Abstract：Listeria monocytogenes is an important food-borne pathogen due to its widespread distribution. Consumption of food contaminated with this pathogen can lead to listeriosis. This condition has high rates of morbidity and mortality (25%~30% over all). The organism can endure adverse conditions such as freezing, drying, mild heat and anaerobic conditions. It can also grow at temperatures used in refrigeration (-1.5~4 oC). Due to these characteristics, L. monocytogenes is a great concern for food safety. In this study, the expression timing of Listeria virulence genes in the viral growth cycle was studied to understand the basis for food poisoning caused by pathogens and the effectiveness of food safety control mechanisms. The growth curves for the pathogen were measured at OD600, and 27 of the Lm 319 virulence genes were detected by PCR. Quantitative Real-time PCR (qRT-PCR) was used to analyze 12 batches of 10 virulence genes. The growth curve showed that the first 2 hours of L. monocytogenes Lm319 growth was the lag phase and 2~8 h was the exponential growth phase. During 8~18 h, cell growth entered stationary phase, followed by the declining phase after 18 h. PCR results for 27 virulence genes of L. monocytogenes Lm319 showed that they explicitly contained 23 virulence genes. Quantification by qRT-PCR indicated that 10 virulence genes had similar expression patterns in a 24 h period, with peak expression levels at about 16~18 h in late stationary phase, which thereafter declined but increased again in the death phase. At the same time, it was also found that the expression levels of the 10 virulence genes were significantly different, with invasion associated protein gene (iap), fibronectin-binding protein gene (fbp), hexose phosphate transporter gene (hpt) and host factor for RNA phage Qβ replicase gene (hfq) having the highest expression levels, actin polymerizing protein gene (actA) and hemolysin gene (hlyA) having the lowest expression levels. This study demonstrated that the major virulence genes of L. monocytogenes Lm 319 peaked in expression during the late-stationary phase of cell growth and maintained the high expression levels through the death phase. This conclusion provides a theoretical basis for L. monocytogenes detection and prevention.
[1]Bai F, Chen J S, Cheng C Y, Chen Q M, Fang W H.Expression Levels of Major Virulence Genes in a Low-pathogenicity Isolate of Listeria monocytogenes[J].Progress in Veterinary Medicine, 2010, 31(S):108-111
[2]Carrero J A, Calderon B and Unanue E R.Listeriolysin O from Listeria monocytogenesis a lymphocyte apoptogenic molecule [J]. The Journal of immunology, 2004, 172:4866-4874.
[3]Chico-Calero I, Suárez M, González-Zorn B, Scortti M, Slaghuis J, Goebel W, Vázquez-Boland J A; European Listeria Genome Consortium.Hpt,a bacterial homolog of the microsomal glucose-6-phosphate translocase,mediates rapid intracellular proliferation in Listeria[J].Proc Natl Acad Sci USA, 2002, 99(1):431-436
[4]de las Heras A, Cain R J, Bielecka M K, Vázquez-Boland J A.Regulation of Listeria virulence: PrfA master and commander [J]. Curr Opin Microbiol. 2011, 14(2):118-27.
[5]Dramsi S, Bourdichon F, Cabanes D, Lecuit M, Fsihi H, Cossart P.FbpA,a novel multifunctional Listeria monocytogenes virulence factor[J].Mol Microbiol, 2004, 53(2):639-649
[6]Feng Y F, Ran L, Zhang L S.Listeriosis cases reported in medical literatures in China,2000-2009[J].Disease Surveillance, 2011, 26(8):654-659
[7]Freitag N E, Port G C, Miner M D.Listeria monocytogenes from saprophyte to intracellular pathogen [J]. Nat Rev Microbiol, 2009, 7: 623-628.）
[8]Gao X, Lorinczi M, Hill KS, Brooks NC, Dokainish H, Ireton K, Elferink LA.Met receptor tyrosine kinase degradation is altered in response to the leucine-rich repeat of the Listeria invasion protein internalin B[J].J Biol Chem, 2009, 284(2):774-
[9]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
