BACKGROUND
mycoplasma pnuemoniae  belongs to the class of the mollicutes and is one of the smallest free-living organisms.
it is a major cause of community-acquired pneumonia  worldwide in all age groups, and can also induce manifestations in extrapulmonary sites involving almost all organs of the human body  <cit> .
with the exception of m. pneumoniae adherence to the host epithelium, molecular mechanisms underlying the pathogenesis of m. pneumoniae infection has long been a mystery  <cit> .
subsequent to cytadherence, m. pneumoniae is believed to cause disease in part through the generation of peroxide  <cit>  and the induction of inflammatory reaction including cytokine productions   <cit> .
simultaneously, autoimmunity developed after m. pneumoniae infection likely contributes to the extrapulmonary complications.
for example, anti-gm <dig> and galactocerebroside antibodies are the primary autoantibodies implicated in the ascending paralysis of guillain-barre syndrome and in encephalitis associated with m. pneumoniae <cit> .
although toxin had not been considered as part of the m. pneumoniae repertoire in previous studies, recent evidence suggested otherwise.
a newly identified exotoxin of m. pneumoniae, named community-acquired respiratory distress syndrome toxin , which has adp-ribosylating and vacuolating activity, has been suggested to be responsible for eliciting extensive vacuolization and ciliostasis of host cells  <cit> .
thus, the pathophysiology of m. pneumoniae infection is likely to be complex and multifactorial, and the underlying molecular mechanisms should involve a large number of genes/proteins participating in various biological pathways  <cit> .
high-throughput technologies including genomics and proteomics can comprehensively and quantitatively decipher gene/protein expression, and therefore, are useful tools in the study of complex systems under the influence of biological perturbations, such as pathogen-host interaction  <cit> .
previously, using a proteomic approach, we had analyzed m. pneumoniae-induced protein expression profile using whole cell lysates, and identified the redox regulatory pathway as a key target during m. pneumoniae infection  <cit> .
however, as noted above, m. pneumoniae-induced immune response is important for m. pneumoniae pathogenesis, and many factors involved in the immune response, such as the cytokines, are so-called secretory proteins, which are part of the “secretome”  <cit> .
secretome proteins include extracellular matrix proteins, growth factors, cytokines and hormones, and other soluble mediators.
it is known that secretory proteins are important for many physiological processes  <cit> .
for example, the matrix metalloproteinases , as extracellular matrix-degrading enzymes, are essential regulators of the cell’s microenvironment governing cell fate and function, such as cell migration, proliferation, apoptosis, invasion and development  <cit> .
moreover, changes in secretory proteins can reflect different conditions of the cells or tissues.
for instance, lietzen et al.
revealed dramatic changes in secretome of macrophages, such as robust secretion of different danger-associated molecular patterns , in response to influenza a infection  <cit> .
arturo et al.
found that muscle secretion pattern varied according to fiber-type constituents , and the secretome could be affected by endurance exercise training  <cit> .
consequently, secretome represent an important source for biomarker and therapeutic target discovery  <cit> .
for that importance, secretomics, a branch of proteomics, focusing on analyzing the profile of all proteins secreted from cells or tissues, has been developed in recent years  <cit> .
in addition, recent studies have showed that secretory proteins are also important for certain disease conditions.
for example, dysregulation of adipocytokines , heparin-binding epidermal growth factor-like growth factor) and adiponectin contributes to the development of a variety of cardiovascular disease  <cit> .
similarly, secretory proteins also play a role in infectious disease.
for instance, changes in the expression of secretory proteins during latent human cytomegalovirus  infection have profound effects on the regulation of the host immune response, such as recruitment of cd4+ t cells by increasing the expression of cc chemokine ligand  <dig>   <cit> .
also, the secreted ifn-induced proteins , ifit <dig>  signal transducer and activator of transcription  <dig> ) were indicated to have important extracellular antiviral functions during herpes simplex virus  <dig>  infection  <cit> .
together, these data indicate the important role of secretory proteins in host-pathogen interaction.
however, although m. pneumoniae infection is a common cause of respiratory disease, secretome change during m. pneumoniae infection had not been thoroughly investigated.
airway epithelial cells form the first line of defense against exposure to infectious agents.
