The Role Of ClpE In The Pathopoiesis Of Streptococcus Pneumoniae

Among the gram-positive opportunistic human pathogens, Streptococcus pneumoniae is a major cause of serious invasive diseases, including pneumonia, otitis media, bacteremia, and meningitis. It remains the high morbidity and mortality throughout the world, particularly in infants, elders, and immunocompromised patients. S. pneumoniae infection is a widespread serious problem because of the increasing antibiotic resistance and defects of the current vaccine. Therefore, we should further understand its molecular pathogenic mechanism to provide new theory and experimental data for more effective drug and vaccine development.HSP100/ ClpATPase ( Heat shock proteins 100 ) are extremely conserved in prokaryotes. Members of the HSP100 family represent proteins that form the largest chaperone and protease complexes in bacterial cells. Previous studies have revealed that ClpATPase are important for not only cell physiology but also regulation of virulence properties of pathogenic bacteria. In Streptococcus pneumoniae, Clp ATP-dependent proteases were composed of a proteolytic subunit, ClpP, and an ATPase subunit (ClpC, ClpE, ClpL, and ClpX) accounting for substrate specificity. ClpC ATPase is the interacting partner of ClpP in many physiologically important processes such as pneumolysin release and adaptation to diverse stress parameters. ClpL not only modulates the virulence gene expression but also affects the adherence and invasion to host cells. Furthermore, in S. pneumoniae R6, depletion of ClpX leads to rapid cell death without overtly affecting cell morphology. Although ClpE of S.pn has been shown to be required for growth at high temperature, the role of ClpE on pathogenesis has not been described so far. The purpose of this study was to investigate the effect of ClpE on the pathogenesis of Streptococcus pneumoniae. This study includes the following two parts:1. Construction of clpE-deletion mutant of S.pneumoniae and investigation of the effect of ClpE on the pathogenesis of Streptococcus pneumoniae.LFH-PCR (Long flanking homology polymerase chain reaction) was introduced to generate a gene disruption construct consisting of erm cassette with long flanking homology regions to the target gene. Then S.pneumoniae D39 was transformed directly with this PCR product. The clpE-deletion mutant was obtained on the TSA agar containing erythromycin and identified by PCR and sequencing. The impact of clpE mutant on the virulence of S. pneumoniae was evaluated in a mouse intraperitoneal challenge model. Our results showed that clpE gene was completely replaced by erm cassette. The virulence of clpE mutant was significantly attenuated in mice intraperitoneal infection model. The results implicated that ClpE was involved in the pathopoiesis of Streptococcus pneumoniae.2. Investigation of the role of ClpE in the pathopoiesis of Streptococcus pneumoniae.Growth of parental strain (D39) and clpE-deletion strain at different temperatures were examined in C+Y media. In addition, we investigated the effect of clpE mutant on adherence and invasion to host cells (human lung epithelial carcinoma A549 cells line and human umbilical vein-derived endothelial cells HUVEC). The underlying molecular mechanism of virulence attenuation induced by the mutation of clpE was further investigated by FQ-PCR (fluorescent quantitative polymerase chain reaction) and two-dimensional protein gel analysis.The growth of clpE mutant was slower than that of the parent at 37°C. The clpE mutants presented a temperature-sensitive growth phenotype at 40°C and 42°C. Cell culture infection experiments indicated that adherence and invasion of isogenic mutants in which the clpE gene was inactivated were both strongly reduced. FQ-PCR analysis demonstrated that the expression of major virulence determinants, such as pneumolysin (ply), pneumococcal surface antigen A (psaA), neuraminidase (nanA), pneumococcal surface protein A (pspA) and major autolysin (lytA) were decreased in clpE mutants. The protein expression analysis led to the identification of hypoxanthine–guanine phosphoribosyltransferase (HPRT), pyrrolidone-carboxylate peptidase 1, formate-tetrahydrofolate ligase, and bifunctional protein PyrR, which were down regulated in the D39ΔclpE and involved in the survival and adhesion of bacteria. These results indicated that ClpE could help S.pneumonia invading and adapting to different environments of the host by participating in the expression of special proteins mentioned above.In a word, ClpE is required for the ability of bacterial surviving, adhering and invading to host cells, and it plays an important role in the pathopoiesis of Streptococcus pneumoniae.