.  https://doi.org/10.20546/ijcmas.2017.603.141 CrossRefGoogle Scholar
  26. Kumar A, Parveen S, Sharma I, Pathak H, Deshmukh MV, Sharp JA, Kumar S (2019) Structural and mechanistic insights into EchAMP: a antimicrobial protein from the Echidna milk. Biochim Biophysica Acta Biomembr 1861:1260–1274.  https://doi.org/10.1016/j.bbamem.2019.03.020 CrossRefGoogle Scholar
  27. Kuruppath S, Bisana S, Sharp JA, Lefevre C, Kumar S, Nicholas KR (2012) Monotremes and marsupials: comparative models to better understand the function of milk. J Biosci 37:581–588CrossRefGoogle Scholar
  28. Liu S et al (2012) High-level expression of bioactive recombinant human lysozyme in the milk of transgenic mice using a modified human lactoferrin BAC. Transgenic Res 21:407–414.  https://doi.org/10.1007/s11248-011-9536-4 CrossRefPubMedGoogle Scholar
  29. Liu X et al (2014) Generation of mastitis resistance in cows by targeting human lysozyme gene to beta-casein locus using zinc-finger nucleases. Proc Biol Sci 281:20133368.  https://doi.org/10.1098/rspb.2013.3368 CrossRefPubMedPubMedCentralGoogle Scholar
  30. Liu E, Liang T, Wang X, Ban S, Han L, Li Q (2015) Apoptosis induced by farrerol in human gastric cancer SGC-7901 cells through the mitochondrial-mediated pathway. Eur J Cancer Prev Off J Eur Cancer Prev Organ (ECP) 24:365–372.  https://doi.org/10.1097/cej.0000000000000104 CrossRefGoogle Scholar
  31. Maga EA, Anderson GB, Cullor JS, Smith W, Murray JD (1998) Antimicrobial properties of human lysozyme transgenic mouse milk. J Food Prot 61:52–56CrossRefGoogle Scholar
  32. Maga EA, Cullor JS, Smith W, Anderson GB, Murray JD (2006) Human lysozyme expressed in the mammary gland of transgenic dairy goats can inhibit the growth of bacteria that cause mastitis and the cold-spoilage of milk. Foodborne Pathog Dis 3:384–392.  https://doi.org/10.1089/fpd.2006.3.384 CrossRefPubMedGoogle Scholar
  33. Munday BL, Whittington RJ, Stewart NJ (1998) Disease conditions and subclinical infections of the platypus (Ornithorhynchus anatinus). Philos Trans R Soc Lond Ser B Biol Sci 353:1093–1099.  https://doi.org/10.1098/rstb.1998.0268 CrossRefGoogle Scholar
  34. Naruse K, Yoo SK, Kim SM, Choi YJ, Lee HM, Jin DI (2006) Analysis of tissue-specific expression of human type II collagen cDNA driven by different sizes of the upstream region of the beta-casein promoter. Biosci Biotechnol Biochem 70:93–98.  https://doi.org/10.1271/bbb.70.93 CrossRefPubMedGoogle Scholar
  35. Newman J, Sharp JA, Enjapoori AK, Bentley J, Nicholas KR, Adams TE, Peat TS (2018) Structural characterization of a novel monotreme-specific protein with antimicrobial activity from the milk of the platypus. Acta Crystallogr Sect F Struct Biol Commun 74:39–45.  https://doi.org/10.1107/s2053230x17017708 CrossRefGoogle Scholar
  36. Park HR et al (2014) Characterisation of Pseudomonas aeruginosa related to bovine mastitis. Acta Vet Hung 62:1–12.  https://doi.org/10.1556/AVet.2013.054 CrossRefPubMedGoogle Scholar
  37. Peel E, Cheng Y, Djordjevic JT, Kuhn M, Sorrell T, Belov K (2017) Marsupial and monotreme cathelicidins display antimicrobial activity, including against methicillin-resistant Staphylococcus aureus. Microbiology (Reading, England) 163:1457–1465.  https://doi.org/10.1099/mic.0.000536 CrossRefGoogle Scholar
  38. Sadashiv SO, Kaliwal BB (2014) Isolation, characterization and antibiotic resistance of Bacillus sps. from bovine mastitis in the region of north Karnataka, India. Int J Curr Microbiol Appl Sci 3:14Google Scholar
  39. Sani RN, Mahdavi A, Moezifar M (2015) Prevalence and etiology of subclinical mastitis in dairy ewes in two seasons in Semnan province, Iran. Trop Anim Health Prod 47:1249–1254.  https://doi.org/10.1007/s11250-015-0855-y CrossRefGoogle Scholar
  40. Schulz J, Friese A, Klees S, Tenhagen BA, Fetsch A, Rosler U, Hartung J (2012) Longitudinal study of the contamination of air and of soil surfaces in the vicinity of pig barns by livestock-associated methicillin-resistant Staphylococcus aureus. Appl Environ Microbiol 78:5666–5671.  https://doi.org/10.1128/aem.00550-12 CrossRefPubMedPubMedCentralGoogle Scholar
  41. Sharma N, Singh SG, Sharma S, Misri J, Gupta SK, Hussain K (2018) Mastitis occurrence pattern in dairy cows and importance of related risk factors in the occurrence of mastitis. J Anim Res 8:12.  https://doi.org/10.30954/2277-940x.04.2018.23 CrossRefGoogle Scholar