epithelial cells are known to kill or neutralize microorganisms through the production of enzymes, permeabilizing peptides, collectins, and protease inhibitors during the innate immune response  <cit> .
epithelial cells are also essential in regulating adaptive immune responses in the airways by expressing pattern-recognition receptors  to trigger host defense response, by activating dendritic cells to regulate ag sensitization, and by releasing cytokines to recruit effector cells  <cit> .
thus, airway epithelial cells are important for the initiation, maintenance, and regulation of both innate and adaptive immune responses, as well as modulating the transition from innate to adaptive immunity.
as the interaction of m. pneumoniae with respiratory epithelial cells is a critical early step of pathogenesis  <cit> , and considering the importance of secretory proteins, a large-scale study on m. pneumoniae-induced protein secretion will help elucidate the molecular mechanisms related to m. pneumoniae infection.
therefore, in the current study, we applied liquid chromatography-tandem mass spectrometry  based label-free quantitative shotgun proteomics approach for global profiling of the m. pneumoniae-infected human alveolar epithelial carcinoma a <dig> cell secretome, in an effort to provide a better view of host-pathogen interaction and identify novel molecules/biomarkers for m. pneumoniae infection.
as reported here, we have identified  <dig> proteins affected by m. pneumoniae infection.
furthermore, we evaluated the clinical application of one identified protein, il- <dig>  as a “proof of concept” example, and the result showed that it could help to distinguish m. pneumoniae pneumonia  patients from non-m. pneumoniae patients.
RESULTS
label-free quantitative shotgun proteomic analysis of cell secretome upon m. pneumoniae infection
the study design is outlined in figure  <dig>  both cell viability and apoptosis assay revealed that serum free medium  did not significantly affect cell integrity and secretion capacity within 24 h , and thus serum-free culture for 24 h was chosen as the time point for secretome collection.
based on the lc-ms/ms data,  <dig> proteins were identified in control a <dig> cells, with  <dig> being identified from all three biological replicates , indicating a relatively good reproducibility.
similarly,  <dig> proteins were identified in m. pneumoniae-infected a <dig> cells, with  <dig> being identified from all three biological replicates .
thus, a total of  <dig> proteins were identified, among which  <dig> proteins were detected in both groups, with  <dig> and  <dig> proteins being uniquely secreted by control cells and m. pneumoniae-infected cells, respectively .
complete protein identification lists for control and m. pneumoniae-infected cells were provided in additional files  <dig> and 5: datasheet s <dig> and table s <dig>
for the identified proteins, label-free quantitative comparison performed by decyder™ ms differential software revealed that  <dig> proteins were significantly affected by m. pneumoniae infection  .
specifically, there were  <dig> up-regulated and  <dig> down-regulated proteins in m. pneumoniae-infected a <dig> cells, among which  <dig> were uniquely expressed in m. pneumoniae-treated a <dig> and  <dig> in control a <dig> cells.
for all  <dig> differential proteins, the number of peptides for each protein used for quantification varied from  <dig> to  <dig>  among them,  <dig> proteins were quantified on the basis of two or more peptides, with average coefficient of variation  of the fold changes for peptides as  <dig> % , demonstrating a rational reproducibility of the quantitative data.
the rest  <dig> proteins were quantified with only one peptide by the decyder software.
validation of proteins with changed expression during m. pneumoniae infection
to verify the proteomic results, real-time pcr and western blot analysis were performed on several identified proteins.
the real-time pcr results demonstrated that the gene expression levels of  <dig> secretory proteins exhibited the same trend of changes as the quantitative ms results .
also, western blot data showed that protein levels of six secretory proteins were significantly increased in the cm and total cell lysates after m. pneumoniae-infection, which were consistent with the proteomic results .
therefore, from the rt-pcr and western blot results, we found that these six secretory proteins  were overexpressed in m. pneumoniae-infected a <dig> cells at mrna and protein levels.
cellular localization of the identified proteins
the  <dig> identified proteins were first categorized as classical secretory proteins or non-classical secretory proteins based on singalp and secretomep analysis.
of the  <dig> proteins,  <dig> were categorized as classical secretory proteins and  <dig> as non-classical secretory proteins .
to determine whether some of the proteins could also be released via exosomes, the exocarta exosome database were searched  <cit> .
the results showed that among the proteins identified,  <dig> proteins were also listed in the exosomal protein database .
