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Objective To analyze the activity of ribose-5-phosphate isomerase B (RpiB) encoded by lmo0736 and explore its effect on the infection of Listeria monocytogenes (LM). Methods The recombinant protein Lmo0736 was obtained by prokaryotic expression and purification, and its catalytic activity for substrates was verified by the enzyme activity assay. The LM strain with lmo0736 knockout (LM Δlmo0736) and the complementary strain (LM CΔlmo0736) were constructed by bacterial homologous recombination. The growth curves of bacteria in vitro were plotted. The adhesion, invasion, and intercellular migration of bacteria were evaluated by in vitro cell infection models (Caco-2 intestinal epithelial cells and L929 fibroblasts). The ICR mouse infection model was used to measure the 7 d survival rate and 48 h organ load of each strain, and thus the pathogenicity of strains in mice was evaluated. Results Lmo0736 had typical RpiB activity and catalyzed the conversion of d-ribose-5-phosphate to d-ribulose-5-phosphate, with Vmax=0.366 mmol/(L·min), Km=4.489 mmol/L, kcat=12.300 s-1, and kcat/Km=2.740 L/(mmol·s). The growth rate of LM Δlmo0736 was not significantly different from that of the wild type EGD-e and LM CΔlmo0736in vitro, indicating that the deletion of lmo0736 did not affect the basic growth of bacteria. LM Δlmo0736 demonstrated significantly decreased adhesion and invasion in Caco-2 cells and intercellular migration in L929 cells and weakened colonization in mice, which indicated that lmo0736 regulated the pathogenicity of LM through a RpiB-dependent metabolic pathway. Conclusion This study reveals for the first time that the Lmo0736 of LM has typical RpiB activity. Although the functional loss of Lmo0736 does not directly affect the basic growth of the bacteria, it significantly attenuates the pathogenicity by weakening the adhesion, invasion, and intracellular migration in host cells and the colonization in vivo. The results accumulate experimental data for in-depth exploration of the biological functions of RpiB in LM. From the perspective of the association between metabolism and virulence, this study provides an experimental basis for delving into the infection mechanism of foodborne pathogens.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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