Abstract:

The aim of this study was to investigate the antibacterial effect of Tae4 amidase, a virulent effector protein of Type Ⅵ secretion systems, on Staphylococcus aureus (S. aureus) and Listeria monocytogenes (L. monocytogenes). The recombinant plasmid pET-28a-Tae4 was transformed into E. coli expression strain BL21 to induce expression, and the induction time, concentration and temperature of IPTG were optimized. Finally, the purified Tae4 protein was tested by Agar pore diffusion test, minimal inhibitory concentration test and antibacterial test combining with antibiotic on S. aureus and L. monocytogenes, respectively. SDS-PAGE results showed that the optimal expression conditions were as follows: the final concentration of IPTG was 0.5 mmol·L^-1 at 16 ℃ for 16 h, the size of the recombinant protein was about 21 ku, and existed in the form of soluble supernatant. The results of Agar pore diffusion test showed that when the concentration of Tae4 protein was 500 μg·mL^-1, S. aureus strains showed bacteriostatic zones above 13mm, and the effect was related to the serotype of strains, and L. monocytogenes strains showed bacteriostatic zones above 18 mm. Minimum inhibitory concentration test showed that MIC of Tae4 against S.aureus was about 250 μg·mL^-1, MIC of Tae4 against L. monocytogenes was about 125 μg·mL^-1, which were consistent with spot plate method and microbroth dilution method. The combination test showed that the bacteriostatic effect of Tae4 protein combined with penicillin was better than that of penicillin alone. Furthermore, the antibacterial effect of L. monocytogenes was better than that of S. aureus. In summary, Tae4 protein has bacteriostatic effect on S.aureus and L. monocytogenes, and the bacteriostatic effect combined with penicillin has been enhanced, which provides a theoretical basis for subsequent antibiotic substitution.

Key words: Tae4, type Ⅵ secretion system, S. aureus, Listeria monocytogenes, alternative to antibiotics