we next analyzed the ontology of the identified proteins based on cellular compartment.
the results showed majority of the proteins belong to more than one go class .
most of the proteins have a nuclear distribution .
functional annotation clustering analysis by david  <dig>  showed that when considering only cellular compartment distribution, the proteins of the extracellular region, vesicle and extracellular matrix were over-represented  of  <dig> ,  <dig> , and  <dig> , respectively) .
similarly, the classification based on the cellular organelle of the differentially expressed proteins also showed that m. pneumoniae infection did not induce protein secretion from any specific cell organelle, but rather, altered the overall secretion of proteins from all the main organelles, including mitochondrion and lysosome .
enrichment in proteins residing in the extracellular region, especially extracellular matrix, extracellular space, and membrane-bound vesicle was observed .
moreover, when p value <  <dig>  was set as the threshold,  <dig> go terms for up-regulated proteins and  <dig> for down-regulated proteins were obtained, respectively .
functional classification of the differentially expressed secretory proteins
to better understand the nature of the differentially expressed proteins, the kegg database was used for pathway analysis, which evaluates the relative importance of the change in a pathway/function in response to treatment and/or change in physiological state.
eleven pathways were listed in the kegg database  after m. pneumoniae infection, of which  <dig> were significantly over-represented  .
the significantly over-represented kegg pathways were related to metabolism, infection, and proliferation .
mycoplasma pneumoniae
infection
p
in addition, an enrichment analysis was performed by bingo for the differentially expressed proteins.
for the up-regulated proteins, go analysis revealed  <dig> molecular function go categories associated with oxidoreductase activity,  <dig> related to protein binding, and  <dig> linked to enzyme regulator activity , while for the down-regulated proteins,  <dig> go terms were identified, which were mainly associated with enzyme inhibitor activity and hydrolase activity .
biological processes 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 .
for the down-regulated proteins, system development seemed to be the most relevant biological process .
based on the bingo biological process analysis, we identified  <dig> up-regulated proteins that were involved in immune response, and  <dig> up-regulated proteins might act as endogenous danger signals  <cit>  .
mycoplasma pneumoniae
infection
protein-protein interaction network analysis during m. pneumoniae infection
as mentioned earlier, pathogen-host interaction is a very complex process and many proteins are involved.
also, biological association network changes in protein expression are not isolated events  <cit> .
therefore, in this study, we want to know how differentially expressed proteins interact with each other and how they affect cell’s function during m. pneumoniae infection.
the biological associations among the differentially expressed proteins were investigated using the string software.
the predicted protein-protein associations were queried through a vast number of databases derived in different ways   <cit> .
as shown in figure  <dig>  for the  <dig> up-regulated proteins, three main networks of protein interactions were identified, including stress response proteins , signaling pathway associated proteins , and cellular metabolic proteins .
for the  <dig> down-regulated proteins, two major networks of the associated proteins were found, including the glucose catabolic proteins  and biological process negative regulation associated proteins  .
clinical examination of il- <dig> expression in plasma and balf
to explore the potential clinical application of secretomic data, we selected one of the identified proteins, il- <dig>  and examined its expression in the plasma and balf of mpp patients, using age-matched patients with foreign body as comparison cohort.
patient information was listed in table  <dig>  first it was shown that il- <dig> secretion was induced in a <dig> cells by m. pneumoniae infection .
results from the measurements of patient samples also showed that il- <dig> level was significantly higher in both plasma and balf of mpp patients than those in patient with foreign body .
to further evaluate whether the increased plasma il- <dig> levels had any potential clinical significance as a possible biomarker for helping distinguish mpp patients from controls, a receiver operating characteristic  curve was constructed by plotting sensitivity vs. specificity.
the area under the roc curve , a commonly used indicator for estimating the diagnostic efficacy of a potential biomarker, was subsequently calculated.
for differentiating mpp patients from controls, the auc was determined to be  <dig>   for plasma il- <dig> .
when a cutoff value of  <dig>  pg/ml was set for plasma il- <dig>  the sensitivity and specificity for discriminating mpp patients from controls were  <dig> % and  <dig> %, respectively.
p
data were expressed as mean ± sd.
these variables were compared using student’s t-test or mann–whitney u test.
