Processing and Presentation of a Mycobacterial Antigen 85B Epitope by Murine Macrophages Is Dependent on the Phagosomal Acquisition of Vacuolar Proton ATPase and In Situ Activation of Cathepsin D

Abstract

Mycobacterium tuberculosis (strain H37Rv) and bacillus Calmette-Guérin (BCG) vaccine inhibit phagosome maturation in macrophages and their effect on processing, and presentation of a secreted Ag85 complex B protein, Ag85B, by mouse macrophages was analyzed. Macrophages were infected with GFP-expressing mycobacterial strains and analyzed for in situ localization of vacuolar proton ATPase (v-ATPase) and cathepsin D (Cat D) using Western blot analysis and immunofluorescence. H37Rv and BCG phagosomes excluded the v-ATPase and maintained neutral pH while the attenuated H37Ra strain acquired v-ATPase and acidified. Mycobacterial phagosomes acquired Cat D, although strains BCG and H37Rv phagosomes contained the inactive 46-kDa form, whereas H37Ra phagosomes had the active 30-kDa form. Infected macrophages were overlaid with a T cell hybridoma specific for an Ag85B epitope complexed with MHC class II. Coincident with active Cat D, H37Ra-infected macrophages presented the epitope to T cells inducing IL-2, whereas H37Rv- and BCG-infected macrophages were less efficient in IL-2 induction. Bafilomycin inhibited the induction of macrophage-induced IL-2 from T cells indicating that v-ATPase was essential for macrophage processing of Ag85B. Furthermore, the small interfering RNA interference of Cat D synthesis resulted in a marked decrease in the levels of macrophage-induced IL-2. Thus, a v-ATPase-dependent phagosomal activation of Cat D was required for the generation of an Ag85B epitope by macrophages. Reduced processing of Ag85B by H37Rv- and BCG-infected macrophages suggests that phagosome maturation arrest interferes with the efficient processing of Ags in macrophages. Because Ag85B is immunodominant, this state may lead to a decreased ability of the wild-type as well as the BCG vaccine to induce protective immunity.

Dormant Mycobacterium tuberculosis Fails To Block Phagosome Maturation and Shows Unexpected Capacity To Stimulate Specific Human T Lymphocytes

“…In agreement with previous data, confocal microscopy showed most R-Mtb in compartments where vATPase was excluded (28). Conversely, D-Mtb was found colocalized with vATPase with a frequency that increased in the time course of infection (Fig.…”

Mycobacterium bovis Bacillus Calmette-Guérin Secreting Active Cathepsin S Stimulates Expression of Mature MHC Class II Molecules and Antigen Presentation in Human Macrophages

“…It is generally accepted that the maturation status of the microbial phagosome dictates its ability to generate peptide-MHC class II complexes in human macrophages (36,37) and this concept would apply also to infection with the vaccine strain BCG. In fact, given the innate tendency of BCG to prevent phagosome maturation (12,57) and acidification (37) one might expect that this vaccine may only present a part of its Ag repertoire in vivo, depending on its ability to block maturation.…”

“…Maturation of microbial phagosomes to phagolysosomes contributes significantly to the normal processing and presentation of Ags to elicit adaptive immunity (4,(35)(36)(37). However, Mtb and BCG strains often block this process to ensure persistence and replication within the macrophage (12,38).…”

Human V-ATPase gene can protect or predispose the host to pulmonary tuberculosis

“…The pathogen inhibits phagosome maturation excluding V-ATPase from the phagosome. 6,7 Bafilomycin A 1 (an inhibitor of V-ATPase) reduces significantly the intracellular killing of phagocytosed M tuberculosis 8 and the production of reactive oxygen species. 8 Both these activities contribute significantly to the antimicrobial activity of the phagocytic cell.…”

“…8 Exclusion of V-ATPase from phagosomes also blocks the processing of Mycobacterium antigens by the macrophages. [7][8][9] Thus, M. tuberculosis curbs both the functions of macrophages, as phagocytic cells and as antigenpresenting cells. In addition, V-ATPase suppresses the expression of macrophage-derived pro-inflammatory cytokines, in particular of TNF-a, 10 which is essential for the control of M. tuberculosis infection: too little as well as too much production of this cytokine predisposes to pulmonary tuberculosis.…”