Screening And Subcellular Location Of Pneumococcal In Vivo-induced Genes Regulated By Transformation

ObjectiveAmong the gram-positive opportunistic pathogen, Streptococcus pneumoniae is a major cause of serious invasive diseases, including pneumonia, meningitis and otitis media. S. pneumoniae infection is a widespread and serious problem which results from the increasing antibiotic resistance and the defects of the current vaccine. Hence it takes a profound understanding of the molecular mechanism that causes the disease to fundamentally solve the prevention and cure of S. pneumoniae.Transformation refers to the process of formation of competence and uptake exogenous deoxyribose nucleic acid. The transformation of bacteria is the start and root of many important physiological phenomenon. The transformation of S. pneumoniae is most efficient. A great deal of proofs have shown that transformation can lead to the change of bacterial virulence and drug-resistance. Yet it's still not clear how transformation regulates the virulence.After the bacteria enter the body, they have to express some genes that strengthen their own virulence or aggravate the damage to host. Therefore, the genes that the bacteria express in the body of host are often those closely related to causing the disease. In the early period, our team selected several S. pneumoniae in vivo-induced genes through in vivo expression technology and differential fluorescence induction, but the functions of some genes are unknown. The expression of genes with unknown functions in the body is increased and is closely related to virulence. Are they also regulated by transformation and related to conditional pathogenesis of S. pneumoniae? In order to make a comprehensive analysis of conditional pathogenesis of S. pneumoniae, a transformation-deficient strain will be constructed to select the S. pneumoniae in vivo-indeced genes that are controlled by transformation.To further study the functions of these genes, we chose spd₀873 regulated by transformation and dnaJ to subcellularly locate their proteins. Protein CodY is constantly expressed in cytoplasm and can be used to evaluate subcellular compositions. Firstly we obtained their recombinant protein through prokaryotic expression, and then produced their polyclonal antibody to analyze the subcellular location of these two proteins.Methods1. Building the transformation-deficient strain. comE is the key factor influencing the formation of S. pneumoniae competence. Deficient of comE makes it impossible for bacteria to transform. So the D39 comE-deficient strain (D39â–³comE) made through insertion inactivation is the transformation-deficient strain.2. Screening the in vivo genes of S. pneumoniae controlled by transformation. The BALB/c mouse was used as test animal and injected with D39 wild type and D39 comE-deficient strain via intraperitoneal injection respectively. The mice blood was obtained about 24 hours after the injection through posterior orbital venous plexus approach. The bacteria induced in vivo were collected from the blood and their RNA were extracted to measure the mRNA expression levels of 13 in vivo- induced gene by RT-PCR. 3. Prokaryotic expression of Spd₀873 and DnaJ. We expressed and purified recombinant protein Spd₀873 and CodY through constructing PW28/0873,PW28/codY.4. Preparing polyclonal antibody. We immunized the New Zealand white rabbits and BALB/c mice respectively with purified recombinant protein Spd₀873 and CodY and obtained their polyclonal antibodies.5. Separating the compositions of D39 and the subcellular location. We separated the supernatants, cell walls and protoplasts of D39 and analyzed the subcellular location of Spd₀873 and DnaJ using western blot and flow cytometry.Results1. As verified by Polymerase Chain Reaction and sequencing, the construction of D39 comE-deficient was successful.2. The differences of the expressions of 8 in vivo-induced genes in D39 and D39 comE-deficient strain were statistically significant (P<0.05) and in which spd₀300, spd₀414, spd₀622, spd₁663, spd₁719, spd₀235, spd₀873 were up-regulated and meanwhile spd₁672 was down-regulated.3. Construcion of recombinant express vector PW28/0873 and PW28/codY were proved to be successful through sequencing and restriction enzyme digestion.4. The polyclonal antibody of Spd₀873,DnaJ were obtained through immuning the New Zealand white rabbits and BALB/c mice.5. The protein Spd₀873,DnaJ were expressed on the surface of S. pneumoniae analysed by flow cytometry and protein DnaJ was located to cell walls and protoplasts identified by western blot.Conclusions1. 8 in vivo-induced genes spd₀300, spd₀414, spd₀622, spd₁663, spd₁719, spd₀235, spd₀873 and spd₁672 controlled by transformation were screened out and they may participate in the processes such as growth regulation, temperature reception, carbohydrate metabolism, lipoids metabolism and so on. The bacterial transformation may enhance the virulence via regulating some in vivo-induced genes expression and it indicates that transformation can strengthen the bacterial virulence through modifying the expressions of certain in vivo-induced genes.2. The protein Spd₀873 has expressions on the surface of S. pneumoniae, but whether it has expressions in other parts of the bacteria needs to be further studied. The protein DnaJ is located in the cell walls and protoplasts of S. pneumoniae. These findings provide a preliminary basis for further functional studies.