By using comprehensive MS-based proteomics combined with label-free quantitation algorithms, we examined the secretome of M. pneumoniae-infected and uninfected A549 cells. This proteomic approach allows the simultaneous observation of alternation in protein expression, which may represent a forecast to disease development or a consequence of the disease . As reported here, a total of 256 proteins were identified, among which 113 were differentially secreted by M. pneumoniae-infected A549 cells versus control. This result is similar to a study conducted by Brioschi et al., in which 273 proteins were identified and 112 differentially expressed in the endothelial cell secretome upon reductase inhibitor treatment .
Among the identified proteins, 152 proteins were designated as putative secretory proteins by using SignalP and SecretomeP. Interestingly, 69 out of the 152 proteins were categorized as non-classical secretory proteins, suggesting that the unconventional protein release is also a major mechanism. More importantly, as exosomal release is also regarded as a non-classical secretion mechanism , it was shown that 74% (190 out of 256) of the identified proteins in our study can be found in the ExoCarta database, highlighting a critical role for exosome in cell-cell communication . In summary, up to 92% (236 out of 256) of the identified proteins could be transported to the extracellular space by at least one of the above mechanisms. Since no significant apoptosis or necrosis was observed in our study (see Additional file 2: Figure S2), those proteins, which were not classified as secretory proteins using the computational approach (SignalP and SecretomeP), should be released mainly by intracellular secretion (e.g. exosome) rather than cell lysis . Furthermore, among the 113 differentially expressed proteins, about 80% (91) were found in the ExoCarta database, suggesting that exosomal protein release might be a major mechanism by which M. pneumoniae-infected cells communicate with other cells. Similarly, exosome-mediated release of proteins in influenza A virus-infected human macrophages has also been reported, underlining the importance of the exosome-mediated non-classical pathway in cell-to-cell communication during microbial infection .
Based on STRING bioinformatics analysis, several clusters of proteins were identified (Figure 5 and 6), suggesting that these proteins often act in cooperation with each other rather than alone during M. pneumoniae infection. Furthermore, the functions of those differential expressed proteins were found to be mainly associated with biological processes including immune response, metabolic process, and stress response (see Additional file 7: Figure S4D and S4E). Indeed, a number of studies have highlighted the importance of host-dependent inflammatory response to M. pneumoniae infection, such as IL-12 and IFN-γ production, as well as the Th1 type T-cell responses in a mouse model [4, 31–34]. Previously we have also shown that the reactive oxygen species (ROS) induced by M. pneumoniae infection attributed in part to the cytopathology of the respiratory epithelium , and M. pneumoniae infection could influence host’s sphingolipid metabolism, including the generation of new ceramide and sphingomyelin species . These reports, together with many other reports, supported the finding from this secretomic study that M. pneumoniae infection systematically alters the biological process of the host, which may partially explain the wide clinical manifestation of M. pneumoniae infection .
Cells under stress are known to actively secrete or passively release endogenous danger signal molecules, which include proteins and other endogenous molecules, such as ATP and uric acid [23, 36]. Interestingly, we have found 36 out of the 113 differentially expressed proteins were associated with stress and may act as endogenous danger signals (Table 2) [23, 24], including heat shock protein beta-1 (HSPB1), galectin-1 (Gal-1), galectin-3-binding protein (LGALS3BP), SERPINE1, disintegrin and metalloproteinase domain-containing protein 9 (ADAM9), peroxiredoxin-4 (PRDX4), and PRDX1. Several of these danger signal proteins, such as HSPs, galectins, and redox-related members, were also secreted during influenza A virus or HSV-1 infection of human macrophages [10, 18]. Therefore, the secretion of such danger signal proteins might be a general host response to pathogen infection. Some of these danger signal molecules were involved in regulating the cellular oxidative status, such as ADAM9, Gal-1 and SERPINE1 [37–39]. In line with such observation, M. pneumoniae is known to induce ROS production and reduce glutathione levels in lung and lung carcinoma cells [3, 40]. Furthermore, M. pneumoniae can inhibit host cell catalase, which could result in the toxicity of hydrogen peroxide in skin fibroblast and ciliated epithelial cells . Together, these results implicate that the enhanced ROS production should be recognized as an important mechanism in the pathogenesis of M. pneumoniae infection .
In addition, many identified proteins were involved in extracellular matrix formation (Figure 4 and see Additional file 7: Figure S4A). Extracellular matrix plays an important role in regulating many cellular functions like adhesion, cell shape, migration, proliferation, polarity, differentiation, and apoptosis . For example, SERPINE1, as a multifaceted proteolytic factor, not only functions as an inhibitor of the serine protease, but also plays an important role in signal transduction, cell adhesion, and migration . Similarly, ADAM9, a member of the ADAM family, is involved in the proteolytic processing of multiple transmembrane proteins, as well as cell adhesion, migration, and signal transduction . Gal-1 also displays diverse biological activities including cell adhesion, B cell development, mRNA splicing, angiogenesis and tissue differential/homeostasis, and inflammation . Thus, targeting the interplay between host cells and microenviroment might be another important mechanism for M. pneumoniae pathogenesis.
Finally, we were interested in the potential clinical application of such secretomic study, e.g. biomarker or therapeutic target discovery . To do that, we chose one of the identified proteins, IL-33, and conducted a “proof-of-concept” experiment. IL-33, a crucial amplifier of the innate immunity in infectious diseases as well as in autoimmune processes, is also a recently identified DAMP [46–48]. It has been shown that IL-33 plays an important role in driving antiviral CD8+ T cell responses in lymphocytic choriomeningitis virus-infected mice . During the experimental intestinal nematodes (Trichuris muris) infection in mice, IL-33 was markedly elevated soon after infection . Schmitz and co-workers demonstrated that injection of IL-33 into mice induced a profound eosinophilia with associated pathologic changes , and had potent effects on eosinophil, including the induced production of superoxide anion and IL-8, degranulation and eosinophil survival . We found M. pneumoniae significantly increased IL-33 production in A549 cells, and IL-33 levels were significantly higher in MPP patients, implying an important role for IL-33 in M. pneumoniae-elicited immune response (Figure 7). Further ROC analysis revealed that IL-33 could help distinguish MPP patients from patients with foreign objects. Thus, manipulation of IL-33 might represent a promising new therapeutic strategy for treating the inflammatory disorder during M. pneumoniae infection.