Mass Spectrometry-Based Identification of Urinary Antimicrobial Peptides in Dairy Cows

Author(s): Ambika Sharma*, Rajesh Nigam, Ashish Kumar, Simmi Singh

Journal Name: Protein & Peptide Letters

Volume 27 , Issue 3 , 2020


Become EABM
Become Reviewer
Call for Editor

Graphical Abstract:


Abstract:

Background: Urine is considered one of the biological fluids in which antimicrobial peptides are secreted or expressed. Cow urine has not been investigated for the presence of these peptides using MALDI-TOF-MS.

Objective: The aim of this study is to isolate, identify and assess the antimicrobial activity of urinary antimicrobial peptides from healthy normal cycling cows.

Methods: We analyzed the urine sample using diafiltration, ion exchange chromatography, Reverse Phase High-Performance Liquid Chromatography (RP-HPLC), acid urea polyacrylamide gel electrophoresis (AU-PAGE) coupled with identification through Peptide Mass Fingerprinting (PMF) by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDITOF- MS). The in vitro antimicrobial effects of purified fractions were assessed using Radial Diffusion Assay (RDA) and microtitre broth dilution assay against Gram-positive and Gramnegative bacteria.

Results: Proteins corresponding to the peaks were identified using SWISSPROT protein database. This study revealed constitutive expression of β-Defensin-1 (DEFB1), β-Defensin-4A (DFB4A), Neutrophil Defensin-1 (DEF1), Neutrophil Defensin-3 (DEF3) in cow urine. The identified peptides are cationic antimicrobial peptides of the defensin family. The purified fractions exhibited antimicrobial effects in radial diffusion assay and MIC values in the range of 2.93-29.3 µM/L.

Conclusion: This study concludes that cow urine, previously unexplored with regard to antimicrobial peptides, would be a promising source of highly potent AMPs and an effective alternative to the resistant antibiotics.

Keywords: Urine, AU-PAGE, MALDI-TOF-MS, antimicrobial peptides, defensins, innate immunity, radial diffusion assay.