  42. Shevchenko A, Wilm M, Vorm O, Mann M (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal Chem 68:850–858CrossRefGoogle Scholar
  43. Sordillo LM, Streicher KL (2002) Mammary gland immunity and mastitis susceptibility. J Mammary Gland Biol Neoplasia 7:135–146CrossRefGoogle Scholar
  44. Suojala L, Kaartinen L, Pyorala S (2013) Treatment for bovine Escherichia coli mastitis—an evidence-based approach. J Vet Pharmacol Ther 36:521–531.  https://doi.org/10.1111/jvp.12057 CrossRefPubMedGoogle Scholar
  45. Tuchscherr LP, Buzzola FR, Alvarez LP, Caccuri RL, Lee JC, Sordelli DO (2005) Capsule-negative Staphylococcus aureus induces chronic experimental mastitis in mice. Infect Immun 73:7932–7937.  https://doi.org/10.1128/iai.73.12.7932-7937.2005 CrossRefPubMedPubMedCentralGoogle Scholar
  46. Van Keuren ML, Gavrilina GB, Filipiak WE, Zeidler MG, Saunders TL (2009) Generating transgenic mice from bacterial artificial chromosomes: transgenesis efficiency, integration and expression outcomes. Transgenic Res 18:769–785.  https://doi.org/10.1007/s11248-009-9271-2 CrossRefPubMedPubMedCentralGoogle Scholar
  47. Vanderhaeghen W, Hermans K, Haesebrouck F, Butaye P (2010) Methicillin-resistant Staphylococcus aureus (MRSA) in food production animals. Epidemiol Infect 138:606–625.  https://doi.org/10.1017/s0950268809991567 CrossRefPubMedGoogle Scholar
  48. Wang J et al (2011) Ancient antimicrobial peptides kill antibiotic-resistant pathogens: Australian mammals provide new options. PLoS ONE 6:e24030.  https://doi.org/10.1371/journal.pone.0024030 CrossRefPubMedPubMedCentralGoogle Scholar
  49. Warren WC et al (2008) Genome analysis of the platypus reveals unique signatures of evolution. Nature 453:175–183.  https://doi.org/10.1038/nature06936 CrossRefPubMedPubMedCentralGoogle Scholar
  50. Whittington CM, Belov K (2014) Tracing monotreme venom evolution in the genomics era. Toxins 6:1260–1273.  https://doi.org/10.3390/toxins6041260 CrossRefPubMedPubMedCentralGoogle Scholar
  51. Whittington RJ, McColl KA (1983) Aspiration pneumonia in a wild platypus Ornithorhynchus anatinus. Aust Vet J 60:277.  https://doi.org/10.1111/j.1751-0813.1983.tb07107.x CrossRefPubMedGoogle Scholar
  52. Wright GD (2016) Antibiotic adjuvants: rescuing antibiotics from resistance: (Trends in Microbiology 24, 862–871; October 17, 2016). Trends Microbiol 24:928.  https://doi.org/10.1016/j.tim.2016.07.008 CrossRefPubMedGoogle Scholar
  53. Younis A, Krifucks O, Heller ED, Samra Z, Glickman A, Saran A, Leitner G (2003) Staphylococcus aureus exosecretions and bovine mastitis. J Vet Med B Infect Dis Vet Public Health 50:1–7CrossRefGoogle Scholar
  54. Younis S, Javed Q, Blumenberg M (2016) Meta-analysis of transcriptional responses to mastitis-causing Escherichia coli. PLoS ONE 11:e0148562.  https://doi.org/10.1371/journal.pone.0148562 CrossRefPubMedPubMedCentralGoogle Scholar
  55. Yu G, Baeder DY, Regoes RR, Rolff J (2018) Predicting drug resistance evolution: insights from antimicrobial peptides and antibiotics. Proc Biol Sci.  https://doi.org/10.1098/rspb.2017.2687 CrossRefPubMedPubMedCentralGoogle Scholar
  56. Zhang J et al (2008) Expression of active recombinant human lactoferrin in the milk of transgenic goats. Protein Expr Purif 57:127–135.  https://doi.org/10.1016/j.pep.2007.10.015 CrossRefPubMedGoogle Scholar
  57. Zheng J, Watson AD, Kerr DE (2006) Genome-wide expression analysis of lipopolysaccharide-induced mastitis in a mouse model. Infect Immun 74:1907–1915.  https://doi.org/10.1128/iai.74.3.1907-1915.2006 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Manjusha Neerukonda
    • 1
    • 2
  • Sivapriya Pavuluri
    • 1
  • Isha Sharma
    • 1
    • 3
  • Alok Kumar
    • 1
  • Purnima Sailasree
    • 1
  • Jyothi  B Lakshmi
    • 1
  • Julie A. Sharp
    • 4
  • Satish Kumar
    • 1
    • 5
    Email author
  1. 1.Centre for Cellular and Molecular BiologyHyderabadIndia
  2. 2.University Medical CentreJohannes Gutenberg UniversityMainzGermany
  3. 3.Northwestern UniversityChicagoUSA
  4. 4.Institute for Frontier MaterialsDeakin UniversityWaurn PondsAustralia
  5. 5.Department of Biotechnology, School of Life SciencesCentral University of HaryanaJant-Pali, MahendergarhIndia

Personalised recommendations