discussion
by using comprehensive ms-based proteomics combined with label-free quantitation algorithms, we examined the secretome of m. pneumoniae-infected and uninfected a <dig> 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  <cit> .
as reported here, a total of  <dig> proteins were identified, among which  <dig> were differentially secreted by m. pneumoniae-infected a <dig> cells versus control.
this result is similar to a study conducted by brioschi et al., in which  <dig> proteins were identified and  <dig> differentially expressed in the endothelial cell secretome upon reductase inhibitor treatment  <cit> .
among the identified proteins,  <dig> proteins were designated as putative secretory proteins by using signalp and secretomep.
interestingly,  <dig> out of the  <dig> 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  <cit> , it was shown that 74%  of the identified proteins in our study can be found in the exocarta database, highlighting a critical role for exosome in cell-cell communication  <cit> .
in summary, up to 92%  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 , those proteins, which were not classified as secretory proteins using the computational approach , should be released mainly by intracellular secretion  rather than cell lysis  <cit> .
furthermore, among the  <dig> differentially expressed proteins, about 80%  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  <cit> .
based on string bioinformatics analysis, several clusters of proteins were identified , 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 .
indeed, a number of studies have highlighted the importance of host-dependent inflammatory response to m. pneumoniae infection, such as il- <dig> and ifn-γ production, as well as the th <dig> type t-cell responses in a mouse model  <cit> .
previously we have also shown that the reactive oxygen species  induced by m. pneumoniae infection attributed in part to the cytopathology of the respiratory epithelium  <cit> , and m. pneumoniae infection could influence host’s sphingolipid metabolism, including the generation of new ceramide and sphingomyelin species  <cit> .
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  <cit> .
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  <cit> .
interestingly, we have found  <dig> out of the  <dig> differentially expressed proteins were associated with stress and may act as endogenous danger signals   <cit> , including heat shock protein beta- <dig> , galectin- <dig> , galectin-3-binding protein , serpine <dig>  disintegrin and metalloproteinase domain-containing protein  <dig> , peroxiredoxin- <dig> , and prdx <dig>  several of these danger signal proteins, such as hsps, galectins, and redox-related members, were also secreted during influenza a virus or hsv- <dig> infection of human macrophages  <cit> .
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 adam <dig>  gal- <dig> and serpine <dig>  <cit> .
in line with such observation, m. pneumoniae is known to induce ros production and reduce glutathione levels in lung and lung carcinoma cells  <cit> .
furthermore, m. pneumoniae can inhibit host cell catalase, which could result in the toxicity of hydrogen peroxide in skin fibroblast and ciliated epithelial cells  <cit> .
together, these results implicate that the enhanced ros production should be recognized as an important mechanism in the pathogenesis of m. pneumoniae infection  <cit> .
in addition, many identified proteins were involved in extracellular matrix formation .
extracellular matrix plays an important role in regulating many cellular functions like adhesion, cell shape, migration, proliferation, polarity, differentiation, and apoptosis  <cit> .
for example, serpine <dig>  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  <cit> .
similarly, adam <dig>  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  <cit> .
gal- <dig> also displays diverse biological activities including cell adhesion, b cell development, mrna splicing, angiogenesis and tissue differential/homeostasis, and inflammation  <cit> .
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  <cit> .
to do that, we chose one of the identified proteins, il- <dig>  and conducted a “proof-of-concept” experiment.
il- <dig>  a crucial amplifier of the innate immunity in infectious diseases as well as in autoimmune processes, is also a recently identified damp  <cit> .
it has been shown that il- <dig> plays an important role in driving antiviral cd8+ t cell responses in lymphocytic choriomeningitis virus-infected mice  <cit> .
during the experimental intestinal nematodes  infection in mice, il- <dig> was markedly elevated soon after infection  <cit> .
schmitz and co-workers demonstrated that injection of il- <dig> into mice induced a profound eosinophilia with associated pathologic changes  <cit> , and had potent effects on eosinophil, including the induced production of superoxide anion and il- <dig>  degranulation and eosinophil survival  <cit> .
we found m. pneumoniae significantly increased il- <dig> production in a <dig> cells, and il- <dig> levels were significantly higher in mpp patients, implying an important role for il- <dig> in m. pneumoniae-elicited immune response .
further roc analysis revealed that il- <dig> could help distinguish mpp patients from patients with foreign objects.
thus, manipulation of il- <dig> might represent a promising new therapeutic strategy for treating the inflammatory disorder during m. pneumoniae infection.
