Nucleotide Sequence And Phylogenetic Analysis Of LIPI-1Gene Cluster And Pathogenic Analysis From Three Listeria Monocytogenes With Different Virulence

Listeria monocytogenes(Lm) is an important food-born pathogen that can cause septicaemia,encephalitis, meningitis and gastroenteritis in humans,with the mortality about25%. Listeriamonocytogenes encompasses a diversity of strains with varping pathogenic potential. While manyListeria monocytogenes strains could be of high pathogenicity, others are less virolent or evenavirulent. Contamination to food by Listeria monocytogenes and its serious consequence havegained considerable attention in the worldwild.For the study of analyze the genetic relationship of Lm virulence genes, and explore themolecular mechanism of its virulence attenuation. Six LIPI-1gene clusters from three LM isolateswith different virulence and serotypes were amplified by PCR and subsequently cloned. Based onthe analysis of the sequence of three LM isolates with significant virulent difference, the molecularcharacterization and genetic variance were analyzed as follows. Sequence analysis and phylogenyamong the six LIPI-1sequencesin this study and others already published clearly demonstratedthatthey had different sequence identities; Comparing attenuated isolate N21with the other two strains,the regulatory sequences prfA have only one silent mutations;The virulence attenuation of stainN21may be due to one amino acid change on the PEST motif of hly sequence. The stability codingregion of mpl has an amino acid change;It is shown that actA sequence difference of the twoisolates is significant, Comparing Lmo0586and N13with the attenuated strain N21,105bpnucleotide was missing in proline-rich region;After prediction and analysis of signal peptides of theLIPI-1of three isolates, except prfA, proteins of other five genes have an average of amino acidslocated at the N-terminal region. Only ActA protrin have transmembrane region in C-terminusregion, and the transmembrane region position of the three isolates position was consistent.For the study of the pathogenic differences of different virulence LM on the body, based onthe analysis of the virulence phenotype of two LM isolates with significant virulent difference,establishing the different virulence of the same strain and the same virulence of different strains ofmouse infection model(group A, low-dose attenuated strain N21, group B, median lethal doseattenuated strain N21, group C median lethal dose strength strain Lmo0586, group D, high-dosestrength strain Lmo0586): The distribution in artificial infectious mice disease model for Lm wasmonitoring; The histopathological of the mice was observed; The liver cell apoptosis was detectionby TUNEL; In order to study the roles of caspase8、caspase3、NF-κB、bax、bcl-2in liver cellapoptosis, we have established a real-time PCR to detect mRNA expression by SYBR Green I; The expression of IL-12、TNF-α、INF-γ was detected in the cultured supernatant of serum of the miceinfected with different virulence isolates. The results show that, comparing virulent isolateLmo0586with attenuated isolate N21, the latter have similar hemolytic activity and greater fatsoluble activity; In group D, Lm DNA could be detected in artificial infectious mouse at2hfollowing infection, it each had one mouse could not detect Lm DNA in artificial in group A andgroup B at4h following infection, Lm DNA couble be detected in the encephalon at6h followinginfection in one mouse of group D, it had one subject mice could not detect Lm DNA inencephalon in group at2d following infection, it showed that the different virulence Lm haddifferent migration in mice; Morphological observation showed that significant histoPathologicalchanges were observed following infection, the degree of injury and bacterial virulence waspositively correlated; it showed that the four groups of mice had different degrees of liver apoptosiswith TUNEL; it showed that the detection method was established successfully and verified,all thestandard curve correlation coefficient (r2) was between0.99-1, all the amplification efficiency wasbetween0.8and1.2, indicating that the Q-PCR had system stability, the result had high credibility,so we could analysis dates by double standard curve methods; Four groups the mice the expressionamount of caspase-8had similar trend in the four groups of the mice, the difference was notsignificant (p>0.05), group D was reaching peak at1d following infection,it was faster than theother three groups, group had lower expression amount than others, The relative expressionchanges of caspase3is similar to caspase8, the change trend of NF-κB was relatively flat, and itwas contrary to the first two apoptotic factors, especially, neither bax and bcl-2did increasesignificantly(p>0.05), Lm-induced liver apoptosis dependented death receptor pathway, but did notcause severe mitochondrial apoptosis; in the serum, after Lm infected, the expression of IL-12hadsimilar trend in group A,B,C, was reaching peak at8h, group D was reaching peak at4h, theexpression of TNF-α had similar trend in the four groups of the mice(p>0.05)，and INF-γ even hada relatively high expression level in the late stage of infection, it showed that the attenuated straincan also activate a certain intensity innate immunity.The aim of this study is to analyze the genetic relationship of Lm virulence genes, and explorethe molecular mechanism of its virulence attenuation. Six LIPI-1gene clusters from three LMisolates with different virulence and serotypes were cloned; Based on the analysis of the virulencephenotype of two LM isolates with significant virulent difference, studyed pathogenic differenceswith different virulence of Lm. In summary, results from these studies have laid good foundationfor researching attenuated mechanism of Lm and developmenting of new vaccine vector.