Study On Contribution Of Streptococcus Pneumoniae Aminopeptidase N To Bacterial Virulence And Its Pathogenic Mechanism

Objective: Streptococcus pneumoniae is a Gram-positive bacterium with high morbidity and mortality worldwide especially among the very young and elderly.Currently,S.pneumoniae resistance continues to increase and the protective effects of the current vaccines are not satisfactory.Therefore,attention has focused on discovery of novel S.pneumoniae virulence proteins and their pathogenic mechanisms to provide new ideas for prevention and treatment of these infections.Previous studies have shown that S.pneumoniae aminopeptidase N(PepN)interacts with the pneumococcal virulence protein heat shock protein ClpE,inhibits cytotoxic T lymphocyte effector function.These studies provide evidence that PepN is most likely a virulence factor of S.pneumoniae.However,its role in S.pneumoniae virulence and its pathogenic mechanism remains to be confirmed.The aim of the present research was to explore the potential role of PepN in the virulence and pathogenesis of S.pneumoniae.Methods: Western blotting was used to detect the expression of PepN in S.pneumoniae.The pepN gene in the S.pneumoniae TIGR4 genome was replaced with the erm gene encoding erythromycin resistance by homologous recombination using the long flanking homology-polymerase chain reaction technique.Transformants were selected on Columbia blood agar plates containing erythromycin.Simulating the natural infection route of S.pneumoniae,the pathogenicity was investigated by infecting mice nasally with TIGR4 and TIGR4△pepN.Colonization experiments of mice in vivo and host cell of adhesion and invasion experiments in vitro were employed to elucidate the role of PepN in the bacterial pathogenic process.The effects of PepN protein on other virulence proteins of pneumococcus were explored by quantitative real-time PCR.The lung inflammation of TIGR4 and TIGR4△pepN infected mice were detected and mouse model of tracheal instillation of rPepN was constructed to evaluate the pro-inflammatory effects of PepN in vivo.Primary peritoneal exudate macrophages from mouse were used to determine the effect of PepN on host innate immunity and further study the related signaling pathways in vitro.Results: In this study,we found that PepN was detected in all strains we examined and PepN is a protein present in the medium supernatant,cell wall and intracellular of S.pneumoniae.we also found that the virulence of the TIGR4ΔpepN was significantly attenuated in the mouse pneumonia model resulting in increased survival of mice,reduced bacterial loads in the respiratory tract and accelerated bacterial clearance.In vitro experiments showed that the adhesion and invasion of TIGR4 was significantly reduced with the deletion of pepN.In addition,the pepN deletion mutant displayed decreased levels of other virulence factors including lytA,nanA and ply.Here we shown that the PepN protein could induce a strong inflammatory response in vivo and in vitro and induced secretion of IL-6 and TNF-α by primary peritoneal macrophages via the phosphorylation of MAPK and PI3K/AKT signaling pathways and this was confirmed using specific pathway inhibitors.Conclusion: PepN is a novel virulence factor that is essential for the virulence of S.pneumoniae and induces host innate immunity via MAPK and PI3K/AKT signaling.