CONCLUSIONS
in the current study, we identified many differentially expressed secretory proteins during m. pneumoniae infection using the quantitative label-free ms method, through which complex regulatory networks have been revealed.
some of the proteins could be used as lead candidates for further functional and preclinical evaluation for their roles in m. pneumoniae infection.
such information will shed new light into the study of host response during m. pneumoniae infection for better understanding the underlying molecular mechanisms.

 mycoplasma pnuemoniae belongs to the class of the mollicutes and is one of the smallest free-living organisms. it is a major cause of community-acquired pneumonia worldwide in all age groups, and can also induce manifestations in extrapulmonary sites involving almost all organs of the human body. with the exception of m. pneumoniae adherence to the host epithelium, molecular mechanisms underlying the pathogenesis of m. pneumoniae infection has long been a mystery. subsequent to cytadherence, m. pneumoniae is believed to cause disease in part through the generation of peroxide and the induction of inflammatory reaction including cytokine productions. simultaneously, autoimmunity developed after m. pneumoniae infection likely contributes to the extrapulmonary complications. for example, anti-gm and galactocerebroside antibodies are the primary autoantibodies implicated in the ascending paralysis of guillain-barre syndrome and in encephalitis associated with m. pneumoniae. although toxin had not been considered as part of the m. pneumoniae repertoire in previous studies, recent evidence suggested otherwise. a newly identified exotoxin of m. pneumoniae, named community-acquired respiratory distress syndrome toxin, which has adp-ribosylating and vacuolating activity, has been suggested to be responsible for eliciting extensive vacuolization and ciliostasis of host cells. thus, the pathophysiology of m. pneumoniae infection is likely to be complex and multifactorial, and the underlying molecular mechanisms should involve a large number of genes/proteins participating in various biological pathways. high-throughput technologies including genomics and proteomics can comprehensively and quantitatively decipher gene/protein expression, and therefore, are useful tools in the study of complex systems under the influence of biological perturbations, such as pathogen-host interaction. previously, using a proteomic approach, we had analyzed m. pneumoniae-induced protein expression profile using whole cell lysates, and identified the redox regulatory pathway as a key target during m. pneumoniae infection. however, as noted above, m. pneumoniae-induced immune response is important for m. pneumoniae pathogenesis, and many factors involved in the immune response, such as the cytokines, are so-called secretory proteins, which are part of the “secretome”. secretome proteins include extracellular matrix proteins, growth factors, cytokines and hormones, and other soluble mediators. it is known that secretory proteins are important for many physiological processes. for example, the matrix metalloproteinases, as extracellular matrix-degrading enzymes, are essential regulators of the cell’s microenvironment governing cell fate and function, such as cell migration, proliferation, apoptosis, invasion and development. moreover, changes in secretory proteins can reflect different conditions of the cells or tissues. for instance, lietzen et al. revealed dramatic changes in secretome of macrophages, such as robust secretion of different danger-associated molecular patterns, in response to influenza a infection. arturo et al. found that muscle secretion pattern varied according to fiber-type constituents, and the secretome could be affected by endurance exercise training. consequently, secretome represent an important source for biomarker and therapeutic target discovery. for that importance, secretomics, a branch of proteomics, focusing on analyzing the profile of all proteins secreted from cells or tissues, has been developed in recent years. in addition, recent studies have showed that secretory proteins are also important for certain disease conditions. for example, dysregulation of adipocytokines, heparin-binding epidermal growth factor-like growth factor) and adiponectin contributes to the development of a variety of cardiovascular disease. similarly, secretory proteins also play a role in infectious disease. for instance, changes in the expression of secretory proteins during latent human cytomegalovirus infection have profound effects on the regulation of the host immune response, such as recruitment of cd4+ t cells by increasing the expression of cc chemokine ligand. also, the secreted ifn-induced proteins, ifit signal transducer and activator of transcription ) were indicated to have important extracellular antiviral functions during herpes simplex virus infection. together, these data indicate the important role of secretory proteins in host-pathogen interaction. however, although m. pneumoniae infection is a common cause of respiratory disease, secretome change during m. pneumoniae infection had not been thoroughly investigated. airway epithelial cells form the first line of defense against exposure to infectious agents. epithelial cells are known to kill or neutralize microorganisms through the production of enzymes, permeabilizing peptides, collectins, and protease inhibitors during the innate immune response. epithelial cells are also essential in regulating adaptive immune responses in the airways by expressing pattern-recognition