Function Research Of A Streptococcus Pneumoniae Teichoic Acid Synthesis Related Protein SPD1672

Background In our previous research, we have screened out some in vivo induced genes(IVI genes) of streptococcus pneumonia which can be related to bacterial infections disease. Spd1672 is one of these IVI genes with product of SPD1672 which contain lipid A core-O antigen enzyme domain, indicating spd1672 gene may be involved in synthesis of cell wall polysaccharide. In this study we will identify the biology function of spd1672 gene and investigate its role in pathogenic process.Methods The spd1672-deletion mutant was constructed by LFH-PCR technology. The thickness of capsule was observed by transmission electron microscopy.The change of teichoic acid content was observed by C-reactive protein bind experiment and complement deposit experiment. LTA and LTA was separated, and then the content was quantitied by western blot to preliminary identify the function of spd1672.The tolerance ability of D39 and D39â–³1672 to high osmotic pressure were observed by growth in high Mg2 + environment. Meanwhile the pathogenic abilities of wild strain and the spd1672-deletion mutant were compared, following with colonization experiment, bactericidal assays and cytokines secretion experiment to understand the virulence mechanisms of spd1672.Results Compared to the D39 wild strain,spd1672-deletion mutant grew much more slowly in culture. There was no obvious difference in the capsule thickness between D39â–³1672 and D39 examined by transmission electron microscope. The results of C-reactive protein binding test showed that the binding of D39â–³1672 with CRP was significantly less than that of D39. C3 complement deposit experiment show that the binding of D39â–³1672 with CRP was significantly less than that of D39.Further Western blot results showed that compared with those of wild type D39, the synthetic quantity of WTA and LTA of D39â–³1672 were both significantly reduced with anti-teichoic acid antibodies. Differences between D39 and D39â–³1672 could also be found when the ion concentration in culture medium changed. When 50mM Mg²⁺ was added the growth rate of D39â–³1672 was obviously higher compared with that in Mg²⁺ free culture medium (P <0.05). However in that case the growth of D39 was almost not affected. When the concentration of Mg²⁺ was increased to 100mM or 200mM in culture medium, the proliferation of D39â–³1672 was significantly inhibited (P <0.05), while that of D39 was just slightly affected (P> 0.05). Mice infected by intraperitoneal route with D39â–³1672 had less bacterial load in blood than mice infected with D39(P <0.05), showing that the virulence of D39â–³1672 was significantly decreased compared with D39. At the same time, the bacterial load of D39â–³1672 colonized in nasopharynx and lung were significantly less than that of D39 (P <0.05). Whole blood bactericidal assay results showed that resistance of D39â–³1672 to bactericidal effect was significantly weaker than that of D39 (P <0.05).The results of cytokines detected show that the mice infected with D39 induced higher levels of IL12 than D39â–³1672.Conclusion This research shows that the gene spd1672 is vital for synthesis of teichoic acid. Deletion of this gene will lead to significant decrease of bacterial teichoic acid synthesis. What's more, after deletion of spd1672, the bacterial proliferation capacity diminishes, and the capacity of colonization and invasion of bacteria declines, as well as with decreased resistance to bactericidal effect of white blood cells. Besides the spd1672-deletion mutant is more sensitive to changes in external environment such as the change of Mg2 + osmotic pressure. These results suggest that this gene is a key virulence factor in bacterial infection process.