Mtb antigens can be processed via a cytosolic pathway [16], potentially accessing the cytosol through a porous phagosome [17],[18]. After polymerization, 60- to 80-nm thin sections were cut on a Reichert ultramicrotome, stained 5 min in lead citrate, rinsed, poststained 30 min in uranyl acetate, rinsed, and dried. to LBPs (Fig. In accordance with this, we detected HLA-E:peptide complexes, but fewer HLA-B44:CFP102–11 complexes within the Mtb phagosome. This would give Mtb antigens access to the cytosol where they would be processed as endogenously expressed antigens. bovis BCG with the CaMKII-dependent oscillations on phagosomes is a crucial event in the M. tuberculosis phagosome maturation arrest. 3, A-C, green bar). Transfected RAW 264.7 cells grown on 25-mm glass coverslips were allowed to phagocytose latex beads or M. tuberculosis var. Early magnetic bead phagosomes were either LAMP-1lo/−/HLA-I+, LAMP-1+/HLA-Ilo/−, or double negative (Figure 3B). Arrows indicate the gated population. Division of Infectious Diseases, Department of Pediatrics, Oregon Health & Science University, Portland, Oregon, United States of America, Affiliations bovis BCG. This excludes phagosomal escape as a confounding factor in our interpretations. concomitant association with any surrounding vesicles. With some variation in the length of phase I*, the oscillatory increases and decreases of P40PX-EGFP and 2xFYVE-EGFP binding to phagosomal membrane, which were associated Our finding of divergent processing pathways during Mtb infection, and the enhanced availability of HLA-E in the phagosome suggests that HLA-E may play a specialized role in the presentation of phagosomal antigens. First discovered in 1882 by Robert Koch, M. tuberculosis has an unusual, waxy coating on its cell surface primarily due to the presence of mycolic acid. Infection of DC with adenovirus-ICP47 led to a 25–40% reduction of cell surface HLA-I compared to mock or empty vector transduced cells based on geometric mean fluorescent intensity, while cell surface HLA-II expression was not significantly affected (data not shown). However, it is intriguing to note that HLA-E and the murine ortholog Qa-1b are less stable than classical MHC-I molecules and have a higher turnover rate [36],[37]. had sealed, albeit such LBPs seemed to remain juxtaposed and possibly tethered to the plasma membrane. 9 (panels M-R) shows that KN62 did not prevent phase I burst but did prevent phase II PI3P waves (Fig. Previous studies have shown that mycobacterial phagosomes display a reduced association with EEA1 (Fratti et al. Additionally, these data are not consistent with simple contamination. No, Is the Subject Area "Mycobacterium tuberculosis" applicable to this article? https://doi.org/10.1371/journal.ppat.1000374.g001. the endomembranes in macrophages. demonstration that treatments with phosphatidylinositol 3-kinase inhibitors prevent phagosomal maturation (23). 8, D-F). 2016 Jun;19(6):579-84. phagosomes was determined using methods of unbiased counting (27). Phagosomes were purified by passing the postnuclear supernatant over a Miltenyi MS column on ice. In the last decade, it has been established that the tuberculosis-causing bacterium, Mycobacterium tuberculosis, produces two pili types: curli and type IV pili. 7, A-C) but was well visible on MBPs (Fig. 2016 Oct;162(10):1784-1796. doi: 10.1099/mic.0.000368. fluorescence intensity of probe. “0” denotes closure of the phagosomal cup. Overall, these data indicate that while both classically and non-classically restricted Mtb antigens are processed in the cytosol, these antigens utilize distinct presentation pathways. Two inhibitors, significance of phosphatidylinositol (PI) derivatives in phagosome and phagolysosome biogenesis. The addition of freeze-thawed, magnetically-purified Mtb phagosomes to D160 1-23 led to IFN-γ production (Figure 7D), demonstrating the presence of loaded HLA-E:peptide complexes in the Mtb phagosome. Alternatively, enzymes converting PI3P into phosphatidylinositol 3,5 bisphosphate, Acid-fast stains such as Ziehl-Neelsen, or fluorescent stains such as auraminea… Clipboard, Search History, and several other advanced features are temporarily unavailable. of chase indicated. In addition to HLA-I (Figure 3), the Mtb phagosome contained loading accessory molecules TAP1, TAP2, and PDI, as well as proteins containing the KDEL ER retrieval sequence (Figure 4A and 4B). average maximum amplitude corresponding to 41% (± 4%) of the phase I peak (corrected for a background photobleaching decline) Proteins can access the cytosol through the phagosomal acquisition of the retrotranslocation machinery from the ER, which serves to transport antigens into the cytosol [11].