receptors to trigger host defense response, by activating dendritic cells to regulate ag sensitization, and by releasing cytokines to recruit effector cells. thus, airway epithelial cells are important for the initiation, maintenance, and regulation of both innate and adaptive immune responses, as well as modulating the transition from innate to adaptive immunity. as the interaction of m. pneumoniae with respiratory epithelial cells is a critical early step of pathogenesis, and considering the importance of secretory proteins, a large-scale study on m. pneumoniae-induced protein secretion will help elucidate the molecular mechanisms related to m. pneumoniae infection. therefore, in the current study, we applied liquid chromatography-tandem mass spectrometry based label-free quantitative shotgun proteomics approach for global profiling of the m. pneumoniae-infected human alveolar epithelial carcinoma a cell secretome, in an effort to provide a better view of host-pathogen interaction and identify novel molecules/biomarkers for m. pneumoniae infection. as reported here, we have identified proteins affected by m. pneumoniae infection. furthermore, we evaluated the clinical application of one identified protein, il- as a “proof of concept” example, and the result showed that it could help to distinguish m. pneumoniae pneumonia patients from non-m. pneumoniae patients.  label-free quantitative shotgun proteomic analysis of cell secretome upon m. pneumoniae infection the study design is outlined in figure  both cell viability and apoptosis assay revealed that serum free medium did not significantly affect cell integrity and secretion capacity within 24 h, and thus serum-free culture for 24 h was chosen as the time point for secretome collection. based on the lc-ms/ms data, proteins were identified in control a cells, with being identified from all three biological replicates, indicating a relatively good reproducibility. similarly, proteins were identified in m. pneumoniae-infected a cells, with being identified from all three biological replicates. thus, a total of proteins were identified, among which proteins were detected in both groups, with and proteins being uniquely secreted by control cells and m. pneumoniae-infected cells, respectively. complete protein identification lists for control and m. pneumoniae-infected cells were provided in additional files and 5: datasheet s and table s for the identified proteins, label-free quantitative comparison performed by decyder™ ms differential software revealed that proteins were significantly affected by m. pneumoniae infection. specifically, there were up-regulated and down-regulated proteins in m. pneumoniae-infected a cells, among which were uniquely expressed in m. pneumoniae-treated a and in control a cells. for all differential proteins, the number of peptides for each protein used for quantification varied from to among them, proteins were quantified on the basis of two or more peptides, with average coefficient of variation of the fold changes for peptides as %, demonstrating a rational reproducibility of the quantitative data. the rest proteins were quantified with only one peptide by the decyder software. validation of proteins with changed expression during m. pneumoniae infection to verify the proteomic results, real-time pcr and western blot analysis were performed on several identified proteins. the real-time pcr results demonstrated that the gene expression levels of secretory proteins exhibited the same trend of changes as the quantitative ms results. also, western blot data showed that protein levels of six secretory proteins were significantly increased in the cm and total cell lysates after m. pneumoniae-infection, which were consistent with the proteomic results. therefore, from the rt-pcr and western blot results, we found that these six secretory proteins were overexpressed in m. pneumoniae-infected a cells at mrna and protein levels. cellular localization of the identified proteins the identified proteins were first categorized as classical secretory proteins or non-classical secretory proteins based on singalp and secretomep analysis. of the proteins, were categorized as classical secretory proteins and as non-classical secretory proteins. to determine whether some of the proteins could also be released via exosomes, the exocarta exosome database were searched. the results showed that among the proteins identified, proteins were also listed in the exosomal protein database. we next analyzed the ontology of the identified proteins based on cellular compartment. the results showed majority of the proteins belong to more than one go class. most of the proteins have a nuclear distribution. functional annotation clustering analysis by david showed that when considering only cellular compartment distribution, the proteins of the extracellular region, vesicle and extracellular matrix were over-represented of,, and, respectively). similarly, the classification based on the cellular organelle of the differentially expressed proteins also showed that m. pneumoniae infection did not induce protein secretion from any specific cell organelle, but rather, altered the overall secretion of proteins from all the main organelles, including mitochondrion and lysosome. enrichment in proteins residing in the extracellular region, especially extracellular matrix, extracellular space, and membrane-bound vesicle was observed. moreover, when p value <  was set as the threshold, go terms for up-regulated proteins and for down-regulated proteins were obtained, respectively. functional classification of the