Abstract: The aim of this paper was to construct a lipoteichoic acid synthase ltaS gene deletion strain to investigate the effect of the ltaS gene on the pathogenicity of Listeria monocytogenes. In this study, we used homologous recombination to construct a strain of Listeria monocytogenes with a deletion of the ltaS gene. The effect of the ltaS gene on the growth and bacterial morphology of Listeria monocytogenes was explored by growth curves assay and observation of bacterial morphology. The anchoring of InlA and InlB on the bacterial surface of wild strain EGDe-prfA* and deletion strain ΔltaS were investigated by Western blot assay. The effect of ltaS gene on the ability of Listeria monocytogenes to infest using DF-1 for cell adhesion invasion assay and mice RAW264.7 for phagocytosis assay with multiplication assay was determined. Effect of ltaS gene deletion on pathogenicity of Listeria monocytogenes as assessed by mice organ bacterial load test and survival assay. The results showed: A strain of Listeria monocytogenes with a deletion of the ltaS gene was successfully constructed by homologous recombination technology. Growth curve assay and light microscopic observation revealed that the ltaS gene did not affect normal growth in BHI and bacterial morphology of Listeria monocytogenes. Deletion of the ltaS gene resulted in a significant reduction of the bacterial surface virulence factors InlA and InlB anchor quantification, as confirmed by Western blot analysis. The results of the cell adhesion rate and invasion rate assay showed that the adhesion rate and the invasion rate of ΔltaS to DF-1 was highly significantly lower than that of the parental strain EGDe-prfA* (P<0.01). Phagocytosis assay revealed that the phagocytosis of ΔltaS by RAW264.7 was not significantly different from that of the parental strain EGDe-prfA* (P>0.05), the amount of ΔltaS multiply in RAW264.7 was significantly different from that of the EGDe-prfA* (P<0.05).The results of the mice pathogenicity virulence test showed that the bacterial load in the liver and spleen of mice infected by ΔltaS was significantly lower than that of EGDe-prfA* (P<0.01). And all mice infected with EGDe-prfA* died within 96 h, while the survival rate of mice infected with the ΔltaS was 80%. The LD₅₀ of parental strain EGDe-prfA* was 1.7×10⁴ CFU and the LD₅₀ of deletion strain ΔltaS was 7.49×10⁶ CFU. In summary, ltaS gene deletion significantly reduced the anchoring quantities of surface InlA and InlB and diminished the ability of infection ability, pathogenicity of Listeria monocytogenes.

Key words: Listeria monocytogenes, teichoic acid, lipoteichoic acid synthase, gene deletion, pathogenicity