单增李斯特菌膜裂解相关基因缺失突变株的构建及生物学特性鉴定

doi:10.11843/j.issn.0366-6964.2016.04.018

Abstract

Abstract:

We attempted to gain insights into the possible mechanism of one Listeria monocytogenes strain M7 with naturally low virulence.Splicing by overlap extension-PCR and homologous recombination were utilized to construct the mutant strains with full deletion of vacuolar lysis related genes hly or (and) plcB.Subsequently，comparisons among mutants and their parent strain M7 together with the reference strain EGD were investigated in terms of hemolytic activity，phospholipase activity，adhesion and intracellular growth in HeLa cells，plaque forming assay in L929 cells，cytotoxicity against Caco-2 cells，as well as virulence to immunocompromised ICR mice.PCR amplification and reverse transcriptional PCR of the target genes indicated the successful construction of the mutants M7-Δhly，M7-ΔplcB and M7-Δhly/plcB.Furthermore，hemolytic and phospholipase activity were devoid in mutants M7-Δhly and M7-ΔplcB，respectively.Neither hemolytic nor phospholipase activity could be seen in the double mutant M7-Δhly/plcB.Besides，mutants M7-Δhly and M7-ΔplcB exhibited similar adhesiveness and intracellular growth in vitro (P＞0.05).Cytotoxicity assay under multiplicity of infection (MOI) ratio 1 000 revealed that the double mutant M7-Δhly/plcB showed the lowest cytotoxic to the host Caco-2 cells with the ratio of 11.10%，followed by mutant M7-Δhly with the ratio of 23.53%.Significant difference could be found between the aforementioned two mutants and other test strains (P<0.05).Plaque forming assay pinpointed that mutant M7-ΔplcB together with its parent strain M7 could form plaques only in the medium without gentamycin.The full deletion of hly or (and) plcB resulted in even less pathogenicity in immunocompromised ICR mice.This study presents some clues as the low virulence of strain M7 probably result from the over-expression of genes hly and plcB.Thereafter，the M7 strain might be exposed to the host immune responses due to its detrimentally stronger cytotoxicity.

Key words: Listeria monocytogenes, vacuolar lysis related genes, cytotoxicity, biological characteristics

CLC Number:

R378.99.4

JIANG Ling-li，GAO You-ling，ZHOU Xiang-yang，BAI Fan，FANG Chun，QIAN Guo-ying，FANG Wei-huan. Construction and Characterization of Mutant Strains from Listeria monocytogenes with Deletion of Vacuolar Lysis Related Genes[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(4): 779-788.

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Construction and Characterization of Mutant Strains from Listeria monocytogenes with Deletion of Vacuolar Lysis Related Genes

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