Live Mycobacterium tuberculosis persists in macrophage phagosomes by interfering with phagolysosome biogenesis. Here, using four-dimensional microscopy and in vitro assays, we report the principal difference between phagosomes containing live and dead mycobacteria. Phosphatidylinositol 3-phosphate (PI3P), a membrane trafficking regulatory lipid essential for phagosomal acquisition of lysosomal constituents, is retained on phagosomes harboring dead mycobacteria but is continuously eliminated from phagosomes with live bacilli. We show that the exclusion of PI3P from live mycobacterial phagosomes can be only transiently reversed by Ca 2؉ fluxes, and that live M. tuberculosis secretes a lipid phosphatase, SapM, that hydrolyzes PI3P, inhibits phagosome-late endosome fusion in vitro, and contributes to inhibition of phagosomal maturation.macrophage ͉ phagosome ͉ tuberculosis ͉ lysosome ͉ phosphatidylinositol 3-phosphate T he infectious cycle of Mycobacterium tuberculosis rests upon the ability of this potent pathogen to parasitize host mononuclear phagocytic cells (1). In infected macrophages, M. tuberculosis resides within a phagosome that avoids the default maturation pathway leading to phagolysosome formation (2). The salient characteristics of the mycobacterial phagosome include (i) paucity of vacuolar H ϩ ATPase (3), (ii) attendant inefficient luminal acidification (3); and (iii) inadequate levels of mature lysosomal hydrolases (3, 4). These and additional (4-6) properties of the M. tuberculosis phagosome promote intracellular survival and growth of the tubercle bacilli and help avoid their immunological detection (1).The arrest of M. tuberculosis phagosome maturation has been studied at the membrane-trafficking level (2), with a focus on the small GTP-binding proteins, including Rab GTPases (7-9). Rabs direct intracellular trafficking by regulating activity and recruitment to organellar membranes of Rab-interacting partners and downstream effectors (10). The initial analyses of Rabs on mycobacterial phagosomes have indicated that the M. tuberculosis phagolysosome biogenesis arrest occurs between the stages controlled by the early endosomal GTPase Rab5 and its late endosomal counterpart Rab7 (7). A number of follow-up studies have indicated critical contributions of Rab5 effectors in mycobacterial phagosome maturation arrest, with a prominent role for the phosphatidylinositol 3-kinase (PI3K) hVPS34, its product phosphatidylinositol 3-phosphate (PI3P), and an array of PI3P-binding proteins (11)(12)(13)(14). PI3P affects localization and function of proteins containing the PI3P-binding domains (FYVE, PH, and PX) (15). These proteins in turn execute various steps in membrane trafficking, endosomal protein sorting, and multisubunit enzyme assembly at the membrane, including phagosomal maturation (11, 16), early endosomal homotypic fusion (17), delivery of internalized plasma membrane receptors to late endosomes (18), formation of internal vesicles within late endosomal multivesicular bodies involved in termination of...

Cytokines are key regulators of adequate immune responses to infection with Mycobacterium tuberculosis. We demonstrate that the p110δ catalytic subunit of PI3K acts as a downstream effector of the TLR family member RP105 (CD180) in promoting mycobacteria-induced cytokine production by macrophages. Our data show that the significantly reduced release of TNF and IL-6 by RP105−/− macrophages during mycobacterial infection was not accompanied by diminished mRNA or protein expression. Mycobacteria induced comparable activation of NF-κB and p38 MAPK signaling in wild-type (WT) and RP105−/− macrophages. In contrast, mycobacteria-induced phosphorylation of Akt was abrogated in RP105−/− macrophages. The p110δ-specific inhibitor, Cal-101, and small interfering RNA–mediated knockdown of p110δ diminished mycobacteria-induced TNF secretion by WT but not RP105−/− macrophages. Such interference with p110δ activity led to reduced surface-expressed TNF in WT but not RP105−/− macrophages, while leaving TNF mRNA and protein expression unaffected. Activity of Bruton’s tyrosine kinase was required for RP105-mediated activation of Akt phosphorylation and TNF release by mycobacteria-infected macrophages. These data unveil a novel innate immune signaling axis that orchestrates key cytokine responses of macrophages and provide molecular insight into the functions of RP105 as an innate immune receptor for mycobacteria.

Glycosylation is the most abundant post-translational polypeptide chain modification in nature. Although carbohydrate modification of protein antigens from many microbial pathogens constitutes important components of B cell epitopes, the role in T cell immunity is not completely understood. Here, using ELISPOT and polychromatic flow cytometry, we show that O-mannosylation of the adhesin, Apa, of Mycobacterium tuberculosis (Mtb) is crucial for its T cell antigenicity in humans and mice after infection. However, subunit vaccination with both mannosylated and non-mannosylated Apa induced a comparable magnitude and quality of T cell response and imparted similar levels of protection against Mtb challenge in mice. Both forms equally improved waning BCG vaccine-induced protection in elderly mice after subunit boosting. Thus, O-mannosylation of Apa is required for antigenicity but appears to be dispensable for its immunogenicity and protective efficacy in mice. These results have implications for the development of subunit vaccines using post-translationally modified proteins such as glycoproteins against infectious diseases like tuberculosis.

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.