| Listeria monocytogenes(L. monocytogenes, LM) can cause significant foodborne zoonosis. It exists generally in natural environment and food processing environment and resists the acid and salt, which can breed under low temperature. Because it can cause meningitis, septicaemia, gastroenteritis of humans and animals and its mortality approaching 30%, that brings major harm to humans and animals health. The virulence diversity of different source listeria monocytogenes is large, so the study of virulence factor of LM is always the hot spot since The 1980s. Internlin C2 (InlC2) is one of the L. monocytogenes internlin family and exists in the major pathogenic L. monocytogenes. The internlin gene inlC2 is L. monocytogenesspecific and forms an internalin cluster with inlD, inlE and, in some cases, inlG between ascB and dapE to play its function. The recent study shows that InlC2 plays important action in humoral immune together with InlA. To understand the affect of InlC2 in L. monocytogenes infection and the relation with other important internlin gene, the several aspects were studied. The results are as below:1、A PCR-based approach revealed the distribution of internlin gene inlA, inlB, inlC, inlC2, inlD, inlE, and inlG and the structure of the internalin cluster between ascB and dapE from 134 source different L. monocytogenes strains. The results showed that internlin gene inlA and inlB existed in all serovars L. monocytogenes but serovars 1/2b strain S10. Internlin gene inlC existed in all lineageⅠ,Ⅱ(121/121) strains and 53.8%(7/13) lineageⅢstrains. Internlin gene inlD existed in all lineageⅠ(55/55) strains,25.7% (17/66)lineageⅡstrains and 69.2%(9/13)lineageⅢstrains. Internlin gene inlE existed in all lineageⅠ,Ⅱ(121/121) strains and 30.7%(4/13) lineageⅢstrains.InlG did not exist in lineageⅠ(0/55) strains and 74.2%(49/66) lineageⅡstrains and 30.7%(4/13) lineageⅢstrains contained this gene.Among 134 strains L. monocytogenes there were 110 strains (82.1%) harbored inlC2. All lineageⅠ(serovars 1/2b and 4b) strains contained inlC2 which was embedded into the inlC2DE cluster except one serovar 1/2b strain bearing inlGC2DE.Among lineageⅡstrains,47 of the 49 serovar 1/2a strains carried inlC2 within inlC2DE (17/47) or inlGC2DE (30/47) clusters, while only 1 of the 16 serovar 1/2c strains harbored this gene within inlGC2DE. Of 13 lineageⅢstrains,6 contained inlC2 within inlGC2DE cluster (4/6) or other structures (2/6). In addition, other five Listeria species lacked this gene. Overall, the L. monocytogenes-specific internalin gene inlC2 presents in all lineageⅠand majority of serovar 1/2a strains, and forms internalin cluster with other internalin genes inlD, inlE and, in some cases, inlG. The polymorphism of ascB-dapE internalin cluster may be molecule marker for L. monocytogenes lineage/serovar distinguish.2、The homology arms of both ends of inlC2 gene were amplified and fused. InlC2 deletion mutant of L. monocytogenes parent strain LM681 and L10 were constructed by homologous recombination and verified by amphemycin screening, side primer amplification and sequencing.3、To examine the influence of inlC2 deletion mutant to L. monocytogenes virulence,we detected the growth cultivation of L. monocytogenes strain LM681 and L10 and their corresponding inlC2 deletion mutant, the proliferation in BALB/c mouse, ICR mouse virulence test, pathologic anatomy and routine tissue staining. The results revealed that the growth cultivation between parent strain and mutant had no obvious diversity,but the survival in mouse spleen of inlC2 mutant was ten fold than parent strain (P<0.01) and about seven fold increment in the liver than parent strain. The LD50 disparity was not significant in ICR mouse virulence test. The tissue stain results displayed that pathological change caused by inlC2 mutate was obvious than that of parent strain, especially in brain, spleen and liver. This indicated that inlC2 was related with L. monocytogenes pathogenecity to mouse and the loss of inlC2 appeared to enhance the pathogenecity.4.To investigate whether the putative infection-related internalin gene inlC2 contributed to internalization of L. monocytogenes by epithelial cells, we compared the adhesion and invasion abilities of LM681, L10 and LM681-ΔinlC2 and L10-ΔinlC2 in HeLa cells. Interestingly adherence ofΔinlC2 to HeLa cells was 1.3-fold higher than that of the parent strain (P<0.05), and invasion of DinlC2 into HeLa cells was around 2.7-fold higher than that of its parent strain (P<0.01;). A 1.7-fold increase in intracellular growth of inlC2 deletion mutant was also observed. These data indicated that the loss of inlC2 appeared to enhance the internalization of L. monocytogenes in HeLa cells.5. To determine whether the increased internalization of inlC2 mutate was related with the level of inlA and inlB, we evaluated expression of inlA at the transcriptional level by qRT-PCR and at posttranscriptional level by western blot.The results suggested that disruption of inlC2 increased the production of InlA and inlB without changing inlA and inlB transcript level.6. In order to clarify whether inlC2 was infection-related, we tested the transcriptional levels of inlA, inlB, inlC2, inlD and inlE when exposed to synthetic human gastric fluid (PH 2.5) and BHI. Transcriptional analysis showed that levels of inlC2, inlA and inlB transcripts in synthetic human gastric fluid significantly increased as compared to those in BHI (pH 7; P\0.01). In contrast, inlD and inlE transcripts remained unchanged These data support that (1) inlC2 is induced in human gastric fluid, representing another LPXTG internalin gene possibly invovled in the infection, and (2) its expression is monocistronic, independently of inlD and inlE expression.Overall, this study presents supportive evidence that deletion of inlC2 gene may strengthen the pathogenicity of L. monocytogenes and its internalization is a complex network through the interplay of distinct internalins and possibly other infection-related factors, e.g. sortase A (encoded by strA) that is implicated in translocation of proteins bearing LPTGX motif.These study have important significance in theory and practice for exploring deeply the molecule mechanism of functional gene, regulation mechanism, pathogenicity and environmental suitability of L. monocytogenes. |
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