[Construction and characterization of a mutant strain of Listeria monocytogenes with a deletion of actA and plcB].
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 10(3)-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.


CONCLUSIONS
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. Furthermore, it provided the possibilities to elucidate the molecular mechanisms of pathogenesis and immune response triggered by L. monocytogenes.
Yin
Yuelan
Y
Jiangsu Key Laboratory of Zoonosis, Yangzhou University, Yangzhou 225009, China. lanyy31@yahoo.com.cn
Zhu
Guoqiang
G
Geng
Shizhong
S
Hu
Maozhi
M
Jiao
Xi'nan
X
D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics*; D001428:Bacterial Vaccines; D015153:Blotting, Western; D004274:DNA, Recombinant / Q000235:genetics; D017353:Gene Deletion; D007928:Lethal Dose 50; D008089:Listeria monocytogenes / Q000235:genetics* / Q000276:immunology; D008565:Membrane Proteins / Q000235:genetics*; D051379:Mice; D008807:Mice, Inbred BALB C; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D013154:Spleen / Q000276:immunology; D016176:T-Lymphocyte Subsets / Q000378:metabolism; D010738:Type C Phospholipases / Q000235:genetics*; D037521:Virulence Factors / Q000235:genetics
D001426:Bacterial Proteins; D001428:Bacterial Vaccines; D004274:DNA, Recombinant; D008565:Membrane Proteins; D037521:Virulence Factors; C072980:actA protein, Listeria monocytogenes; D010738:Type C Phospholipases; C105598:phosphatidylcholine-specific phospholipase C