differentially expressed secretory proteins to better understand the nature of the differentially expressed proteins, the kegg database was used for pathway analysis, which evaluates the relative importance of the change in a pathway/function in response to treatment and/or change in physiological state. eleven pathways were listed in the kegg database after m. pneumoniae infection, of which were significantly over-represented. the significantly over-represented kegg pathways were related to metabolism, infection, and proliferation. mycoplasma pneumoniae infection p in addition, an enrichment analysis was performed by bingo for the differentially expressed proteins. for the up-regulated proteins, go analysis revealed molecular function go categories associated with oxidoreductase activity, related to protein binding, and linked to enzyme regulator activity, while for the down-regulated proteins, go terms were identified, which were mainly associated with enzyme inhibitor activity and hydrolase activity. biological processes 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. for the down-regulated proteins, system development seemed to be the most relevant biological process. based on the bingo biological process analysis, we identified up-regulated proteins that were involved in immune response, and up-regulated proteins might act as endogenous danger signals. mycoplasma pneumoniae infection protein-protein interaction network analysis during m. pneumoniae infection as mentioned earlier, pathogen-host interaction is a very complex process and many proteins are involved. also, biological association network changes in protein expression are not isolated events. therefore, in this study, we want to know how differentially expressed proteins interact with each other and how they affect cell’s function during m. pneumoniae infection. the biological associations among the differentially expressed proteins were investigated using the string software. the predicted protein-protein associations were queried through a vast number of databases derived in different ways. as shown in figure  for the up-regulated proteins, three main networks of protein interactions were identified, including stress response proteins, signaling pathway associated proteins, and cellular metabolic proteins. for the down-regulated proteins, two major networks of the associated proteins were found, including the glucose catabolic proteins and biological process negative regulation associated proteins. clinical examination of il- expression in plasma and balf to explore the potential clinical application of secretomic data, we selected one of the identified proteins, il- and examined its expression in the plasma and balf of mpp patients, using age-matched patients with foreign body as comparison cohort. patient information was listed in table  first it was shown that il- secretion was induced in a cells by m. pneumoniae infection. results from the measurements of patient samples also showed that il- level was significantly higher in both plasma and balf of mpp patients than those in patient with foreign body. to further evaluate whether the increased plasma il- levels had any potential clinical significance as a possible biomarker for helping distinguish mpp patients from controls, a receiver operating characteristic curve was constructed by plotting sensitivity vs. specificity. the area under the roc curve, a commonly used indicator for estimating the diagnostic efficacy of a potential biomarker, was subsequently calculated. for differentiating mpp patients from controls, the auc was determined to be for plasma il-. when a cutoff value of  pg/ml was set for plasma il- the sensitivity and specificity for discriminating mpp patients from controls were % and %, respectively. p data were expressed as mean ± sd. these variables were compared using student’s t-test or mann–whitney u test. discussion by using comprehensive ms-based proteomics combined with label-free quantitation algorithms, we examined the secretome of m. pneumoniae-infected and uninfected a 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 proteins were identified, among which were differentially secreted by m. pneumoniae-infected a cells versus control. this result is similar to a study conducted by brioschi et al., in which proteins were identified and differentially expressed in the endothelial cell secretome upon reductase inhibitor treatment. among the identified proteins, proteins were designated as putative secretory proteins by using signalp and secretomep. interestingly, out of the 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% 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% 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, those proteins, which were not classified as secretory proteins using the computational approach, should be released mainly by intracellular secretion rather than cell lysis. furthermore, among the differentially expressed proteins, about 80% 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, 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. indeed, a number of studies have highlighted the importance of host-dependent inflammatory response to m. pneumoniae infection, such as il- and ifn-γ production, as well as the th type t-cell responses in a mouse model. previously we have also shown that the reactive oxygen species 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. interestingly, we have found out of the differentially expressed proteins were associated with stress and may act as endogenous danger signals, including heat shock protein beta-, galectin-, galectin-3-binding protein, serpine disintegrin and metalloproteinase domain-containing protein, peroxiredoxin-, and prdx several of these danger signal proteins, such as hsps, galectins, and redox-related members, were also secreted during influenza a virus or hsv- infection of human macrophages. 