Evaluation of kinetic stability and anti-staphylococcal activity of recombinant LasA protein produced in Escherichia coli

Document Type : Original Article

Authors

1 Department of Microbiology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

2 Department of Medical Biotechnology, School of Medicine, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

3 Zoonotic Diseases Research Center, Department of Food Hygiene and Safety, School of Public Health, Shahid Sadoughi University of Medical Sciences, Yazd, Iran

Abstract

Objective(s): Staphylococcus aureus has become a major clinical concern due to the growing prevalence of multi-drug resistant (MDR) strains. Enzybioticts are peptidoglycan hydrolases that are recently introduced as an alternative agent to confront the MDR strains with a more effective mechanism than conventional antibiotics.  In this regard, our study aimed to evaluate the kinetic stability of LasA protease as a potent enzybiotic in the specific destruction of the S. aureus cell wall.
Materials and Methods: The catalytic domain of the Codon-optimized LasA gene was sub-cloned into pET28a vector, and BL21 DE3 cells were used for protein expression. Recombinant LasA protein was affinity purified by Ni-NTA column and staphylolytic activity of the LasA protein against methicillin-resistant strains was evaluated by disk diffusion and MIC test. The kinetic stability was evaluated in different temperatures during 48 hr.
Results: Our results revealed that LasA protein can completely prevent the growth of Methicillin-resistant S. aureus (MRSA) strain and inhibit the examined strain at the amount of 4 µg. furthermore, the catalytic domain of LasA protein can tolerate higher temperatures as well.
Conclusion: With regard to the failure of conventional antibiotics in treatment of MRSA infections, novel agents to combat multidrug-resistant strains are needed. The present study shows that LasA protein can eradicate MRSA strains, so it can be promising for the treatment of antibiotic-resistant staphylococci infection. The kinetic stability of LasA has also confirmed the possibility of industrial-scale manufacturing for the subsequent use of the enzyme clinically.

Keywords

References

1. Rao Q, Shang W, Hu X, Rao X. Staphylococcus aureus ST121: a globally disseminated hypervirulent clone. J Med Microbiol 2015; 64:1462-1473.
2. Haaber J, Penadés JR, Ingmer H. Transfer of antibiotic resistance in Staphylococcus aureus. Trends Microbiol 2017; 25:893-905.
3. Alibayov B, Baba-Moussa L, Sina H, Zdeňková K, Demnerová K. Staphylococcus aureus mobile genetic elements. Mol Biol Rep 2014; 41:5005-5018.
4. Kapoor G, Saigal S, Elongavan A. Action and resistance mechanisms of antibiotics: a guide for clinicians. J Anaesthesiol Clin Pharmacol 2017; 33:300-305.
5. Elward AM, McAndrews JM, Young VL. Methicillin-sensitive and methicillin-resistant Staphylococcus aureus: preventing surgical site infections following plastic surgery. Aesthet Surg J 2009; 29:232-244.
6. Appelbaum PC. Microbiology of antibiotic resistance in Staphylococcus aureus. Clin Infect Dis 2007; 45:S165-S170.
7. Hojckova K, Stano M, Klucar L. phiBIOTICS: catalogue of therapeutic enzybiotics, relevant research studies and practical applications. BMC Microbiol 2013; 13:53.
8. Bebell LM, Muiru AN. Antibiotic use and emerging resistance: How can resource-limited countries turn the tide? Glob Heart 2014; 9:347-358.
9. Pai A, Sudhakar G, Kamath V. Enzybiotics-A review. Int J Pharm Res 2013; 3:69-71.
10. Wu H, Lu H, Huang J, Li G, Huang Q. EnzyBase: A novel database for enzybiotic studies. BMC Microbiol 2012; 12:54.
11. Veiga-Crespo P, Sanchez-Perez A, Villa TG. Enzybiotics: The rush toward prevention and control of multiresistant bacteria (MRB). Antimicrobial Compounds: Springer; 2014. p. 215-235.
12. Kessler E, Safrin M, Gustin JK, Ohman DE. Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides. J Biol Chem 1998; 273:30225-30231.
13. Andrews JM. Determination of minimum inhibitory concentrations. J Antimicrob Chemother 2001; 48:5-16.
14. Jaśkiewicz M, Neubauer D, Kazor K, Bartoszewska S, Kamysz W. Antimicrobial activity of selected antimicrobial peptides against planktonic culture and biofilm of Acinetobacter baumannii. Probiotics Antimicrob Proteins 2019; 11:317-324.
15. Sanchez-Ruiz JM. Protein kinetic stability. Biophys Chem 2010; 148:1-15.
16. Ventola CL. The antibiotic resistance crisis: part 1: causes and threats. P T 2015; 40:277-283.
17. Goldberg JB, Ohman DE. Activation of an elastase precursor by the lasA gene product of Pseudomonas aeruginosa J Bacteriol 1987; 169:4532-4539.
18. Schad PA, Iglewski BH. Nucleotide sequence and expression in Escherichia coli of the Pseudomonas aeruginosa lasA gene. J Bacteriol 1988; 170:2784-2789.
19. Szweda P, Schielmann M, Kotlowski R, Gorczyca G, Zalewska M, Milewski S. Peptidoglycan hydrolases-potential weapons against Staphylococcus aureus. Appl Microbiol Biotechnol 2012; 96:1157-1174.
20. Kessler E, Safrin M, Abrams WR, Rosenbloom J, Ohman DE. Inhibitors and specificity of Pseudomonas aeruginosa LasA. J Biol Chem 1997; 272:9884-9889.
21. Barequet IS, Ben Simon GJ, Safrin M, Ohman DE, Kessler E. Pseudomonas aeruginosa LasA protease in treatment of experimental staphylococcal keratitis. Antimicrob Agents Chemother 2004; 48:1681-1687.
22. Barequet IS, Habot-Wilner Z, Mann O, Safrin M, Ohman DE, Kessler E, et al. Evaluation of Pseudomonas aeruginosa staphylolysin (LasA protease) in the treatment of methicillin-resistant Staphylococcus aureus endophthalmitis in a rat model. Graefes Arch Clin Exp Ophthalmol 2009; 247:913-917.
23. Peters JE, Galloway DR. Purification and characterization of an active fragment of the LasA protein from Pseudomonas aeruginosa: Enhancement of elastase activity. J Bacteriol 1990; 172:2236-2240.
Iranian Journal of Basic Medical Sciences
Volume 24, Issue 6
June 2021
Pages 851-855

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