Proteomic analysis identifies highly antigenic proteins in exosomes from M. tuberculosis‐infected and culture filtrate protein‐treated macrophages

Abstract

Exosomes are small 30–100 nm membrane vesicles released from hematopoietic and non-hematopoietic cells and function to promote intercellular communication. They are generated through fusion of multivesicular bodies with the plasma membrane and release of interluminal vesicles. Previous studies from our laboratory demonstrated that macrophages infected with Mycobacterium release exosomes that promote activation of both innate and acquired immune responses; however, the components present on exosomes inducing these host responses were not defined. The present study used LC-MS/MS to identify 41 mycobacterial proteins present on exosomes released from M. tuberculosis-infected J774 cells. Many of these proteins have been characterized as highly immunogenic. Further, since most of the mycobacterial proteins identified are actively secreted, we hypothesized that macrophages treated with M. tuberculosis culture filtrate proteins (CFP) would release exosomes containing mycobacterial proteins. We found 29 M. tuberculosis proteins in exosomes released from CFP-treated J774 cells, the majority of which were also present on exosomes isolated from M. tuberculosis-infected cells. The exosomes from CFP-treated J774 cells could promote macrophage and dendritic cell activation as well as activation of naïve T cells in vivo. These results suggest that exosomes containing M. tuberculosis antigens may be alternative approach to developing a tuberculosis vaccine.

Exosomes: New Tuberculosis Biomarkers – Prospects From the Bench to the Clinic

“…Several known secreted proteins previously undetected in exosomes, including KatG (Rv1908c), the Ag85 complex (Rv3804c, Rv1886c and Rv0129c), GroES (Rv3418c) and CFP10 (Rv3874), were also demonstrated by western blot analysis (Giri, Kruh et al 2010). In addition to these proteins, we identified roughly forty mycobacterial proteins by MS, of which 95% were previously defined either experimentally or through predictive algorithms to be secreted (Table 2) (Rosenkrands, King et al 2000;Malen, Berven et al 2007;Giri, Kruh et al 2010). One can hypothesize that the mycobacterial proteins released into the phagosome or cytoplasm are transported to an MVB, incorporated into the interluminal vesicles and released from the infected cell via exosomes into various bodily fluids ( Figure 2).…”

“…However, the mining of exosomederived proteins related to infectious diseases has yet to be exploited. The discovery that mycobacterial products could be found in exosomes release from infected animals led to the first full-scale attempt to characterize the Mtb exo-proteome (comprehensive collection of proteins capable of being secreted from Mtb-infected cells within exosomes) (Giri, Kruh et al 2010). The objective of this research is to fuel biomarker discovery utilizing mass spectrometry based approaches.…”

“…Moreover, while ~8 µg of exosomes is required for each western blot (identifying only one protein per blot), a parallel sample subject to MS analysis, allows for the discrimination of hundreds of proteins of both host and bacterial origin. Giri, Kruh et al 2010, for the extended list. The fifteen most abundant mycobacterial proteins are listed, based on the total number of spectral counts from both insolution and in-gel processing (compiled in the aforementioned paper plus additional unpublished experiments).…”

“…Exosomes were purified from Mtb infected macrophages using differential centrifugation and sucrose gradient (Griffiths, Heesom et al 2007;Giri, Kruh et al 2010). The exosomes contained the characteristic size and shape as defined by EM (Figure 1).…”

Proteomic progress in studying tuberculosis from 2010 to 2011

“…Rv3615c [Esx-1 substrate protein C (EspC)], a protein with similar size and sequence to CFP-10 and ESAT-6, was found to be a highly immunodominant RD1 (Region of Difference 1)-dependent secreted antigen specific for Mycobacterium tuberculosis infection through a quantitative proteomics and metabolically labeled mutant and genetically complemented MTB strains [32]. Giri et al [33] used LC-MS/MS to identify 41 mycobacterial proteins present in exosomes released from M. tuberculosis-infected J774 cells. Another quantitative proteomic analysis showed a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur from their culture filtrate [20].…”