The InlF Protein Plays Important Roles In Virulence Of Serotype 4b Listeria Monocytogenes

Listeria monocytogenes(Lm) is an important food-borne pathogen, which can cause zoonotic listeriosis with high mortality, its ability to cross the intestinal, blood-brain, maternofetal barriers and thereby to disseminate in host is closely related to virulence factors. Internalins family, one of the major virulence determinants, induces internalization of Lm into non-phagocytic cells, thus plays key role in the adhesion and invasion, therefore, the intense research on the mechanism of internalins has great significance on revealing the pathogenecity and control of listeriosis. InlF is a 823-amino acid protein with unknown role, its amino acid sequence varies among the strains isolated from clinical rhombencephalitis, food and environment. In this study, the InlF protein of serotype 4b LmNTSN isolated from sheep brain with listeriosis was investigated in non-phagocytic cells, mice and guinea pigs model as well as its potential function on pathogenesis, which laid the theoretical foundation for defining the pathogenecity of Lm.1. The transcriptional level analysis of internalin genes during Lm infections in vivo and in vitroIn this study, the relative transcriptional level of internalin genes(inlA, inlB, inlC, inlC2, inlD, inlF, inll, inlJ and vip) in Lm was assessed by real-time PCR in vitro infection of five non-phagocytic cells (human intestinal epithelial cell line Caco-2, human hepatocyte cell line HepG2, human brain endothelial cell HBMEC, murine hepatocyte cell line BRL3A and murine fibroblast cell line NIH3T3), and in vivo infection of BALB/c mice and guinea pigs. Results showed that the expression of internalin genes(inlF, inll, inlJ and vip) up-regulated significantly after in vitro infection of five non-phagocytic lines except HBMEC. While in oral infection, a concurrent transcription of internalin genes was observed in spleens and livers from mice and guinea pigs. By contrast, transcription in brain was more diverse, inlF, inlI and vip were up-expressed in guinea pigs only. In conclusion, it suggested that inlF gene plays an important role in Lm infections in vitro and in vivo apart from mice brains, and pathogenesis of serotype 4b Lm is great variation between mice and guinea pigs.2. The bioinformatics analysis and prokaryotic expression of internalin InlF proteinThe inlF gene of LmNTSN was amplified by PCR, the structure and function of InlF protein was predicted with bioinformatics software in this study. Results showed that InlF was a surface protein containing a signal peptide of 35 amino acids on its N-terminal, followed by curved LRR domain with 13 leucine-rich repeats (LRRs), and C-terminal LPXTG motif recognized by the sortase A (SrtA) enzyme, which covalently anchors InlF to the cell wall. The amino acids sequence of InlF in LmNTSN was analyzed with MEGA5. The results displayed that the InlF protein in LmNTSN was highly homologous to the strains belonging to lineage I, while it was only 78% homologous with strains belonging to lineage II. The inlF gene was cloned into the prokaryotic expression vector pCold, the expression of InlF protein was identified by SDS-PAGE and Western blotting. The results proved that the InlF protein was expressed in the form of inclusion body, which could specifically react with anti-LmNTSN polyclonal antibody. The immunogenicity of InlF protein combination with Freund’s adjuvant was evaluated in BALB/c mice, indirect ELISA result confirmed that the InlF protein could react the serum antibodies against InlF with a titer of 1:800. The prokaryotic expression and preparation of polyclonal antibody provided the foundation for further studying the InlF protein.3. Construction and biological characteristics of LmNTSN △inlF mutant strainThe recombinant bacteria LmNTSN △inlF was obtained by homology recombination. The inlF gene of the mutant strain couldn’t be efficient transcription as shown in RT-PCR assay, and couldn’t react with anti-InlF polyclonal antibody identified with Western blotting assay. The growth curve, physiological and biochemical characteristics of the wide type (WT) strain and deletion mutant (LmNTSN △inlF) were measured, the results revealed that the deletion of inlF gene was not affect the growth and metabolism of Lm.Adhesion and invasion of Lm in the five humanized and murine non-phagocytic cells (human intestinal epithelial cell line Caco-2, human hepatocyte cell line HepG2, human brain endothelial cell HBMEC, murine hepatocyte cell line BRL3A and murine fibroblast cell line NIH3T3) were evaluated in vitro. Results demonstrated that there was no significant difference between the mutant and WT strains within adhesion and invasion ability to non-phagocytic cells. Furthermore, the infection ability to intestine in vivo and ex vivo of the WT strain and LmNTSN △inlF was analyzed. Interestingly, the colonization of LmNTSN △inlF in intestine reduced significantly after 1h post-infection (P<0.05). LmNTSN △inlF colonized in the Peyer’s patches and mesenteric lymph nodes after 3h and 5h post-infection, respectively. Moreover, the colonization of LmNTSN △inlF in mesenteric lymph nodes decreased significantly after 6h post-infection (P<0.05). The above results suggested that InlF protein plays a vital role in colonization during the early phase of intestine infection.BALB/c mice and guinea pigs were inoculated via orally infection route with WT strain and △inlF respectively to compare the pathogenicity. Results showed that LmNTSN △inlF strain displayed 2-logio decreases in colonization of mice liver (P<0.05) after 4h post-infection compared to the WT strain, while for spleen, intestine and mesenteric lymph nodes, no significant difference was observed between the WT strain and LmNTSN △inlF. Besides, there was no difference between the two strains in colonization of mice after 72h infection. The LmNTSN △inlF strain showed 1-and 1.5-logio decreases in liver and spleen of guinea pigs, respectively after 6h post-infection (P<0.05, P<0.01). The bacterial counts of the LmNTSN △ inlF strain reduced by 2-log10 in liver and spleen of guinea pigs (P<0.001) after 72h post-infection. The results of histopathologic sections also confirmed the reduced virulence of LmNTSN △inlF in guinea pigs. Above all, the InlF protein makes contribution to colonization and pathogenicity during crossing the intestinal barrier with infection in guinea pigs.In conclusion, the research on InlF protein of LmNTSN demonstrated that it posed a great influence on adhesion and invasion during crossing the intestinal barrier of mice and guinea pigs. Furthermore, comparing with mice, InlF protein played a more important role in colonization of spleen and liver of guinea pigs. The results above indicated that InlF protein is an important virluence factor of serotype 4b strains. The study on function of InlF protein laid the theoretical foundation for researching its mechanism during crossing the intestinal barrier and blood-brain barrier.