Characterization Of Lmo1059, A DsbA Like Protein, In Listeria Monocytogenes

Listeria monocytogenes (Lm) is a Gram-positive intracellular foodbornepathogen, causinglisteriosis in human and animals. It can break through the host’s gastrointestinal barrier,blood-brain barrier, placental barrier and proliferate in the cells. Lmfaces a variety of oxidativestresses in the gastrointestinal tract of the host. How Lmmaintains the disulfide homeostasis underoxidative stress conditions in host niche environment is still largely unknown. Lmo1059is adisulfide bond forming protein (DsbA-like), containing a typical CXXC motif, as annotated in thesequenced genome. Therefore, to unveil the survival mechanism of Lm under oxidative stressconditions, the function of Lmo1059was characterized in this study. Firstly, Lmo1059wasexpressed in the prokaryotic expression system and purified by chromatography. The disulfideisomerase activity of Lmo1059was analyzed in the presence of dithiothreitol (DTT) as a substrate.The Michaelis-Menton constant (Km) of Lmo1059isomerase catalytic activity was22.94mM.The disulfide reductase activity of Lmo1059was evaluated by insulin reduction assay. Thisshowed that Lmo1059had no disulfide reductase activity. To further investigate the function ofLmo1059in vivo, the lmo1059in-frame deletion mutant was constructed by homologousrecombination. No growth defect occurred in solid, semi-solid and liquid medium for lmo1059mutant compared with the wild type strain. Furthermore, hydrogen peroxide (H2O2) stressexperiment showed that lmo1059had no significant phenotype alternations in the presence ofH2O2stress, indicating that Lmo1059might not be involved in repair of hydrogenperoxide-induced DNA/protein damage. The adhesion and invasion abilities of lmo1059strainto epithelial cellCaco-2and macrophage Raw264.7were investigated, respectively. No significantdifferences between lmo1059and the wild type strain were found for adhesion and intracellularsurvival. Therefore, Lmo1059might not be a regular DsbA-like protein as annotated in genome,but rather an isomerase, needing to be investigated further. Nevertheless, the Dsb system of L.monocytogenes, lmo1609, lmo1903and lmo2830in-frame deletion mutants were also constructedin this study. This will pave the way for a more detailed exploration of Dsb-like proteins in theantioxidative defense mechanisms of the intracellular Lm.