Abstract:

The aim of this experiment was to construct a double-deletion strain of antitranscriptional terminator (Lm4b_02325) and unknown protein (Lm4b_02326) in Listeria pathogenicity island 4 (LIPI-4) of foodborne Listeria monocytogenes (LM). In order to study the effect of Lm4b_02325/26 gene on partial biological characteristics of LM. LM928ΔLm4b_02325/26 double-deletion strain was constructed by homologous recombination technology. The D_{600 nm} of LM928 and LM928ΔLm4b_02325/26 double-deletion strains were detected under BHI, different EtOH and NaCl concentrations, different pH and different temperatures, and the growth curves were plotted. The transcription level of some virulence factors of LM in BHI culture were detected by RT-qPCR. The adhesion, invasion and intracellular proliferation of LM-infected murine macrophages RAW264.7 were measured by plate count. And the bacterial load in liver, spleen and brain of C57BL/6 mice infected with LM intraperitoneal injection were detected. The results showed that, a genetically stable double-deletion strain LM928Δ Lm4b_02325/26 was successfully constructed. In the medium containing 0.5%~10% NaCl or 3%~4.5% EtOH, there was no significant difference between the growth of LM928ΔLm4b_02325/26 and LM928. The growth rate of LM928ΔLm4b_02325/26 strain was significantly higher than that of LM928 under the condition of pH5, and the growth rate of LM928Δ Lm4b_02325/26 strain was significantly lower than that of LM928 under the condition of pH9.In BHI medium at different temperatures (4 ℃, 37 ℃, 42 ℃), there was no significant difference between the growth of double-deficient strains and wild strains. The hly, inlC and PlcA genes of LM928Δ Lm4b_02325/26 strain in BHI were significantly upregulated (P < 0.01), while mpl, inlB, actA, inlP, PlcB, prfA, SigB, iap, and inlA genes were significantly downregulated (P < 0.01). The adhesion rate (14.86%) and invasion rate (2.23%) of LM928Δ Lm4b_02325/26 strain to RAW264.7 cells were significantly lower than those of wild strains (with adhesion rate of 22.93% and invasion rate of 4.28%) (P < 0.01). The number of bacteria in RAW264.7 cells infected by LM928Δ Lm4b_02325/26 strain was significantly lower than that of LM928 strain at 3, 6 and 12 h after infection (P < 0.01). In animal experiment, after the infection of mice, there was no significant difference in bacterial load between the two strains in the liver or spleen of mice, but in the brain tissue, the bacterial load of LM928Δ Lm4b_02325/26 strain was 10⁴, which was higher than that of the LM928 10^3.07, and the difference was extremely significant (P < 0.01). In summary, the Lm4b_02325 and Lm4b_02326 genes of LIPI-4 are involved in the expression of LM virulence factors and the colonization ability of brain tissues, and are related to adaptability under weak acid and strong base conditions and the adhesion, invasion and intracellular proliferation of LM to RAW264.7 cells. This study lays a base for the functional study of LIPI-4.

Key words: Listeria monocytogenes, double deletion strain, adhesion invasion, intracellular proliferation, bacterial load, growth curve