The Role Of LicC In Pneumocaccal Infection

Streptococcus pneumoniae that universally colonizes the mucosal surfaces of the host nasopharynx and upper airway, is one of the important pathogenic bacteria of human being. It is a major pathogen causing pneumonia, otitis media, meningitis, bacteremia and nasosinusitis. And the meningitis and bacteremia caused by Streptococcus pneumoniae have a higher mortality. The invasion of Streptococcus pneumoniae principally occurs in children under 2 and old people upper 65. It is one of the most important causes of children's death worldwide. In addition, the antibiotic resistance of Streptococcus pneumoniae has developed rapidly and the isolation rate has increased obviously ever since the first strain of Penicillin-Resistant Streptococcus pneumoniae (PRSP) was isolated in 1967. In some countries and areas, PRSPs are 40%-50% among the all isolated strains. And the phenomenon of multidrug resistance is become increasingly severe. There are 60%-90% PRSPs which resist Chloramphenicol, Clindamycin, Sulfamethoxazole, Erythromycin, and Tetracycline. In recent years, with the increase of antibiotic-resistant Streptococcus pneumoniae and the lack of effective vaccine, discovering the new target of antibiotics is become more and more important, and the corresponding genes relevant to pathogenicity are more concerned than ever before.A mutant library of streptococcus pneumonia had been constructed, in which a new gene licC relevant to pathogenicity was screened. Pneumococcal licC gene encodes CCT (CTP: phosphocholine cytidylyltransferase) which is a special member of the nucleoside triphosphate transferase superfamily and catalyzes the transfer of a cytidine monophosphate from CTP to phosphocholine to form CDP-choline. So, Pneumococcal LicC is one of the key enzymes to synthesize phosphocholine (PCho). Previous researches on Streptococcus pneumoniae have denoted that the choline metabolism plays an important role in the courses of cell division, transformation, autolysis and pathogenicity. PCho not only is an important virulence factor, but also binds choline binding proteins (Cbps) which are essential factors in adhesion and invasion of Streptococcus pneumoniae. We constructed an insertion mutant strain whose inserted site locates at 78bp of 3'end of licC. The results of research on this licC mutant strain indicated that the virulence was attenuated significantly, the growth was inhibited greatly and the licC was associated with several virulence factors. Consequently, the licC gene which is conservative in Streptococcus pneumoniae and not homologous with corresponding gene in other eukaryotic and prokaryotic cell maybe developed as an ideal antibiotic target.Based on our preliminary research, the mechanism of the attenuation of licC mutant was further clarified. At first, the recombinant plasmids pQE80-licC and pQE80-?licC were constructed, which contain licC and ?licC respectively. In ?licC, truncated licC gene, the 78bp of 3'end of licC are lost. And the deletion is in accordance to the insertion mutation in STM mutant strain. The target proteins were expressed in E.coli BL21 under isopropy-β-D-thiogalactoside(IPTG)induction and the soluble proteins were obtained through affinity chromatograph. The activities of the proteins were determined using home made bioluminescence test based on firefly luciferase assay system. It was showed that the activity of ?LicC was equal to about 6% of LicC's. The close relationship between the lost sequence of ?LicC and enzymatic activity was demonstrated. Because the lost sequence includes Glu216 which can bind to Mg2+ during the course of catalyzing substrates, we suppose that the markedly lower activity of ?LicC is related to the deletion of Glu216. It is suggested that the deleted part is the cause resulting in the lower growth rate and the attenuation of virulence in STM mutant strain and is the important functional site of LicC.Construction of mutation strain in which licC was deleted, and the subsequently comparison with wild strain, are the important approaches to study the functions of LicC on the pneumococcal virulence. We constructed several mutants which consist of upstream homologous sequence, antibiotic screening sequence and downstream homologous sequence to transform the Streptococcus pneumoniae R6. After the homologous recombination of homologous sequence, the whole mutant should have been integrated into genome DNA and licC deleted mutation strain should have been obtained. But the expected mutation strain could not be successed even 6 different mutants screened were on behalf of different homologous sequence and different antibiotic screening sequence. In this research it was found that the homologous sequence including deleted licC sequence can't be integrated into genome DNA, but the other homologous sequence can. Actually, this approach for constructing deletion mutation strain by homologous recombination has been proved to be a convenient, efficient and matured method in many researches. It was proved again that licC gene is the essential gene of Streptococcus pneumoniae and a promising drug target.Although the licC deletion mutation could not be construted, we finally obtained a mutant strain named ?R6. The sequencing analysis showed that the antibiotic screening sequence was integrated into genome DNA resulting from the homologous recombination of downstream homologous sequence which do not contain partial licC gene. We suppose that the following causes lead to the occurrence of ?R6. First, one end of mutant was inserted into genome DNA because of the homologous recombination mediated by downstream homologous sequence. Second, upstream homologous sequence which contains partial licC gene cannot trigger homologous recombination with licC in genome DNA because the pneumococcal licC gene is essential for survival. Third, antibiotic screening sequence was integrated into genome DNA under the pressure of antibiotic. The sequencing analysis of ?R6 showed that it was an insertion mutant strain of dprA gene which located at the downstream of licC. The results of relevant experiments showed that in comparison with wild R6, the ability of adhesion to A549 was significantly impaired (decreased by 70%), the curve of hydrophobicity analysis changed and one of the choline binding proteins (about 100KD) was lost. At the first time, it was demonstrated the dprA gene is related to the pneumococcal virulence. The specific mechanism need to be further studied. Because the dprA is the essential gene of the natural transformation of Streptococcus pneumoniae, these experimental results lay foundation to study pneumococcal natural transformation and virulence together.It is well known that the Antisense RNA technology is an effective strategy of inhibiting gene expression. In this research, the licC low-level expression strains were constructed by transforming the recombinant plasmids into wild strain R6. The recombinant plasmids contain three Antisense RNAs being half on different length, i.e. 321bp, 506bp and 708bp of licC 5'end. The result of western blot showed that the three ASRNAs have certain effect on inhibiting the licC expression. The licC inhibition strain obtained in this research will compensate for the failure of constructing licC deletion mutation by homologous recombination and has an important significance to study the biological characteristics and pathogenicity and to lay the foundation of the whole-cell antibacterial discovery strategy aimed at licC gene.To sum up, in this research the cause of the changes of the biological characteristics and virulence of STM mutant strain was characterized. It was identified that 78bp of 3'end was the essential site of licC function. In addition, the licC was demonstrated to be an essential gene in Streptococcus pneumoniae, and this is the theoretic foundation of the whole-cell antibacterial discovery strategy aimed at licC gene. Finally, the licC low-level expressive strains which are important to study the biological characteristics and pathogenicity were constructed by Antisense RNA technology. Exceeds our expectations, we also found that the dprA gene is related to the pneumococcal pathogenicity. Also the inhibiting the expression of dprA could cause the attenuation of pneumococcal virulence and the decrease of the ability of natural transformation.