Construction and characterization of a mutant strain of Listeria monocyto-genes with a deletion of actA and plcB

Abstract:

The virulence of Listeria monocytogenes is directly related to its ability to spread from cell to cell without leaving the intracellular milieu. Among the bacterial factors involved in cell-to-cell spread, actin-polymerizing protein ActA is required for bacterial spread to adjacent cells, while the broad-range phospholipase C (PC-PLC) contributes to bacterial escape from secondary vacuoles. [Methods] Based on homologous recombination technology, we constructed a double gene-mutant strain by deleting two virulence factors, ActA and PC-PLC, [Objective] its toxicity and immunopotency was evaluated in murine model. [Results] And then, the mutant strain was further verified the absence of ActA and PC-PLC using Western blot analysis and phospholipase activity assay. It showed highly attenuated and its 50% lethal dose in BLAB/c mice was increased at least 103-fold compared to the parent strain. Nevertheless, mice preimmunized with the mutant strain elicited strong T-cell responses and fully protected against a lethal challenge with the virulent strain. [Con-clusion] These results not only verified that ActA and PC-PLC were essential virulence factors for L. monocytogenes, but also demonstrated that the mutant strain possessed good immunogenicity with higher safety. The mutant strain could be further studied as a vaccine candidate to prevent listeriosis and used as a vector to deliver heterologous antigens. Fur-thermore, it provided the possibilities to elucidate the molecular mechanisms of pathogenesis and immune response trig-gered by L. monocytogenes.