| Haemophilus parasuis is a commensal Gram-negative bacterium in the swine upper respiratory tract, but virulent strains can invade host and cause severe Glasser’s disease under certain conditions, such as stress, hypoimmunity, and coinfection with other pathogens, characterized by fibrinous polyserositis, arthritis and meningitis. H. parasuis has emerged gradually as a major cause of morbidity and mortality in the pig industry. So far, however, the virulence factors and the mechanism of the pathogenicity in H. parasuis are incompletely understood due to the backwardness and the lack of the effective genetic manipulation system, which impedes the prevention and development of the novel vaccines against this disease.In the current study, we selected two H. parasuis isolates from clinical diseased pigs, SH0165(serotype5) and7140(serotype4), and demonstrated their significant difference on the pathogenicity through the animal infection, between which SH0165was a highly virulent strain and7140was low virulent. By the approach of genomic subtraction hybridization, PCR and dot-blot verification of the subtractive library,33subtractive fragments were finally shown to be present in the highly virulent strain SH0165, while absent from low virulent strain7140. Having analyzed these33subtractive gene fragments by sequencing, we found that they can be classified into8categories, including the sclB family, restriction modification system, phage-related products, transport system, outer membrane proteins, metabolism, and DNA synthesis and insertion sequences. We next investigated the distribution of21fragments excluding the phage-related products in15H. parasuis reference strains and42clinical isolates with different virulence background. The data showed that six genes, including sclB4, sclB7, sclBll, nhaC, ABC-type transport system and fhuA, were more prevalent in highly virulent strains than that in the strains with low virulence based on the statistical analysis, indicating a potential association between these differential genes and the virulence in H. parasuis strains.Combined with other existing reports involving the identification of H. parasuis virulence factors, we chose a novel factor, capD gene, for further functional investigation. The capD gene encodes a polysaccharide biosynthesis protein, this protein shares52%amino acid identity with the protein WbfY of Vibrio vulnificus, which is encoded by the group1capsular polysaccharide operon, suggesting a possible involvement of the CapD protein in the function of the capsular polysaccharide. The capD gene has been identified previously as a differential gene between H. parasuis virulent strain Nagasaki and avirulent strain SW114. Here, we did not find this gene in our genomic subtractive library, but our following PCR demonstrated that this capD gene existed in strain SH0165but not in7140, indicating that capD is a potential virulence-related gene. However, the characteristics of this gene involving in H. parasuis infection remain unclear. In the current study, by construction of the capD gene deletion mutant△capD and its complement strain C-capD, we provided the first direct evidence that capD is involved in the pathogenicity of H, parasuis. This was shown by the demonstration that (1) The deletion of capD gene significantly attenuated the virulence of the wild type strain SH0165, while the gene complementation largely recovered its pathogenicity to piglets.(2) After the challenge of the wild type strain SH0165, deletion mutant△capD and the complement strain C-capD, both SH0165and C-capD strains were able to invade the systemic tissues, and the bacteria can be recovered from most tissues. But the mutant△capD could not invade and cause systemic infection; thereby no bacteria were successfully recovered. This indicates a decreased invasiveness of the strain after capD deletion.(3) The△capD strain exhibited an extreme sensitivity to the serum complement-mediated killing compared with wild type SH0165, while its serum-resistance ability largely restored with the capD gene complementation. Taken together, these data present the evidence that the capD gene is a novel pathogenicity-associated determinant and involved in the serum-resistance ability of H. parasuis. Noticeably, our following transmission electron microscopy implied that this gene may be only related to the structure of the capsular polysaccharide, but not the presence or absence of the capsule, indicating that the involvement of CapD protein in serum-resistance ability of the strain may not be totally related to the capsule. Subsequently, we compared the proteomic differences between the wild type SH0165and the△capD mutant by the proteome two-dimensional electrophoresis, and identified18proteins that were up-regulated and18down-regulated after capD deletion. Further analysis of these regulated proteins will promote a better understanding of their possible interactions with CapD, and their contributions to the pathogenicity of H. parasuis.Bacterial meningitis continues to be an important cause of high mortality and morbidity, which needs to be better controlled urgently. One of the typical characteristics of H. parasuis infection is the development of mainigitis, and it has been reported that H. parasuis virulrnt strain can invade the brain microvascular endothelial cells and break through the blood-brain barrier (BBB), leading to the central nervous system (CNS) infection. However, the mechanism involved in H. parasuis penetration of the BBB is completely unknown, and some key molecular targets in this process need to be identified. At present, a good meningitis-research platform, especially the appropriate cell line model for pathogens penetration of the BBB, is not available domestically. In our current research, we co-laboratorially worked with another international meningitis-studying group, using E. coli K1strain which is the most common gram-negative bacillary causing meningitis as the representative CNS-infecting strain, and explored the key targets affecting the meningitis-causing strains penetration of the BBB.Here, we provide the first direct evidence that sphingosine1-phosphate (SIP) and epidermal growth factor receptor (EGFR) play essential roles in meningitis-causing E. coli strain RS218penetration of the BBB in vitro and in vivo. Meningitis-causing strain RS218invasion of human brain micro vascular endothelial cell (HBMEC) can activate the SphK2/S1P/S1P2signaling, which thereby leads to HB-EGF-mediated EGFR transactivation. This was shown by the demonstration that (1) RS218invasion of HBMEC induced EGFR activation, and pretreatment of the cells by EGFR inhibitor gefitinib or transfection with EGFR dominant-negative plasmid significantly reduced RS218invasion of HBMEC. Administration of gefitinib to neonatal mice significantly reduced the circulating RS218strain entering the brain.(2) RS218infection can also activate the sphingosine kinase SphK2, and both SphK2inhibitor and S1P receptor S1P2antagonist blocked the RS218invasion of HBMEC. Meanwhile, the circulating RS218strain invading into the brain of SphK2-/-mice was significantly reduced compared with that of wild type mice.(3) The same E. coli factors (OmpA, FimH and N1pI) were shown to be involved in the infection-induced EGFR activation, S1P generation and SphK2activation.(4) In the process of RS218invasion of HBMEC, SphK2/S1P/S1P2signaling cascade was upstream of EGFR activation, it transactivated EGFR through the EGFR ligand HB-EGF.(5) SphK2/SlP/S1P2-EGFR signaling cascade led to the activation of c-Src and the ERM proteins, therefore contributing to the meningitis-causing strain penetration of BBB. Taken together, we demonstrate firmly that the SIP and EGFR represent the novel and potent targets utilized by the meningitis-causing E. coli K1strain RS218in the development of CNS infection, pathogens can help themselve to penetrate into the BBB by activating these two signal molecules. Moreover, our finding suggests that counteracting such targets provides a novel approach for the prevention and controlling bacterial meningitis in the era of emerging antimicrobial resistant pathogens. |
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