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 adam gal- and serpine. in line with such observation, m. pneumoniae is known to induce ros production and reduce glutathione levels in lung and lung carcinoma cells. 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. extracellular matrix plays an important role in regulating many cellular functions like adhesion, cell shape, migration, proliferation, polarity, differentiation, and apoptosis. for example, serpine 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, adam 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- 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- and conducted a “proof-of-concept” experiment. il- a crucial amplifier of the innate immunity in infectious diseases as well as in autoimmune processes, is also a recently identified damp. it has been shown that il- plays an important role in driving antiviral cd8+ t cell responses in lymphocytic choriomeningitis virus-infected mice. during the experimental intestinal nematodes infection in mice, il- was markedly elevated soon after infection. schmitz and co-workers demonstrated that injection of il- 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- degranulation and eosinophil survival. we found m. pneumoniae significantly increased il- production in a cells, and il- levels were significantly higher in mpp patients, implying an important role for il- in m. pneumoniae-elicited immune response. further roc analysis revealed that il- could help distinguish mpp patients from patients with foreign objects. thus, manipulation of il- might represent a promising new therapeutic strategy for treating the inflammatory disorder during m. pneumoniae infection.  in the current study, we identified many differentially expressed secretory proteins during m. pneumoniae infection using the quantitative label-free ms method, through which complex regulatory networks have been revealed. some of the proteins could be used as lead candidates for further functional and preclinical evaluation for their roles in m. pneumoniae infection. such information will shed new light into the study of host response during m. pneumoniae infection for better understanding the underlying molecular mechanisms. 
mycoplasma pneumoniae is one of the major etiological agents for community-acquired pneumonia in all age groups. however, no comprehensive analysis of the secretory proteins has been conducted to date regarding the host response upon m. pneumoniae infection. cellular localization analysis of the secretome data revealed % of the proteins were considered as “putative secretory proteins”. the present study provided systematic information about the changes in the expression of secretory proteins during m. pneumoniae infection, which is useful for the discovery of specific biomarkers and targets for pharmacological intervention. we employed the liquid chromatography-tandem mass spectrometry -based label-free quantitative proteomic technology to identify and characterize the members of the human alveolar epithelial carcinoma a cell secretome during m. pneumoniae infection. the early host response to m. pneumoniae infection relies on the concerted release of proteins with various biological activities. functional analysis revealed that the proteins affected upon m. pneumoniae infection were mainly related to metabolic process, stress response, and immune response. a total of proteins were identified, with being differentially expressed, among which were only expressed in control cells, only in m. pneumoniae-treated cells, while were up-regulated and down-regulated by m. pneumoniae. the result showed that il- levels were significantly higher in the plasma and bronchoalveolar lavage fluid of m. pneumoniae pneumonia patients.
 mycoplasma pneumoniae is one of the major etiological agents for community-acquired pneumonia in all age groups. 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.  we employed the liquid chromatography-tandem mass spectrometry -based label-free quantitative proteomic technology to identify and characterize the members of the human alveolar epithelial carcinoma a cell secretome during m. pneumoniae infection. a total of proteins were identified, with being differentially expressed, among which were only expressed in control cells, only in m. pneumoniae-treated cells, while were up-regulated and down-regulated by m. pneumoniae. the changed expression of some of the identified proteins was validated by rt-pcr and immunoblot analysis. cellular localization analysis of the secretome data revealed % of the proteins were considered as “putative secretory proteins”. functional analysis revealed that the proteins affected upon m. pneumoniae infection were mainly related to metabolic process, stress response, and immune response. we further examined the level of one up-regulated protein, il- in clinical samples. the result showed that il- levels were significantly higher in the plasma and bronchoalveolar lavage fluid of m. pneumoniae pneumonia patients.  the present study provided systematic information about the changes in the expression of secretory proteins during m. pneumoniae infection, which is useful for the discovery of specific biomarkers and targets for pharmacological intervention. mycoplasma pneumoniaesecretomeil-33biological pathway 