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.

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.

0
/ / Recommend

Add to citation manager EndNote|Reference Manager|ProCite|BibTeX|RefWorks

URL: https://www.xmsyxb.com/EN/10.11843/j.issn.0366-6964.2016.04.018

https://www.xmsyxb.com/EN/Y2016/V47/I4/779

References

［1］LECHOWICZ J，KRAWCZYK-BALSKA A.An update on the transport and metabolism of iron in Listeria monocytogenes： the role of proteins involved in pathogenicity［J］.Biometals，2015，28(4)：587-603.
［2］DESHAYES C，BIELECKA M K，CAIN R J，et al.Allosteric mutants show that PrfA activation is dispensable for vacuole escape but required for efficient spread and Listeria survival in vivo［J］.Mol Microbiol，2012，85(3)：461-477.
［3］SCORTTI M，MONZÓ H J，LACHARME-LORA L，et al.The PrfA virulence regulon［J］.Microbes Infect，2007，9(10)：1196-1207.
［4］VASANTHAKRISHNAN R B，DE LAS HERAS A，SCORTTI M，et al.PrfA regulation offsets the cost of Listeria virulence outside the host［J］.Environ Microbiol，2015，17(11)：4566-4579.
［5］STACHOWIAK R，LYZNIAK M，BUDZISZEWSKA B K，et al.Cytotoxicity of bacterial metabolic products，including listeriolysin O on leukocyte targets［J］.J Biomed Biotechnol，2012，2012：954375.
［6］STACHOWIAK R，?Y?NIAK M，GRABOWSKA M，et al.Cytotoxicity of purified listeriolysin O on mouse and human leukocytes and leukaemia cells［J］.BMC Biotechnol，2014，14：77.
［7］PUSHKAREVA V I，ERMOLAEVA S A.Listeria monocytogenes virulence factor Listeriolysin O favors bacterial growth in co-culture with the ciliate Tetrahymena pyriformis，causes protozoan encystment and promotes bacterial survival inside cysts［J］.BMC Microbiol，2010，10：26.
［8］ASSIS P A，ESPÍNDOLA M S，PAULA-SILVA F W，et al.Mycobacterium tuberculosis expressing phospholipase C subverts PGE2 synthesis and induces necrosis in alveolar macrophages［J］.BMC Microbiol，2014，14：128.
［9］CHEN J，XIA Y，CHENG C，et al.Genome sequence of the nonpathogenic Listeria monocytogenes serovar 4a strain M7［J］.J Bacteriol，2011，193(18)：5019-5020.
［10］FANG C，CAO T，CHENG C，et al.Activation of PrfA results in overexpression of virulence factors but does not rescue the pathogenicity of Listeria monocytogenes M7［J］.J Med Microbiol，2015，64(8)：818-827.
［11］BAI F，CHEN J，CHEN Q，et al.A single substitution in 5′-untranslated region of plcB is involved in enhanced broad-range phospholipase C activity in Listeria monocytogenes strain H4［J］.Acta Biochim Biophys Sin (Shanghai)，2011，43(4)：275-283.
［12］江玲丽，陈健舜，李爱云，等.单核细胞增多性李斯特菌弱毒株的生物学特性鉴定［J］.畜牧兽医学报，2014，45(2)：274-280.
JIANG L L，CHEN J S，LI A Y，et al.Characterization of Listeria monocytogenes strains with low virulence［J］.Acta Veterinaria et Zootechnica Sinica，2014，45(2)：274-280.(in Chinese)
［13］HECKMAN K L，PEASE L R.Gene splicing and mutagenesis by PCR-driven overlap extension［J］.Nat Protoc，2007，2(4)：924-932.
［14］ONO T，PARK K S，UETA M，et al.Identification of proteins secreted via Vibrio parahaemolyticus type III secretion system 1［J］.Infect Immun，2006，74(2)：1032-1042.
［15］JIANG L L，XU J J，CHEN N，et al.Virulence phenotyping and molecular characterization of a low-pathogenicity isolate of Listeria monocytogenes from cow’s milk［J］.Acta Biochim Biophys Sin (Shanghai)，2006，38(4)：262-270.
