Tuberculosis toxin blocking phagosome maturation inhibits a novel Ca2+/calmodulin-PI3K hVPS34 cascade

Abstract

The capacity of Mycobacterium tuberculosis to infect latently over one billion people and cause two million fatalities annually rests with its ability to block phagosomal maturation into the phagolysosome in infected macrophages. Here we describe how M. tuberculosis toxin lipoarabinomannan (LAM) causes phagosome maturation arrest, interfering with a new pathway connecting intracellular signaling and membrane trafficking. LAM from virulent M. tuberculosis, but not from avirulent mycobacteria, blocked cytosolic Ca2+ increase. Ca2+ and calmodulin were required for a newly uncovered Ca2+/calmodulin phosphatidylinositol (PI)3 kinase hVPS34 cascade, essential for production of PI 3 phosphate (PI3P) on liposomes in vitro and on phagosomes in vivo. The interference of the trafficking toxin LAM with the calmodulin-dependent production of PI3P described here ensures long-term M. tuberculosis residence in vacuoles sequestered away from the bactericidal and antigen-processing organelles in infected macrophages.

Figure 1.

M. tuberculosis LAM inhibits [Ca²⁺]_c rise. J774 cells were preincubated 30 min without or with 20 μg/ml M. tuberculosis LAM (Mt LAM) or M. smegmatis LAM (Ms LAM). (A) Ratio (340/380 nm) images at different time points after the addition of 1 μM A23187 untreated (control), Mt LAM–treated, or Ms LAM–treated J774 cells. (B) [Ca²⁺]_c kinetics (mean ± SEM, n = 30 cells). Lines: black, untreated cells; red, Mt LAM-treated cells; green, Ms LAM–treated cells. (C) Rate of [Ca²⁺]_c increase ± SEM (n = 3 independent experiments, mean of 30 cells per experiment). (D) Maximum initial [Ca²⁺]_c increase ± SEM (n = 3 independent experiments, mean of 30 cells per experiment). (E) [Ca²⁺]_c kinetics after FcR cross-linking (refer to Materials and Methods) on J774 cells treated with 10 μM DMS (blue line), 20 μg/ml Mt LAM (red line), or control (black line). Mean of 65 cells (same field). FcR clustering revealed by Texas Red anti–mouse F(ab')₂ fragment in presence (E”) or absence (E') of Mt LAM. (F) [Ca²⁺]_c increase upon FcR cross-linking in the absence or presence of 10 μM DMS or 20 μg/ml Mt. LAM. Four independent experiments, mean of 65 cells per experiment, are shown. P-values, paired t test (www.graphpad.com).

Figure 2.

Ca²⁺/calmodulin and CaMKII positively regulate EEA1 recruitment to phagosomes. (A) Immunoblotting analysis and quantitation (mean ± SE) of EEA1 on purified LBC isolated from 25 μM W7-treated or -untreated macrophages. (B–E) Immunofluorescence images of EEA1 recruited to phagosomes (B and D) and corresponding phase contrast images (C and E) in W7-treated (D and E) or -untreated (B and C) macrophages. Triangles indicate EEA1 colocalization with phagosomes. (F) Phagosome colocalization quantitation of EEA1 and Syntaxin 8 by immunofluorescence (mean ± SE; n = 1,145 phagosomes, 35 fields). (G) Immunoblotting analysis of EEA1 and Syntaxin 3 on purified phagosomes from 2 μM KN62-treated and -untreated cells.

Figure 3.

Ca²⁺/calmodulin and PI3 kinase–dependent association of EEA1 with liposomes and Ca²⁺-dependent binding of calmodulin to a protein complex containing PI3 kinase hVPS34. (A) Quantitation (mean ± SE) of EEA1 on phosphatidylserine (PS) and phosphatidylinositol (PI) or PS and PI3P liposomes after incubation with J774 cytosol in the presence or absence of 100 nM PI3 kinase inhibitor wortmannin or 100 μM calmodulin inhibitor W7. (B) Immunoblotting analysis of hVPS34 and p150 on calmodulin agarose beads after incubation with J774 cytosol in the presence of EGTA (lane 1), Ca²⁺ (lanes 2, 3, and 4), W7 (lane 3), and W5 (lane 4).

Figure 4.

Calmodulin positively regulates PI3P on phagosomes. RAW 264.7 macrophages, transfected with the PI3P probe p40PX-EGFP, were imaged live while phagocytosing latex beads using an UltraView confocal microscope. After latex bead internalization and determination of PI3P positivity of the LBC, solvent without (Ctrl) or with 25 μM W7 (+W7) were added to the chamber. Panels: a, d, g, and j, Texas Red–labeled latex beads; b, e, h, and k, GFP probe for PI3P; c, f, i, and l, merged red and green channel images. Note localization of PI3P GFP probe on LBC after bead internalization (a–c and g–i), continued presence of the PI3P GFP probe on phagosomes 10 min after the addition of solvent alone (d–f), and the disappearance of the PI3P GFP probe when W7 was added (j–l). Arrows depict representative LBC in each experimental set. Examples of three independent experiments with comparable results are shown. Also, see Video 1 (available at http://www.jem.org/cgi/content/full/jem.20030527/DC1), which corresponds to panels g–l.