The Mycobacterium tuberculosis Phagosome Is a HLA-I Processing Competent Organelle

Abstract

Mycobacterium tuberculosis (Mtb) resides in a long-lived phagosomal compartment that resists maturation. The manner by which Mtb antigens are processed and presented on MHC Class I molecules is poorly understood. Using human dendritic cells and IFN-γ release by CD8+ T cell clones, we examined the processing and presentation pathway for two Mtb–derived antigens, each presented by a distinct HLA-I allele (HLA-Ia versus HLA-Ib). Presentation of both antigens is blocked by the retrotranslocation inhibitor exotoxin A. Inhibitor studies demonstrate that, after reaching the cytosol, both antigens require proteasomal degradation and TAP transport, but differ in the requirement for ER–golgi egress and new protein synthesis. Specifically, presentation by HLA-B8 but not HLA-E requires newly synthesized HLA-I and transport through the ER–golgi. Phenotypic analysis of the Mtb phagosome by flow organellometry revealed the presence of Class I and loading accessory molecules, including TAP and PDI. Furthermore, loaded HLA-I:peptide complexes are present within the Mtb phagosome, with a pronounced bias towards HLA-E:peptide complexes. In addition, protein analysis also reveals that HLA-E is enriched within the Mtb phagosome compared to HLA-A2. Together, these data suggest that the phagosome, through acquisition of ER–localized machinery and as a site of HLA-I loading, plays a vital role in the presentation of Mtb–derived antigens, similar to that described for presentation of latex bead-associated antigens. This is, to our knowledge, the first description of this presentation pathway for an intracellular pathogen. Moreover, these data suggest that HLA-E may play a unique role in the presentation of phagosomal antigens.

The Mycobacterium bovis Bacille Calmette-Guérin Phagosome Proteome

“…2), consistent with prior reports regarding highly purified latex bead phagosomes from mouse J774 macrophages (14,24). Whether the presence of calreticulin and calnexin in regions of the iodixanol gradient containing BCG phagosomes and latex bead phagosomes reflects a specific contribution of ER to the phagosome, as has been reported in the case of phagosomes containing latex beads, killed bacteria, zymosan, (14, 24 -26), Toxoplasma gondii (27), and M. tuberculosis (28), or instead reflects contamination (29) remains to be determined. Clathrin, present on the plasma membrane and Golgi apparatus (30), was detected in the PNS but was absent from the gradient fractions containing the BCG phagosomes (Fig.…”

“…Most pertinent is the report by Grotzke et al (28) of a specific association of ER components (including transporter associated with antigen processing (TAP) and protein-disulfide isomerase) with the M. tuberculosis phagosome based on flow organellometry and antigen processing and presentation studies.…”

Rv3615c is a highly immunodominant RD1 (Region of Difference 1)-dependent secreted antigen specific for Mycobacterium tuberculosis infection

“…We determined that EspC-derived peptides are recognized by both CD4 + and CD8 + IFN-γ-secreting T cells, induction of the latter being consistent with EspC secretion and access to the cytosolic MHC class I antigen-processing pathway during intracellular infection in vivo (27)(28)(29). The high density of multiple CD4 + and CD8 + epitopes in EspC, with a dominance of CD4 + responses, is reminiscent of ESAT-6 (21, 22, 30, 31) and CFP-10 (21, 22, 30, 31).…”

Antigens for CD4 and CD8 T Cells in Tuberculosis

“…However, most evidence supports that antigenic peptide accesses the cytosol (Behar 2013). Specifically, evidence suggests that in primary human DCs, MTB phagosomes contain class I processing machinery, and antigens contained in the phagosome are retrotranslocated into the cytosol, processed in a proteasome-and TAPdependent manner, and egress to the cell surface through the endoplasmic reticulum Golgi apparatus (Grotzke et al 2009(Grotzke et al , 2010.…”