Molecular cloning of lipocalin-2 into a eukaryotic vector and its expression inbovine mammary epithelial cells as a potential treatment for bovine mastitis

Molecular cloning of lipocalin-2 into a eukaryotic vector and its expression inbovine mammary epithelial cells as a potential treatment for bovine mastitis

Lipocalin-2 (LCN2) is a 25-kDa protein in the lipocalin family that shows antibacterial activity. The aim of this study was to clone human LCN2 into the mammalian-specific pIRES2-AcGFP1 vector and to determine the antibacterial activity of the recombinant pIRES2-AcGFP1-hLCN2 plasmid against major bacterial pathogens that cause bovine mastitis. The amplified hLCN2 was successfully cloned into the vector, and the plasmid was transfected into bovine mammary epithelial stem cells using nucleofection. Immunochemistry, reverse transcription polymerase chain reaction, and ELISA were used to monitor and quantify hLCN2 expression in the cells. After 1 day of incubation, the concentration of hLCN2 secreted by cells in the medium (6.85 ± 0.11 μg) was higher than that stored within the cells (22 ± 2.52 ng). After 4 days of culture, the amount of hLCN2 was 300-fold higher in the medium than within the cells. The recombinant clone showed potential antibacterial activity against mastitis-causing bacteria, with greater inhibitory activity against Escherichia coli than against Staphylococcus aureus. Antibacterial activity remained strong after 4-5 h of culture. Thus, pIRES2-AcGFP1 was identified as an efficient vector for the delivery of the target gene to the mammary cells, and recombinant hLCN2 showed strong antibacterial activity against major mastitis-causing bacteria.

