| Background and objectives:Mycoplasma pneumoniae (M. pneumoniae) is one of the major etiological agents causing respiratory infection worldwide in all age groups. Generally, M. pneumoniae infection is considered as a self-limiting disease with good prognosis. However, more and more severe and refractory M. pneumoniae pneumonia (MPP) cases have been reported in recent years, and some of them even developed life-threatening complications. Although the pathogenesis of M. pneumoniae infection has been studied extensively, and certain mechanisms including cytadherence, intracellular localization, cytotoxicity and immune response have been proposed, the molecular mechanisms underlying M. pneumoniae pathogenesis still remain elusive. The attachment of M. pneumoniae to respiratory epithelium is a prerequisite step of M. pneumoniae infection, however, little is known about the response of epithelial cells after the adhesion of M. pneumoniae. Of note, the early host response to M. pneumoniae infection relies on the concerted release of proteins with various biological activities. However, no comprehensive analysis of the secretory proteins has been conducted to date regarding the host response upon M. pneumoniae infection.Therefore, in the current study, we applied liquid chromatography-tandem mass spectrometry (LC-MS/MS) based label-free quantitative shotgun proteomic technique for global profiling of the M. pneumoniae-infected human alveolar epithelial carcinoma A549cell secretome, in effort to provide a better view of host-pathogen interaction and identify novel molecules/biomarkers for M. pneumoniae infection.Methods:1. Human alveolar epithelial carcinoma A549cells were chosen as a cell model for our secretome study in vitro. Cell viability in serum free medium (SFM) was assessed by MTT test and trypan blue exclusion assay, and the cell death was assessed by apoptosis assay.2. The members of the A549cell secretome during M. pneumoniae infection were identified and characterized by the LC-MS/MS-based label-free quantitative proteomic technique.3. The identified proteins were classified on the basis of their cellular compartment by GO annotation. Classical secretory proteins with a signal peptide were predicted by SignalP4.1and non-classical secretory proteins without a signal peptide were predicted by SecretomeP2.0. All the identified proteins were searched against ExoCarta data to determine whether they were present in exosome fractions.4. BiNGO was used to determine over-representation of GO categories (including cellular compartment, molecular function and biological process) for differentially expressed proteins.5. Biological pathways that differentially expressed protein involved were analyzed by the KEGG database.6. STRING was performed to construct a network model for differentially expressed proteins showing protein interaction based on known and predicted protein-protein interactions.7. The changed expressions of six differentially expressed proteins (including ADAM9, SERPNE1, IL-33, IGFBP4, Gal-1and MIF) were detected by Western blot and RT-PCR.8. To investigate the role of Gal-1in M. pneumoniae infection, Gal-1siRNA was designed and utilized to suppress its expression. The silencing effect of siRNA on Gal-1expression was verified by Western blot. For the evaluation of Gal-1function, following performance were taken:1) reactive oxygen species generation detected by flow cytometry;2) yH2AX foci formation confirmed by immunofluorescent microscope;3) yH2AX levels detected by Western blot;4) altered expression of cytokines detected by RT-PCR.9. Peripheral blood and bronchoalveolar lavage fluid (BALF) samples of pediatric patients with MPP were collected. Samples from age-matched children with foreign body in bronchus were used as controls. The levels of IL-33in serum and BALF were determined using the IL-33enzyme-linked immunosorbent assay kits.Results:1. SFM did not significantly affect cell morphology, proliferation rate and cell viability within24h, with a good survival rate above98.5%.2. Based on the LC-MS/MS data, a total of256proteins were identified, among which233proteins were identified in control A549cells and237were identified in M. pneumoniae-infected A549cells. Label-free quantitative comparison performed by DeCyder MS label-free quantitation software revealed that113proteins were significantly affected by M. pneumoniae infection (fold difference≥1.55,p<0.05), among which65were up-regulated and48down-regulated by M. pneumoniae.3. Of the256proteins,83were categorized as classical secretory proteins and69as non-classical secretory proteins based on SingalP and SecretomeP analysis. In addition, among the proteins identified,190proteins were also listed in the exosomal proteins database by searching the ExoCarta exosome database. The GO ontology of the identified proteins based on cellular compartment showed the majority of the proteins belong to more than one GO class (e.g. nucleus, membrane and cytosol).4. The classification based on the cellular organelle of the differentially expressed proteins showed that M. pneumoniae infection did not induce protein secretion from any specific cell organelle, but rather, alter the overall secretion of proteins from all the main organelles, including mitochondrion and lysosome. For the up-regulated proteins, GO analysis revealed their molecular function mainly related with oxidoreductase activity, protein binding and enzyme regulator activity, while for the down-regulated proteins, their molecular function mainly associated with enzyme in inhibitor activity and hydrolase activity. Biological process analysis of up-regulated proteins led to the identification of functional groups related to monosaccharide catabolic process, inflammatory response, cell redox homeostasis, and defense response, while for down-regulated proteins, system development seemed to be the most relevant biological process.5. KEGG database search revealed the differentially expressed proteins were involved in85biological pathways, of which11were significantly over-represented (including pyruvate metabolism, glycolysis/gluconeogenesis and infection pathways).6. STRING analysis revealed that among the113differentially expressed proteins,88proteins can interact with each other. For the65up-regulated proteins, three main networks of proteins interactions were identified, including stress response proteins, signaling pathway associated with proteins, and cellular metabolic proteins. For the48down-regulated proteins, two major networks of the associated proteins were found, including the glucose catabolic proteins and biological process negative regulation associated proteins.7. RT-PCR and Western blot results confirmed that the expressions of the six secretory proteins were significantly increased after M. pneumoniae infection, which were consistent with the proteomic results.8. When Gal-1-knockdown A549cells were infected by M. pneumoniae, the generation of reactive oxygen species (ROS) was decreased, accompanied by reduced yH2AX foci and significantly increased proinflammatory cytokine expression at24h (e.g.IL-8and IL-18).9. Examination of clinical samples showed that IL-33levels were significantly higher in the serum and BALF of MPP patients, when compared to children with foreign body in bronchus.Conclusion:1. The expressions of secretory proteins from A549cells were altered during M. pneumoniae infection. The label-free quantitative shotgun proteomics approach is shown to be a feasible strategy for the investigation of M. pneumoniae pathogenesis.2. The non-classic secretion mechanism, especially exosomal release is an important mechanism through which identified proteins were transported to the extracellular space during M. pneumoniae infection.3. As stress response proteins (e.g. MIF, HSPB1and Gal-1), inflammatory proteins (e.g. YWHAZ, ADAM9and PRDXl) and metabolic proteins (e.g. GPI, LDHB and ENO1) were up-regulated in M. pneumoniae-infected A549cells, we proposed that the host responses caused by M. pneumoniae infection were mainly related to stress reaction, immune response and cell metabolism.4. Gal-1and IL-33may mediate the M. pneumoniae-induced cytotoxic effect. |
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