[1]
Ganz, T. Defensins: Antimicrobial peptides of innate immunity. Nat. Rev. Immunol., 2003, 3(9), 710-720.
[http://dx.doi.org/10.1038/nri1180] [PMID: 12949495]
[2]
Becknell, B.; Schwaderer, A.; Hains, D.S.; Spencer, J.D. Amplifying renal immunity: The role of antimicrobial peptides in pyelonephritis. Nat. Rev. Nephrol., 2015, 11(11), 642-655.
[http://dx.doi.org/10.1038/nrneph.2015.105] [PMID: 26149835]
[3]
Valore, E.V.; Park, C.H.; Quayle, A.J.; Wiles, K.R.; McCray, P.B. Jr.; Ganz, T. Human β-defensin-1: An antimicrobial peptide of urogenital tissues. J. Clin. Invest., 1998, 101(8), 1633-1642.
[http://dx.doi.org/10.1172/JCI1861] [PMID: 9541493]
[4]
Park, C.H.; Valore, E.V.; Waring, A.J.; Ganz, T. Hepcidin, a urinary antimicrobial peptide synthesized in the liver. J. Biol. Chem., 2001, 276(11), 7806-7810.
[http://dx.doi.org/10.1074/jbc.M008922200] [PMID: 11113131]
[5]
Vohora, S.B.; Khan, S.Y. Animal origin drugs used in unani medicine. Inj. J. Pharm., 1978, 10(3), 255.
[6]
Pandey, G.S.; C.K, Bhav Prakash Nighantu. (Indian Materia Medica) of Sri Bhavamisra (c.1600-1600 AD).In: Ath Mutravargh; Chaukhamba Bharati Academy: Varanasi, 2009, Vol. 18, p. 778.
[7]
Prashith, K.T.R.; Nishanth, B.C.; Praveen, K.S.V.; Kamal, D.; Sandeep, M.; Meghraj, H.K. Cow urine concentrate: A potent agent with antimicrobial and anthelmintic activity. J. Pharm. Res., 2010, 1025-1027.
[8]
Decramer, S.; Gonzalez de Peredo, A.; Breuil, B.; Mischak, H.; Monsarrat, B.; Bascands, J-L.; Schanstra, J.P. Urine in clinical proteomics. Mol. Cell. Proteomics, 2008, 7(10), 1850-1862.
[http://dx.doi.org/10.1074/mcp.R800001-MCP200] [PMID: 18667409]
[9]
Pappin, D.J.; Hojrup, P.; Bleasby, A.J. Rapid identification of proteins by peptide-mass fingerprinting. Curr. Biol., 1993, 3(6), 327-332.
[http://dx.doi.org/10.1016/0960-9822(93)90195-T] [PMID: 15335725]
[10]
Millares, P.; Lacourse, E.J.; Perally, S.; Ward, D.A.; Prescott, M.C.; Hodgkinson, J.E.; Brophy, P.M.; Rees, H.H. Proteomic profiling and protein identification by MALDI-TOF mass spectrometry in unsequenced parasitic nematodes. PLoS One, 2012, 7(3)e33590
[http://dx.doi.org/10.1371/journal.pone.0033590] [PMID: 22479418]
[11]
Xu, C.; Shu, S.; Xia, C.; Wang, P.; Sun, Y.; Xu, C.; Li, C. Mass spectral analysis of urine proteomic profiles of dairy cows suffering from clinical ketosis. Vet. Q., 2015, 35(3), 133-141.
[http://dx.doi.org/10.1080/01652176.2015.1055352] [PMID: 26011147]
[12]
Simon, S.L.; Lamoureux, L.; Plews, M.; Stobart, M.; LeMaistre, J.; Ziegler, U.; Graham, C.; Czub, S.; Groschup, M.; Knox, J.D. The identification of disease-induced biomarkers in the urine of BSE infected cattle. Proteome Sci., 2008, 6(1), 23.
[http://dx.doi.org/10.1186/1477-5956-6-23] [PMID: 18775071]
[13]
Porter, E.; Valore, E.V.; Anouseyan, R.; Salzman, N.H. Detection of Antimicrobial (Poly)Peptides with Acid Urea Polyacrylamide Gel Electrophoresis Followed by Western Immunoblot.In: Salmonella: Methods and Protocols; Schatten, H.; Eisenstark, A., Eds.; Springer: New York, NY, 2015, pp. 105-115.
[14]
Mi, H.; Huang, X.; Muruganujan, A.; Tang, H.; Mills, C.; Kang, D.; Thomas, P.D. PANTHER version 11: Expanded annotation data from Gene Ontology and Reactome pathways, and data analysis tool enhancements. Nucleic Acids Res., 2017, 45(D1), D183-D189.
[http://dx.doi.org/10.1093/nar/gkw1138] [PMID: 27899595]
[15]
Bindea, G.; Mlecnik, B.; Hackl, H.; Charoentong, P.; Tosolini, M.; Kirilovsky, A.; Fridman, W-H.; Pagès, F.; Trajanoski, Z.; Galon, J.; Clue, G.O.; Clue, G.O. A Cytoscape plug-in to decipher functionally grouped gene ontology and pathway annotation networks. Bioinformatics, 2009, 25(8), 1091-1093.
[http://dx.doi.org/10.1093/bioinformatics/btp101] [PMID: 19237447]
[16]