［16］KIM Y R，VAN’T OEVER R，LANDAYAN M，et al.Automated red blood cell differential analysis on a multi-angle light scatter/fluorescence hematology analyzer［J］.Cytometry B Clin Cytom，2003，56(1)：43-54.
［17］FANG C，SHAN Y，CAO T，et al.Prevalence and virulence characterization of Listeria monocytogenes in chilled pork in Zhejiang Province，China［J］.Foodborne Pathog Dis，2015，doi：10.10899/fpd.2015.2023.
［18］JIANG L，CHEN J，XU J，et al.Virulence characterization and genotypic analyses of Listeria monocytogenes isolates from food and processing environments in eastern China［J］.Int J Food Microbiol，2008，121(1)：53-59.
［19］JENSEN V B，HARTY J T，JONES B D.Interactions of the invasive pathogens Salmonella typhimurium，Listeria monocytogenes，and Shigella flexneri with M cells and murine Peyer’s patches［J］.Infect Immun，1998，66(8)：3758-3766.
［20］CORR S，HILL C，GAHAN C G.An in vitro cell-culture model demonstrates internalin-and hemolysin-independent translocation of Listeria monocytogenes across M cells［J］.Microb Pathog，2006，41(6)：241-250.
［21］BURRACK L S，HARPER J W，HIGGINS D E.Perturbation of vacuolar maturation promotes listeriolysin O independent vacuolar escape during Listeria monocytogenes infection of human cells［J］.Cell Microbiol，2009，11(9)：1382-1398.
［22］KÖSTER S，VAN PEE K，HUDEL M，et al.Crystal structure of listeriolysin O reveals molecular details of oligomerization and pore formation［J］.Nat Commun，2014，5：3690.
［23］HERNÁNDEZ-FLORES K G，VIVANCO-CID H.Biological effects of listeriolysin O implications for vaccination［J］.Biomed Res Int，2015，2015：360741.
［24］DECATUR A L，PORTNOY D A.A PEST-like sequence in listeriolysin O essential for Listeria monocytogenes pathogenicity［J］.Science，2000，290(5493)：992-995.
［25］CARRERO J A，VIVANCO-CID H，UNANUE E R.Listeriolysin o is strongly immunogenic independently of its cytotoxic activity［J］.PLoS One，2012，7(3)：e32310.
［26］GLOMSKI I J，DECATUR A L，PORTNOY D A.Listeria monocytogenes mutants that fail to compartmentalize listerolysin O activity are cytotoxic，avirulent，and unable to evade host extracellular defenses［J］. Infect Immun，2003，71(12)：6754-6765.
［27］JIANG L L，SONG H H，CHEN X Y，et al.Characterization of a mutant Listeria monocytogenes strain expressing green fluorescent protein［J］.Acta Biochim Biophys Sin (Shanghai)，2005，37(1)：19-24.

[1]	JIANG Ling-li，CHEN Jian-shun，LI Ai-yun，WAN Jing，ZHOU Xiang-yang，QIAN Guo-ying，LI Cai-yan，FANG Wei-huan. Characterization of Listeria monocytogenes Strains with Low Virulence [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2014, 45(2): 274-280.
[2]	YANG Li-hong, ZHANG Zai-chao, MENG Qing-ling, QIAO Jun, CAI Xue-peng,CAI Kuo-jun, WANG Wei-sheng, WANG Jun-wei, CHEN Chuang-fu. Regulating Role of σ^B Factor in the Environmental Stress Response of Listeriamonocytogenes [J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2013, 44(3): 441-446.

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

PDF (PC)

Knowledge

Abstract

Cite this article

share this article

References

Related Articles 2

Recommended Articles

Metrics

Comments