The Roles Of PepO In Activation Of Host Innate Immunity And The Involved Molecular Mechanisms

Background: Streptococcus pneumoniae endopeptidase O(PepO) is a newly discovered and widely expressed pneumococcal virulence protein, contributing to adherence and invasion to host cells. Interaction between virulence factors of Streptococcus pneumoniae and innate immune receptors elicits host defense responses through specific signaling pathways during infection. Whether PepO is involved in the activation of host innate immunity and the related molecular mechanisms remain to be understood. Insights into the signaling events may contribute to the development of anti-bacterial drugs and may broaden the scope of researches on protein vaccines.Methods: rPepO protein was expressed in E. coli BL21. Mouse model of intratracheal instillation of rPepO was made to access the effect of protein on activation of innate immunity in vivo. Mouse peritoneal exudate macrophages(PEMs), MLE-12 cells, and Beas-2B cells were used to explore the cells targeted by rPepO and the related signaling pathways in vitro. Cytokines production was determined by Q-PCR and ELISA. Lung inflammatory change was analyzed by histological assays and hematoxylin-eosin staining. Recruited nucleated cells in bronchial alveolar lavage fluid(BALF) were analyzed using flow cytometry analysis and Wright-Giemsa staining. rPepO- induced activation of intracellular signaling molecules was determined with the use of pharmaceutical inhibitors, western blot, and immunofluorescence assays.Results: Here we demonstrated a significant induction of innate immune response elicited by rPepO, a newer pneumococcal virulence protein, both in vivo and in vitro. Intratracheal instillation of rPepO protein resulted in significant increase of cytokines production and neutrophils infiltration in mouse lungs. TLR2 or TLR4 deficient mice subjected to rPepO treatment showed decreased cytokines production, reduced neutrophils infiltration and intensified tissue injury as compared with WT mice. Upon stimulation, cytokines TNF-α, IL-6, CXCL1, and CXCL10 were produced by peritoneal exudate macrophages(PEMs) in a TLR2 and TLR4 dependent manner. Cytokines induction relied on the rapid phosphorylation of p38, Akt and p65. rPepO enhances IL-6 and CXCL1 production in MLE-12 cells via MAPKs- NF-κB- PI3K/Akt signaling pathway, and promotes IL-8 and IP-10 production in Beas-2B via PI3K/Akt-MAPKs signaling pathway.Conclusion: We prove that rPepO induces strong host defense responses both in vivo and in vitro partially through the TLR2 and TLR4 signaling pathways.