Lehrer, R.I.; Rosenman, M.; Harwig, S.S.; Jackson, R.; Eisenhauer, P. Ultrasensitive assays for endogenous antimicrobial polypeptides. J. Immunol. Methods, 1991, 137(2), 167-173.
[http://dx.doi.org/10.1016/0022-1759(91)90021-7] [PMID: 1901580]
[17]
Wiegand, I.; Hilpert, K.; Hancock, R.E.W. Agar and broth dilution methods to determine the minimal inhibitory concentration (MIC) of antimicrobial substances. Nat. Protoc., 2008, 3(2), 163-175.
[http://dx.doi.org/10.1038/nprot.2007.521] [PMID: 18274517]
[18]
Randhawa, G.K.; Sharma, R. Chemotherapeutic potential of cow urine: A review. J. Intercult. Ethnopharmacol., 2015, 4(2), 180-186.
[http://dx.doi.org/10.5455/jice.20150222100320] [PMID: 26401404]
[19]
Khanuja, S.; Kumar, S.; Shasany, A.; Arya, J.; Darokar, M.; Singh, M.; Sinha, P.; Awasthi, S.; Gupta, S.; Gupta, V. Use of bioactive fraction from cow urine distillate ('Go-Mutra’) as a bio-enhancer of anti-infective, anti-cancer agents and nutrients. US20020164378A1, November 7, 2002.
[20]
Hancock, R.E.W.; Scott, M.G. The role of antimicrobial peptides in animal defenses. Proc. Natl. Acad. Sci. USA, 2000, 97(16), 8856-8861.
[http://dx.doi.org/10.1073/pnas.97.16.8856] [PMID: 10922046]
[21]
Ericksen, B.; Wu, Z.; Lu, W.; Lehrer, R.I. Antibacterial activity and specificity of the six human alpha-defensins. Antimicrob. Agents Chemother., 2005, 49(1), 269-275.
[http://dx.doi.org/10.1128/AAC.49.1.269-275.2005] [PMID: 15616305]
[22]
Selsted, M.E.; Harwig, S.S.; Ganz, T.; Schilling, J.W.; Lehrer, R.I. Primary structures of three human neutrophil defensins. J. Clin. Invest., 1985, 76(4), 1436-1439.
[http://dx.doi.org/10.1172/JCI112121] [PMID: 4056036]
[23]
Lehrer, R.I.; Lichtenstein, A.K.; Ganz, T. Defensins: Antimicrobial and cytotoxic peptides of mammalian cells. Annu. Rev. Immunol., 1993, 11, 105-128.
[http://dx.doi.org/10.1146/annurev.iy.11.040193.000541] [PMID: 8476558]
[24]
Ihi, T.; Nakazato, M.; Mukae, H.; Matsukura, S. Elevated concentrations of human neutrophil peptides in plasma, blood, and body fluids from patients with infections. Clin. Infect. Dis., 1997, 25(5), 1134-1140.
[http://dx.doi.org/10.1086/516075] [PMID: 9402371]
[25]
Ryckman, C.; Vandal, K.; Rouleau, P.; Talbot, M.; Tessier, P.A. Proinflammatory activities of S100: Proteins S100A8, S100A9, and S100A8/A9 induce neutrophil chemotaxis and adhesion. J. Immunol., 2003, 170(6), 3233-3242.
[http://dx.doi.org/10.4049/jimmunol.170.6.3233] [PMID: 12626582]
[26]
Sroussi, H.Y.; Köhler, G.A.; Agabian, N.; Villines, D.; Palefsky, J.M. Substitution of methionine 63 or 83 in S100A9 and cysteine 42 in S100A8 abrogate the antifungal activities of S100A8/A9: Potential role for oxidative regulation. FEMS Immunol. Med. Microbiol., 2009, 55(1), 55-61.
[http://dx.doi.org/10.1111/j.1574-695X.2008.00498.x] [PMID: 19087201]
[27]
Adachi, J.; Kumar, C.; Zhang, Y.; Olsen, J.V.; Mann, M. The human urinary proteome contains more than 1500 proteins, including a large proportion of membrane proteins. Genome Biol., 2006, 7(9), R80.
[http://dx.doi.org/10.1186/gb-2006-7-9-r80] [PMID: 16948836]
[28]
Ganz, T.; Selsted, M.E.; Szklarek, D.; Harwig, S.S.; Daher, K.; Bainton, D.F.; Lehrer, R.I. Defensins. Natural peptide antibiotics of human neutrophils. J. Clin. Invest., 1985, 76(4), 1427-1435.
[http://dx.doi.org/10.1172/JCI112120] [PMID: 2997278]
[29]
Hancock, R.E. Peptide antibiotics. Lancet, 1997, 349(9049), 418-422.
[http://dx.doi.org/10.1016/S0140-6736(97)80051-7] [PMID: 9033483]
[30]
Gough, M.; Hancock, R.E.; Kelly, N.M. Antiendotoxin activity of cationic peptide antimicrobial agents. Infect. Immun., 1996, 64(12), 4922-4927.
[PMID: 8945527]


Rights & PermissionsPrintExport Cite as

Article Details

VOLUME: 27
ISSUE: 3
Year: 2020
Published on: 10 February, 2020
Page: [225 - 235]
Pages: 11
DOI: 10.2174/0929866526666191025105038
Price: $65

Article Metrics

PDF: 26
HTML: 5