[10]Gründler T, Quednau N, Stump C, Orian-Rousseau V, Ishikawa H, Wolburg H, Schroten H, Tenenbaum T, Schwerk C.The surface proteins InlA and InlB are interdependently required for polar basolateral invasion by Listeria monocytogenes in a human model of the blood-cerebrospinal fluid barrier[J].Microbes and Infection, 2013, 15(4):291-301
[11]Grundling A, Gonzalez M D, Higgins D E.Requirement of the Listeria monocytogenes broad-range phospholipase PC-PLC during infection of human epithelial cells[J].J Bacteriol, 2003, 185(21):6295-6307
[12]Henderson C A, Vincent H A, Casamento A, Stone C M, Phillips J O, Cary P D, Sobott F, Gowers D M, Taylor J E, Callaghan A J.Hfq binding changes the structure of Escherichia coli small noncoding RNAs OxyS and RprA，which are involved in the riboregulation of rpoS[J].RNA, 2013, 19(8):1089-1104
[13]Jiang L L, Chen J S, Li AY, Wan J, Zhou X Y, Qian G Y, Li C Y, Fang W H.Characterization of Listeria monocytogenes Strains with Low Virulence[J].Acta Veterinaria et Zootechnica Sinica, 2014, 45(2):274-280
[14]Lam G Y, Fattouh R, Muise A M, Grinstein S, Higgins D E, Brumell J H.Listeriolysin O suppresses phospholipase C-mediated activation of the microbicidal NADPH oxidase to promote Listeria monocytogenes infection[J].Cell Host Microbe, 2011, 10(6):627-634
[15]Lee H Y, Chai L C, Pui C F, Tunung R, Wong W C, Shuhaimi M, Cheah Y K, Farinazleen M G, Nishibuchi M, Son R.Using RAPD-PCR as molecular assessment on the performance of CHROMAgar? Listeria and PALCAM agar on isolation of Listeria spp. and L. monocytogenes from foods. International Food Research Journa, 2011, 18: 498-503
[16]Le Monnier A, Autret N, Join-Lambert O F, Jaubert F, Charbit A, Berche P, Kayal S.ActA is required for crossing of the fetoplacental barrier by Listeria monocytogenes[J].Infect Immun, 2007, 75(2):950-957
[17]Mandin P, Fsihi H, Dussurget O, Vergassola M, Milohanic E, Toledo-Arana A, Lasa I, Johansson J and Cossart.P. VirR,a response regulator critical for Listeria monocytogenes virulence[J].Mol Microbiol, 2005, 57(5):1367-1380
[18]Mead P S, Dunne E F, Graves L, Wiedmann M, Patrick M, Hunter S, Salehi E, Mostashari F, Craig A, Mshar P, Bannerman T, Sauders BD, Hayes P, Dewitt W, Sparling P, Griffin P, Morse D, Slutsker L Swaminathan B.Nationwide outbreak of listeriosis due to contaminated meat [J]. Epidemiol Infect, 2006, 134:744-751.
[19]Milohanic E, Jonquieres R, Cossart P, Berche P, Gaillard J L.The autolysin Ami contributes to the adhesion of Listeria monocytogenes to eukaryotic cells via its cell wall anchor[J].Mol Microbiol, 2001, 39(5):1212-1224
[20]Peng Y L, Qiao J, Meng Q L, Xie K, Chen C, Liu T L, Ma Y, Cai X P, Chen C F.Cloning and Molecular Characteristics of hfq Gene of Listeria monocytogenes[J].Biological technology, 2014, 24(4):1-4
[21]Pilgrim V, Kolb-Maurer A, Gentschev L, Goebel W, Kuhn M.Deletion of the gene encoding p60 in Listeria monocytogenes leads to abnormal cell division and los of actin-based motility[J]. Infect Immun, 2003a, 71:3473–3484
[22]Régnier P，Hajnsdorf E.The interplay of Hfq,poly (A) polymerase I and exoribonucleases at the 3'ends of RNAs resulting from Rho-independent termination: A tentative model[J].RNA Biology, 2013, 10(4):602-609
[23]Scallan E, Hoekstra R M, Angulo F J, Tauxe R V, Widdowson M, Roy S L, Jones JL, Griffin PM.Foodborne illness acquired in the United States--major pathogens. Emerging infectious diseases, 2011, 17:7-15.
[24]Schraff R L.Economic burden from health losses due to foodborne illness in the United States [J]. J. Food Prot, 2012:75(1): 123-131.
[25]Travier L, Guadaqnini S, Gouin E, Dufour A, Chenal-Francisque V, Cossart P, Olivo-Marin JC, Ghigo JM, Disson O, Lecuit M.ActA Promotes Listeria monocytogenes Aggregation,Intestinal Colonization and Carriage[J].PLoS Pathogens, 2013, 9(1):e1003131-
[26]Wei L, Deng X M, Jiang J J, Yan G Q, Ma X, Wang P Y.Construction and Charactetization of Listeria monocytogenes strain with inlB gene deletion[J].Progress in Veterinary Medicine, 2014, 35(10):19-24
[27]Yeung P S, Zagorski N, Marquis H.The metalloprotease of Listeria monocytogenes controls cell wall translocation of the broad-range phospholipase C[J].J Bacteriol, 2005, 187(8):2601-2608
