Role Of DprA In Virulence Of Streptococcus Pneumoniae

Pneumococcal disease is a major public health problem worldwide. Recently, with the prevalence of antibiotic-resistant Streptococcus pneumoniae and the emergence of vaccine escape strains, exploring new targets for antibiotic treatment is becoming more and more important. The corresponding genes relevant to pathogenicity are more concerned than ever before.The protein, DNA processing protein A(DprA) has dual-function in later-phase of pneumococcal natural transformation. Firstly, after exogenous double-stranded DNA is internalized as single strand by competent cells, DprA ensures the loading of RecA onto the single strand DNA(ssDNA) to form presynaptic filaments, by which ssDNA can be protected from DNase and integrated into chromosome relying on the universal bacterial recombinase RecA. Lack of protective contribution of DprA makes internalized ssDNA rapidly degraded and then results in the efficiency of integrating ssDNA into genomic DNA decreased approximately by 104-fold. Furthermore, DprA can shut off the competence via the interaction with activated ComE, which is the response regulator(RR) of a classic two-component regulatory system and can activate the transcription of relevant genes to develop and maintain the competent state of S. pneumoniae. Due to the competent state has possibly toxic effect to bacterial physiology and virulence, the exit pathway from competence depending on DprA may plays a key role in virulence by keeping the competence transient. In addition, recovery from competence to normal state makes the expression of lots of corresponding genes changed. So, DprA may influence the baterial physiology and pathogenesis via regulating the transcription of these genes. A dprA mutant was constructed in preliminary experiment, in which a kanamycin-resistent sequence Janus cassette was inserted at 312 bp site of dprA gene. The result of bacterial adhesion assay showed that the adhesive ability to pulmonary epithelial cells A549 was impaired and a component of choline binding proteins lower expressed, which is an important virulent factor of S. pneumoniae. These changes indicated that DprA may play role in virulence.In this study we’ll further validate the influence of DprA to biologically pneumococcal characteristics, identify the role of DprA in virulence and explore the potential mechanism.The RF domain at C terminal of DprA is as lost domain in partially dprA-missing mutants because RF is essential for DprA’s function in pneumococcal transformation and competent shut-off. The dprA mutants were constructed by natural transformation based on homologous recombination in this study, including RF lost mutants based on D39, R6 and Xen11, DprA complementation strains on RF lost mutants, control strains with inserted Janus cassette at the downstream point of dprA and bioluminenscent strains without RF domain,The characteristics of the mutants were identified by using bacterial growth curve, biofilm formation, adhesion and invasion assay to A549 cell, and CD1 mice infection experiments. By checking the OD620 of the cultures, it was observed that dprA mutants grew much 2-3 hours slower than the wild type strains(p<0.05). The biofilm formation experiments on polystyrene substance showed that in the same growth conditions the biofilm formation of dprA mutant cell was smaller than wild type strain. Comparing with wild strains, the ability of adhesion of the dprA mutant to A549 was significantly impaired, which decreased by 48%-63%. At the same time, the invasion ability of dprA mutants was attenuated, which decreased approximately by 90%-92%. However, the complemented strains which expressed the completed dprA with the strong promoter of pIB166 restored the ability of growing in THY and biofilm formation, and the levels in adherence and invasion to A549 cells recovered to some extent, which almost reaching to 54-62% and 79-82% of wild type, respectively. Moreover, the results of survival curve and bacterial burden assay showed that the lethality rate to CD1 mice and the ability to reproduce in vivo were decreased compared with that of wild type. Furthermore, the attenuation resulting from the partial lost of dprA was verified in co-infection animal model. The clearance of mutant by CD1 mice is strikingly faster than that of wild type in pharynx nasalis, lung tissue and blood. The data from animal experiments indicated that the missing part might be needed during pneumococcal colonization and systemic infection. Accordingly, DprA served as an important virulent factor of S. pneumoniae. Finally, by SDS-PAGE, mass spectrogram and Western blot, we found that choline binding protein A(CbpA) was lower expressed in dprA mutants, which is an important virulence factor in pneumococcal disease. It was indicated that the lower expressed CbpA is a crucial reason to the impaired virulence of dprA mutants.In conclusion, a series of dprA mutants were constructed in this study at first. Then, based on bacterial, cellular and animal experiments, dprA gene was identified as a relevant gene to pneumococcal virulence through regulating the expression of CbpA.