Roles of key amino acid residues of the virulence factor listeriolysin O in Listeria monocytogenes infection

Abstract:

[Objective] To investigate the impacts of the amino acid residues at position 253 (glutamine, Q) and 254 (isoleucine, I) in the β8 sheet of the D3 domain of listeriolysin O (LLO) on the biological functions of Listeria monocytogenes. [Methods] We constructed the mutant proteins LLO_Q253A and LLO_I254A and the mutant strains hly_Q253A and hly_I254A by homologous recombination. After the expression and purification, the mutant proteins examined for the hemolytic activity. Furthermore, the growth, adhesion, invasion, intracellular migration, and proliferation were compared between the mutant strains hly_Q253A and hly_I254A. [Results] After the mutation of the corresponding sites, LLO proteins could be expressed normally. However, the mutant proteins and strains lost hemolytic activity at pH 6.5, and the hemolytic activities of LLO_I254A and hly_I254A were restored at pH 5.5. The mutant strains showed no significant differences in extracellular growth, adhesion, and intracellular proliferation compared with the wild-type strain. However, the invasion and intercellular migration of the mutant strains were significantly lower than that of the wild-type strain. [Conclusion] The mutations of Q253A and I254A in LLO cause the loss of hemolytic activity at pH 6.5 and a reduction in the bacterial infection, the specific mechanisms of which remain to be explored. This study establishes a foundation for deeply understanding the impact of LLO structure on the biological function of L. monocytogenes and holds significance for the construction of point-mutated strains of L. monocytogenes.