Functional Key Sites Identification Of Listeriolysin O And Surface Display System Construction Using Listeria Monocytogenes As A Model Strain

Listeria monocytogenes(LM) is a gram-positive and food-borne intracellular pathogen. The LPXTG motif containing proteins are anchored on the membrane of bacterial cell wall through sortase system. Listeriolysin O(LLO) is a pore-forming toxin belonging to the family of cholesterol-dependent cytolysins(CDCs). Furthermore, LLO could form a diameter of 35 nm pore under the free condition and insert into the host cell membrane, which plays a critical role during LM infection.Here, to characterize the Listeriolysin O(LLO), the gene hly encoding LLO was modified at genomic level to obatain LPXTG motif by using homologous recombination and amino acid mutagenesis approaches. Thus, LLO deletion mutant(ΔLLO) and recombination LLO strain containing LPXTG motif(LLOLPXTG) were constructed. The expression of LLO and LLOLPXTG was analyzed by Western blot. Deletion of hly totally impaired LLO expression, indicating that ΔLLO and LLOLPXTG strains were successfully constructed. The intracellular growth ability of EGD-e ΔLLO and LLOLPXTG strains significantly compromised in RAW264.7. Hemolytic activities of M7 ΔLLO and LLOLPXTG strains were completely lost. Moreover, the recombinant LLO and its derivatives were expressed and purified from E.coli, and showed that only the mutant LLO_N478A/V479 A completely imparied the hemolytic activity. We further showed that LLO contributed to bacterial proliferation inside host cell. Most importantly, we, for the first time, found and confirmed that the N478/V479 of LLO are the key amino acid sites, which are critical to LLO activity.In conclusion, we demonstrate that the amino acids N478V479 of LLO play critical roles in hemolytic activities. The LLOLPXTG strain provides a valuable tool for intracellular antigen presenting, which could be applied to the prevention and treatment of viral diseases, shedding light on the development of intracellular bacteria surface display techniques in presenting viral antigens inside host cell.