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  • Armacki M (2010). Identification and characterization of PKD2 substrates-CIB1A as a substrate for PKD2. PhD, Universitätsklinikum Ulm, Ulm, Germany.Berger T, Togawa A, Duncan GS, Andrew JE, You-Ten A, Wakeham A, Hannah EHF, Carol CC, Tak WM (2005). Lipocalin 2-deficient mice exhibit increased sensitivity to Escherichia coliinfection but not to ischemiareperfusion injury. P Natl Acad Sci USA 106: 1834–1839.Bnyan IA, Bnyan HA, Ali JA (2010). The siderophore production of E. coli isolated from urinary tract infection and fecal isolates. J Babylon Univ/Pure Appl Sci 18: 862–864.Cappuccino JG, Sherman N (1999). Microbiology: A Laboratory Manual. Boston, MA, USA: Addison Wesley Longman Inc.Chan YR, Liu JS, Pociask DA, Zheng M, Mietzner TA, Berger T, Mak TW, Clifton MC, Strong RK, Ray P et al. (2009). Lipocalin 2 is required for pulmonary host defense against Klebsiella infection. J Immunol 182: 4947–4956.Cowland JB, Borregaard N (1997). Molecular characterization and pattern of tissue expression of the gene for neutrophil gelatinase-associated lipocalin from humans. Genomics 45: 17.Cowland JB, Sorensen OE, Sehested M, Borregaard N (2003). Neutrophil gelatinase-associated lipocalin is up-regulated in human epithelial cells by IL-1 beta, but not by TNF-alpha. J Immunol 171: 6630–6639.Dale SE, Doherty-Kirby A, Lajoie G, Heinrichs DE (2004). Role of siderophore biosynthesis in virulence of Staphylococcus aureus: identification and characterization of genes involved in production of a siderophore. Infect Immun 72: 29–37.de Souza BF (2011). Analysis of novel steroidogenic factor-1 targets in the human adrenal gland. PhD, University College London, London. Espitia PJP, Soares NFF, Teofilo RF, Vitor DM, Coimbra JSR, Andrade NJ, Sousa FB, Sinisterra RD, Medeiros EAA (2013). Optimized dispersion of ZnO nanoparticles and antimicrobial activity against foodborne pathogens and spoilage microorganisms. J Nanopart Res 15: 1324–1339.Flo TH, Smith KD, Sato S, Rodriguez DJ, Holmes MA, Strong RK, Akira S, Aderem A (2004). Lipocalin 2 mediates an innate immune response to bacterial infection by sequestrating iron. Nature 432: 917–921.Flower DR (1994). The lipocalin protein family: a role in cell regulation. FEBS Lett 354: 7–11.Friedl A, Stoesz SP, Buckley P, Gould MN (1999). Neutrophil gelatinase-associated lipocalin in normal and neoplastic human tissues: cell type-specific pattern of expression. Histochem J 31: 433.Fukushima T, Allred BE, Sia AK, Nichiporuk R, Andersen UN, Raymond KN (2013). Gram-positive siderophore-shuttle with iron-exchange from Fe-siderophore to apo-siderophore byBacillus cereusYxeB. P Natl Acad Sci USA 110: 13821–13826.Hammer ND, Skaar EP (2011). Molecular mechanisms of Staphylococcus aureusiron acquisition. Ann Rev Microbiol 65: 129–147.Hashemi A, Roohvand F, Ghahremani MH, Aghasadeghi MR, Vahabpour R, Motevali F, Memarnejadian A (2012). Optimization of transfection methods for Huh_7 and Vero cells: a comparative study. Cytol Genet 46: 347–353.Jang S, Krausslich H, Nicklin MJ, Duke GM, Palmenberg AC, Wimmer E (1988). A segment of the 5’ nontranslated region of encephalomyocarditis virus RNA directs internal entry of ribosomes during in vitro translation. J Virol 62: 2636.Johnson T, Case C (1995). Chemical Methods of Control: Laboratory Experiments in Microbiology. 4th ed. Redwood City, CA, USA: Benjamin/Cummings Publishing Co.Kawai S, Nishizawa M (1984). New procedure for DNA transfection with polycation and dimethyl sulfoxide. Mol Cell Biol 4: 1172.Kjeldsen L, Cowland JB, Borregaard N (2000). Human neutrophil gelatinase-associated lipocalin and homologous proteins in rat and mouse. Biochim Biophys Acta 1482: 272–283.Lourenco FR, Pinto TJA (2011). Antibiotic microbial assay using kinetic-reading microplate system. Braz J Pharm Sci 47: 573–584.Maurisse R, Semir DD, Emamekhoo H, Bedayat B,Abdolmohammadi A, Parsi H, Gruener DC (2010). Comparative transfection of DNA into primary and transformed mammalian cells from different lineages. BMC Biotechnol 10: 2–9.McLaren A (1992). The quest for immortality. Nature (Lond) 359: 482–483.Melkonyan H, Sorg C, Klempt M (1996). Electroporation efficiency in mammalian cells is increased by dimethyl sulfoxide (DMSO). Nucleic Acids Res 24: 4356–4357.Miharada K, Hiroyama T, Sudo K, Nagasawa T, Nakamura Y (2005). Lipocalin 2 functions as a negative regulator of red blood cell production in an autocrine fashion. FASEB J 19: 1881–1883. Nairz M, Schroll A, Sonnweber T, Weiss G (2010). The struggle for iron—a metal at the host–pathogen interface. Cellular Microbiol 12: 1691–1702.Nielsen BS, Borregaard N, Bundgaard JR, Timshel S, Sehested M, Kjeldsen L (1996). Induction of NGAL synthesis in epithelial cells of human colorectal neoplasia and inflammatory bowel diseases. Gut 38: 414–420.Raffatellu M, George MD, Akiyama Y, Hornsby MJ, Nuccio SP, Paixao TA, Butler BP, Chu H, Santos RL, Berger T et al. (2009). Lipocalin-2 resistance confers an advantage to Salmonella enterica serotype typhimurium for growth and survival in the inflamed intestine. Cell Host Microbe 5: 476–486.Robinson KM, McHugh KJ, Mandalapu S, Clay ME, Lee B, Scheller EV, Enelow RI, Chan YR, Kolls JK, Alcorn JF (2014). Influenza-A virus exacerbates Staphylococcus aureus pneumonia in mice by attenuating antimicrobial peptide production. J Infect Dis 209: 865–875.Sharma N, Gupta SK, Sharma U, Hussain K (2007). Treatment of clinical mastitis in buffalo—a case report. Buffalo Bull 26: 56–58.Smith KD (2007). Iron metabolism at the host pathogen interface: lipocalin2 and the pathogen-associated iroA gene cluster. Int J Biochem Cell Biol 39: 1776–1780.Thalhamer T (2009). B cell signalling in mechanisms of central and peripheral tolerance. PhD, University of Glasgow, Glasgow, UK.Tiwari JG, Babra C, Tiwari HK, Williams V, Wet SD, Gibson J, Paxman A, Morgan E, Sunagar R, Isloor S et al. (2013). Trends in therapeutic and prevention strategies for management of bovine mastitis: an overview. J Vaccines Vaccin 4: 176.Xu S, Venge P (2000). Lipocalins as biochemical markers of disease. Biochim Biophys Acta 1482: 298–307.Zhang JX, Zhang SF, Wang TD, Guo XJ, Hu RL (2007). Mammary gland expression of antibacterial peptide genes to inhibit bacterial pathogens causing mastitis. J Dairy Sci 90: 5218–5225.
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