Recombinant expression and antimicrobial activity of Aureocin A53 against Staphylococcus aureus isolated from bovine intramammary infections

Document Type : Full paper (Original article)

Authors

1 Biotechnology Center of the Litoral, Faculty of Biochemistry and Biological Sciences, National University of the Litoral, University City, S3000ZAA, Santa Fe, Argentina

2 Department of Physics, Faculty of Biochemistry and Biological Sciences, National University of the Litoral, University City, S3000ZAA, Santa Fe, Argentina

3 Dairy Chain Research Institute (INTA-CONICET), Rafaela Agricultural Experimental Station, National Institute of Agricultural Technology (INTA), Route 34, Km 227, Rafaela 2300, Santa Fe, Argentina

4 Department of Public Health, Faculty of Veterinary Sciences, National University of the Litoral, Kreder 2805, Esperanza 3080, Santa Fe, Argentina

5 Laboratory of Molecular Biology and Applied Immunology, Faculty of Biochemistry and Biological Sciences, National University of the Litoral, University City, S3000ZAA, Santa Fe, Argentina

6 Laboratory of Experimental Immunology, Faculty of Biochemistry and Biological Sciences, National University of the Litoral, University City, S3000ZAA, Santa Fe, Argentina

Abstract

Background: Bovine mastitis causes major economic losses in dairy production. Staphylococcus aureus, the most prevalent causative pathogen of mastitis, is often resistant to antibiotics. Bacteriocins, like Aureocin A53 (A53), are antimicrobial peptides that can provide an alternative treatment. Aims: The present study aimed to express a recombinant peptide (A53) in Escherichia coli and evaluate its antimicrobial activity against S. aureus isolated from bovine mastitis. Methods: The A53 gene was cloned into pET24a and expressed in E. coli strains BL21(DE3), Tuner (DE3), and Rosetta (DE3), in different induction conditions. Results: Among all the evaluated conditions, A53-pET24a in E. coli Rosetta (DE3) induced with IPTG 0.2 mM overnight appeared to yield slightly higher expression levels than the other conditions tested. Cell lysates supernatants containing recombinant A53 showed inhibitory activity against two S. aureus indicator strains in agar diffusion assays. Microtiter plate assays conducted with S. aureus isolated from bovine mastitis showed that purified recombinant A53 significantly reduced bacterial growth after 24 h of incubation, in comparison with non-treated cultures. Conclusion: Our results underscore the feasibility of producing active recombinant A53. Further studies are needed to evaluate its effectiveness against additional S. aureus isolates and to optimize expression and recovery processes. Improving production scalability will be essential to evaluate the stability and efficacy of peptide in in vivo models, such as the bovine mammary gland, thereby supporting its potential therapeutic application.

