The bacterium Mycoplasma pneumoniae colonizes host pulmonary epithelium and is the most common cause of human community-acquired pneumonia. It successfully avoids detection by the host immune system, as the microbe alters its own cell membrane to mimic its host in order to establish chronic respiratory infections. Progression to autoimmune disease is not unknown.
Initial infection by M. pneumoniae stimulates production and release of proteins by the host cell in response to binding to the infectious agent and its internalization. Li et al. (2014) used a label-free shotgun quantitative proteomics approach to investigate the host secretome.1 By characterizing the biologically active proteins released in this initial phase, the researchers hope to elucidate the pathways involved and the roles of these secretory proteins in disease pathogenesis.
Li and colleagues used the human alveolar carcinoma cell line A549 to establish initial proteomic events following infection by M. pneumoniae. They chose airway epithelial cells because these are the most common primary cells colonized by the microorganism upon infection.
The researchers cultured A549 cells either with or without the infectious agent before harvesting the conditioned media. The scientists analyzed the tryptic digests by nano liquid chromatography–tandem mass spectrometry (LC-MS/MS) using an LTQ Velos ion trap mass spectrometer (Thermo Scientific). The authors searched the data obtained against the IPI human protein database v3.60 to identify proteins secreted by the cell cultures.
Initial LC-MS/MS quantification using DeCyder software showed that 113 out of the 256 proteins identified showed at least 1.5-fold differential expression between the control (uninfected) and the infected cell cultures. Of these, 65 were elevated in abundance and 48 were reduced in the infected cells. Nine proteins were found only in the uninfected control cells, whereas 10 were exclusive to the cells post-infection. Interleukin-33 (IL-33) was one of these proteins, and the researchers confirmed increased levels following infection by utilizing an enzyme-linked immunosorbent assay (ELISA).
The researchers confirmed the proteomics results using Western blotting and real-time polymerase chain reaction (PCR) for selected proteins. Immunoblotting identified the same proteins in cell lysates and conditioned media, showing results consistent with the LC-MS/MS findings for proteins ADAM9, SERPINE1, IL-33, IGFBP4, Gal-1, and MIF, which were more abundant in conditioned media from infected cells.
Out of the 256 identified, over 59% (n = 152) of the proteins were classified as either classical secretory (n = 83) or non-classical secretory (n = 69). A total of 190 proteins were associated with exosomes. Further analysis using GO classification showed that the majority were nuclear-associated. The researchers used DAVID 6.7 for functional annotation clustering analysis and found that most proteins were associated with vesicles or were from the extracellular region or matrix.
Using the KEGG database for pathway analysis, the researchers found that those associated with metabolism, infection and proliferation were over-represented in the proteins identified post-infection. Li and co-workers further analyzed their data to discover more about the functional processes targeted by the post-infection secretome, using the BiNGO tool and STRING algorithm to examine differentially expressed proteins. The STRING analysis highlighted clusters involving stress and immune response pathways, among others. In summary, pathway analyses gave the researchers indications of where M. pneumoniae could induce protein secretion that alters the host cell function.
Finally, Li and co-authors repeated their investigation and found similar proteins in broncheoaveolar lavage samples and plasma from patients with confirmed M. pneumoniae infection. When they measured IL-33 in these two samples, they found elevated levels compared with a control group of patients who presented with respiratory foreign body. Statistical testing showed that IL-33 could indeed be used as a diagnostic marker of infection.
Overall, Li et al. are confident that, by characterizing the post-infection secretome, they have uncovered new regulatory pathways in the host response to M. pneumoniae that can be explored further to elucidate disease pathogenesis and treatment options.
1. Li, S., et al. (2014, February) “Global secretome characterization of A549 human alveolar epithelial carcinoma cells during Mycoplasma pneumoniae infection,” BMC Microbiology, 14(27), doi: 10.1186/1471-2180-14-27.
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