The Functional Study Of Lmo0964in Listeria Monocytogenes

Listeria monocytogenes, a Gram-positive bacterium, is an important zoonotic foodborne pathogencausing gastroenteritis, septicemia, meningoencephalitis, abortion and other clinical symptoms. InGram-positive bacteria, LPXTG-anchored proteins, toxins, fimbriae and flagella-associated proteins aremodified by disulfide bond formation proteins (Dsb) prior to being transported across the plasmamembrane by the Sec secretion. In L.monocytogenes, Lmo0964and Lmo1059in genome are annotatedas a DsbA-like protein. But the mechanism of how these disulfide bond related proteins beingtransported across the plasma membrane is largely unknown. Therefore, the functions of Lmo0964andSecY were investigated in this study. First of all, SecY (lmo2612) and Lmo0964were expressed by theprokaryotic expression system. The expression level of these two proteins in the E. coli was0.2mg/mland10mg/ml respectively. The antibody against SecY and Lmo0964was prepared in rabbitsubsequently. The SecY and Lmo0964antibody titers reached to1:1000by Western blot analysis.Protease K localization assay showed that Lmo0964was localized in the cytoplasm, which was differentfrom the membrane anchoring DsbA found in Gram-negative bacteria. To further investigate the roles ofLmo0964, an in-frame deletion mutant (lmo0964) was obtained through the homologous recombinationand a series of phenotypes of Δlmo0964were analyzed. The Δlmo0964strain had no growth defect inBHI medium or under low pH condition, but the motility of the mutant was much slower in semi-solidmedium compared with the wild type strain. The Δlmo0964also exhibited weak oxidative stressresistance when exposed with hydrogen peroxide. Cell adhesion and invasion experiments showed thatΔlmo0964had higher adhesion and invasion capibility for Caco-2epithelial cell, and also had higherproliferation ability in the macrophage Raw264.7. These indicate that Lmo0964is likely to play aninhibition role in the infection cycle of L.monocytogenes, while the exact biological role of Lmo0964remained unknown. This study built a solid foundation for future investigations illuminating themechanism of how L. monocytogenes maintains its redox homeostasis by the Dsb and the Sec system.