Keywords

Main Subjects

References

Acedo, JZ; van Belkum, MJ; Lohans, CT; Towle, KM; Miskolzie, M and Vederas, JC (2016). Nuclear magnetic resonance solution structures of lacticin Q and aureocin A53 reveal a structural motif conserved among leaderless bacteriocins with broad-spectrum activity. Biochemistry, 55: 733-742.
Brunelle, JL and Green, R (2014). One-dimensional SDS-polyacrylamide gel electrophoresis (1D SDS-PAGE). Methods Enzymol., 541: 151-159.
Ceotto, H; Brede, D; Salehian, Z; Dos Santos Nascimento, J; Fagundes, PC; Nes, IF and Do Carmo De Freire Bastos, M (2010). Aureocins 4185, bacteriocins produced by Staphylococcus aureus 4185: potential application in food preservation. Foodborne Pathog. Dis., 7: 1255-1262.
Fagundes, PC; Farias, FM; Santos, OCS; De Oliveira, NEM; Da Paz, JAS; Ceotto-Vigoder, H and Do Carmo De Freire Bastos, M (2016). The antimicrobial peptide aureocin A53 as an alternative agent for biopreservation of dairy products. J. Appl. Microbiol., 121: 435-444.
Guryanova, SV (2023). Immunomodulation, bioavailability and safety of bacteriocins. Life, 13: 1521.
Hammond, K; Lewis, H; Halliwell, S; Desriac, F; Nardone, B; Ravi, J and Ryadnov, MG (2020). Flowering poration—A synergistic multi-mode antibacterial mechanism by a bacteriocin fold. iScience, 23: 101423.
Jaouani, I; Abbassi, MS; Alessandria, V; Bouraoui, J; Ben Salem, R; Kilani, H and Cocolin, L (2014). High inhibition of Paenibacillus larvae and Listeria monocytogenes by Enterococcus isolated from different sources in Tunisia and identification of their bacteriocin genes. Lett. Appl. Microbiol., 59: 17-25.
Krishnamoorthy, P; Suresh, KP; Jayamma, KS; Shome, BR; Patil, SS and Amachawadi, RG (2021). An understanding of the global status of major bacterial pathogens of milk concerning bovine mastitis: A systematic review and meta-analysis. Pathogens, 10: 545.
Lander, AJ; Mercado, LD; Li, X; Taily, IM; Findlay, BL;
Jin, Y and Luk, LYP (2023). Roles of inter- and intramolecular tryptophan interactions in membrane-active proteins revealed by racemic protein crystallography. Commun. Chem., 6: 154.
Marques-Bastos, SLS; Coelho, MLV; de Sousa Santos, IN; Moreno, DSA; Barrias, ES; de Mendonça, JFM and Do Carmo de Freire Bastos, M (2022). Effects of the natural antimicrobial peptide aureocin A53 on cells of Staphylococcus aureus and Streptococcus agalactiae involved in bovine mastitis in the excised teat model. World J. Microbiol. Biotechnol., 39: 5.
Molineri, AI; Camussone, C; Zbrun, MV; Suárez Archilla, G; Cristiani, M; Neder, V and Signorini, M (2021). Antimicrobial resistance of Staphylococcus aureus isolated from bovine mastitis: Systematic review and meta-analysis. Prev. Vet. Med., 188: 105261.
Netz, DJA; Bastos, MDCDF and Sahl, HG (2002b). Mode of action of the antimicrobial peptide aureocin A53 from Staphylococcus aureus. Appl. Environ. Microbiol., 68: 5274-5280.
Netz, DJA; Pohl, R; Beck-Sickinger, AG; Selmer, T; Pierik, AJ; Bastos, MDCDF and Sahl, HG (2002a). Biochemical characterization and genetic analysis of aureocin A53, a new, atypical bacteriocin from Staphylococcus aureus. J. Mol. Biol., 319: 745-756.
Novagen (2011). pET system manual. 11th Edn., Madison, WI, USA, Novagen. PP: 33-38.
Parachin, NS; Mulder, KC; Viana, AAB; Dias, SC and Franco, OL (2012). Expression systems for heterologous production of antimicrobial peptides. Peptides, 38: 446-456.
Perez, RH; Zendo, T and Sonomoto, K (2018). Circular and leaderless bacteriocins: Biosynthesis, mode of action, applications, and prospects. Front. Microbiol., 9: 2085.
Rosano, GL and Ceccarelli, EA (2014). Recombinant protein expression in Escherichia coli: Advances and challenges. Front. Microbiol., 5: 172.
Sivashanmugam, A; Murray, V; Cui, C; Zhang, Y; Wang, J and Li, Q (2009). Practical protocols for production of very high yields of recombinant proteins using Escherichia coli. Protein Sci., 18: 936-948.
Soltani, S; Hammami, R; Cotter, PD; Rebuffat, S; Said, LB; Gaudreau, H and Fliss, I (2021). Bacteriocins as a new generation of antimicrobials: Toxicity aspects and regulations. FEMS Microbiol. Rev., 45: fuaa039.
Studier, FW (2005). Protein production by auto-induction in high density shaking cultures. Protein Expr. Purif., 41: 207-234.
Varella Coelho, ML; Santos Nascimento, JD; Fagundes, PC; Madureira, DJ; Oliveira, SSD; Vasconcelos De Paiva Brito, MA and Do Carmo De Freire Bastos, M (2007). Activity of staphylococcal bacteriocins against Staphylococcus aureus and Streptococcus agalactiae involved in bovine mastitis. Res. Microbiol., 158: 625-630.

Files

Share

How to cite

Statistics