The Role And Mechanisms Of MTORC1 In Staphylococcus Aureus And Peptidoglycan Induced Inflammatory Response

Staphylococcus aureus is a typical Gram-positive pathogen, may occur commonly in the environment. Also, S. aureus is one of the most important causal agents in human and zoonotic infection, and is the most common type of contagious mastitis in dairy herds around the world. Peptidoglycan is a major component of the Staphylococcus aureus cell wall, and the ligand of TLR2 (toll like receptor 2) which recognized by TLR2 resulting in gathered and activation of MyD88 (myeloid differentiation 88) by its intracellular TIR domain, it can raise IRAK (IL-1-receptor kinase) and TRAF6 (TNF-receptor-associated factor 6) and initiate the activation of nuclear transcription factors (nuclear factor kappa-light-chain-enhancer of activated B cells, NF-κB) to promote the expression of proinflammatory cytokines which finally cause inflammatory respose to clear the pathogen from body.mTOR is a very conserved serine/threonine kinase in evolution, it belongs to PIKK (phosphatidylinositol 3-kinase-related kinase) superfamily, and is PI3K downstream proteins. Before studied that PI3K regulated the innate immune response induced by a variety of viruses and bacteria components via TLRs. Lipopolysaccharide (LPS) activates PI3K and induces complex formation of PI3K and MyD88, which regulates the expression of cytokines. Peptidoglycan activates PI3K rapidly through TLR2. mTOR forms two complexes with other proteins, mTORC1 and mTORC2, mTORC1 is sensitive to rapamycin. mTORC1 mediates upstream signals and activates downstream p70S6K (ribosomal protein p70S6 kinase) and 4E-BP1 (transcription initiation factor 4E binding protein 1) to regulate gene expression in a variety of physiological and pathological processes. mTOR has an important role in the innate immune response, such as effecting on the expression of bacteria or other stimulating factor induced cytokines. However, it is unknown whether mTORC1 regulates peptidoglycan induced inflammation. In this study, we plan to explore the mechanism of mTORCl in regulation of peptidoglycan induced inflammatory response using the mice macrophage cell line. Therefore, in the process of pathogen associated pattern molecules induced mTORCl signaling to regulate in immune responses, the changes in protein activation still lack of proteomics data. It is also necessary for some important functional genes to further explore and verify.Firstly, we performed that mTORC1 inhibitor rapamycin were used to block mTORCl signaling pathway in mouse macrophage Ana-1 cell line which stimulated by S. aureus cell wall peptidoglycan. Inflammatory cytokines (TNF-a, IL-6 and IL-10) were detected by ELISA to conform whether mTORCl regulate peptidoglycan induced inflammatory cytokine expression and cell proliferation. The results show that peptidoglycan induces TNF-α, IL-6 and IL-10 expression in a time-dependent and dose-dependent manner. The optimal peptidoglycan stimulated doses and times were 25 μg/ml and 6 hr in Ana-1. Peptidoglycan plays different roles in different condition.25 μg/ml peptidoglycan induces Ana-1 cell proliferation that can be inhibited by rapamycin. Rapamycin increases peptidoglycan induced IL-10 expression, but rapamycin decreases the secretion of peptidoglycan induced TNF-a and IL-6 expression. These results suggest mTORC1 pathway plays an important role in the regulation of peptidoglycan induced inflammation. In order to investigate the molecular mechanisms of mTORC1 pathway in peptidoglycan induced inflammatory response, we study the signaling pathways and transcription factors. In this aim, we determined the expression and phosphorylation of mTOR, and S6,4E-BP1 were detected to confirm whether peptidoglycan activates mTOR signaling pathway; cells were stimulated with peptidoglycan after pretreated with rapamycin to inhibit mTOR, then the activity of mTOR pathway was measured by western blot; also, the activity of mTOR signaling pathway and cytokines expression were detected after TLR1 and TLR2 were blocked through antibodies. Next, the level of protein expressions and phosphorylations of STAT3 and NF-κB p65 were detected. The data shows that peptidoglycan activates mTOR signaling pathway. Peptidoglycan activated mTOR pathway were inhibited by rapamycin; peptidoglycan activates mTOR signaling pathway, STAT3 and NF-κB via a TLR1 and TLR2-dependent mechanism; rapamycin impairs peptidoglycan induced NF-κB and STAT3 activation in mouse macrophage.We can get by S. aureus cell wall peptidoglycan binding to receptors TLR1 and TLR2, and activate mTORC1 signaling pathways and the transcription factors NF-κB and STAT3, then promote the secretion of inflammatory cytokines TNF-α, IL-6 and IL-10. In this process, mTORC1 by modulating the activity of transcription factors to achieve the regulation of cytokine expression.Next, we performed that mTORC1 inhibitor rapamycin were used to block mTORC1 signaling pathway in bovine mammary epithelial cell line (BMECs) which infected by S. aureus. In this study mass spectrometry (iTRAQ) was used to detect peptide sequence in bovine mammary epitheial cell line of non-treated group, S. aureus infected group, rapamycin treatment group, S. aureus infected after rapamycin treatment group, and trying to research the proteins changes between the compared groups in the process of bacterial infection induced and mTORC1-regulated inflammation related cytokines and signaling pathways. BMECs were used as research object, and iTRAQ technology was used as methods, four sets of proteomic data were obtained. Meanwhile, we got four sets of comparison proteomics data via pairwise comparison of proteomics data, and also obtained their GO functional analysis data, and KEGG pathway functional analysis data. Samples from four groups to totally identify 1761 differentially expressed proteins; and 54 proteins were associated with mTORC1, on which can be retrieved 22 proteins functional annotation in the KEGG database; 44 proteins were associated with bacterial infection, on which can be retrieved 16 proteins functional annotation in the KEGG database; 61 proteins associated with bacterial infection and mTORC1, on which can be functionally retrieved 27 proteins in the KEGG database; 58 proteins were associated with bacterial infection and by mTORC1 regulation, on which can be retrieved 22 proteins functional annotation in the KEGG database.In summary, mTORC1 signaling pathway plays an important role in the regulation of peptidoglycan-induced inflammation. Its possible molecular mechanism by TLR1 and TLR2 peptidoglycan activate mTORCl pathway, and activation of the transcription factor NF-κB and STAT3, regulation of inflammatory cytokine expression. Proteomics studies show, mTORC1 may play a regulatory role in the process of Staphylococcus aureus infection in bovine mammary epithelial cells. This study findings enrich the research in the field of knowledge of interaction between pathogen and host cell for prevention and treatment of mastitis and to provide basic data.