Vimentin expressed on Mycobacterium tuberculosis-infected human monocytes is involved in binding to the NKp46 receptor.

We previously showed that human NK cells used the NKp46 receptor to lyse Mycobacterium tuberculosis H37Ra-infected monocytes. To identify ligands on H37Ra-infected human mononuclear phagocytes, we used anti-NKp46 to immunoprecipitate NKp46 from NK cells bound to its ligand(s) on H37Ra-infected monocytes. Mass spectrometry analysis identified a 57-kDa molecule, vimentin, as a putative ligand for NKp46. Vimentin expression was significantly up-regulated on the surface of infected monocytes, compared with uninfected cells, and this was confirmed by fluorescence microscopy. Anti-vimentin antiserum inhibited NK cell lysis of infected monocytes, whereas antiserum to actin, another filamentous protein, did not. CHO-K1 cells transfected with a vimentin construct were lysed much more efficiently by NK cells than cells transfected with a control plasmid. This lysis was inhibited by mAb-mediated masking of NKp46 (on NK cells) or vimentin (on infected monocytes). ELISA and Far Western blotting showed that recombinant vimentin bound to a NKp46 fusion protein. These results indicate that vimentin is involved in binding of NKp46 to M. tuberculosis H37Ra-infected mononuclear phagocytes.

Clinical and molecular analysis of macrolide resistance in Mycobacterium avium complex lung disease.

RATIONALE: The clinical features and outcome of macrolide-resistant Mycobacterium avium complex (MAC) lung disease are not known. OBJECTIVES: Characterize patients, treatment, and isolates in macrolide-resistant MAC lung disease. METHODS: Retrospective chart review, susceptibility testing, molecular fingerprinting, and DNA sequence analyses of resistant MAC isolates. MEASUREMENTS AND MAIN RESULTS: We identified 51 patients over a 15-yr period with clarithromycin-resistant MAC (minimum inhibitory concentration (MIC)>or=32 microg/ml) lung disease at a single referral center. Twenty-four (47%) patients had nodular disease with bronchiectasis and 27 (53%) had upper lobe cavitary disease. Most patients (77%) had M. intracellulare. Sequencing of the 23S r-RNA gene showed 49 of 51 isolates (96%) with the expected mutation in adenine 2058 or 2059. Risk factors for resistance included macrolide monotherapy or combination with a quinolone only (39/51 or 76%). Macrolide resistance developed in 12 of 303 (4.0%) patients started on the American Thoracic Society-recommended two companion drugs, with no risk difference in clarithromycin versus azithromycin and daily versus intermittent therapy. Sputum conversion with macrolide-resistant MAC occurred in 11 of 14 (79%) patients who received more than 6 mo of injectable aminoglycoside therapy and lung resection, compared with 2 of 37 (5%) who did not. The 1-yr mortality in patients who remained culture positive was 34% (13/38) compared with 0% (0/13) of patients who became culture negative (converted). CONCLUSIONS: Macrolide resistance rarely occurs in patients also receiving ethambutol and a rifamycin. Macrolide-resistant MAC lung disease requires aggressive drug and surgical therapy for cure.

Role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with an intracellular bacterium.

We studied the role of NK cell-activating receptors and their ligands in the lysis of mononuclear phagocytes infected with the intracellular pathogen Mycobacterium tuberculosis. Expression of the activating receptors NKp30, NKp46, and NKG2D were enhanced on NK cells by exposure to M. tuberculosis-infected monocytes, whereas expression of DNAX accessory molecule-1 and 2B4 was not. Anti-NKG2D and anti-NKp46 inhibited NK cell lysis of M. tuberculosis-infected monocytes, but Abs to NKp30, DNAX accessory molecule-1, and 2B4 had no effect. Infection of monocytes up-regulated expression of the NKG2D ligand, UL-16 binding protein (ULBP)1, but not expression of ULBP2, ULBP3, or MHC class I-related chain A or chain B. Up-regulation of ULBP1 on infected monocytes was dependent on TLR2, and anti-ULBP1 abrogated NK cell lysis of infected monocytes. The dominant roles of NKp46, NKG2D, and ULBP1 were confirmed for NK cell lysis of M. tuberculosis-infected alveolar macrophages. We conclude that NKp46 and NKG2D are the principal receptors involved in lysis of M. tuberculosis-infected mononuclear phagocytes, and that ULBP1 on infected cells is the major ligand for NKG2D. Furthermore, TLR2 contributes to up-regulation of ULBP1 expression.

Repeat positive cultures in Mycobacterium intracellulare lung disease after macrolide therapy represent new infections in patients with nodular bronchiectasis.

The genomic DNA patterns (genotypes) of 55 episodes of late positive sputum isolates, collected after >or=4 consecutive months of negative sputum cultures, in prospective macrolide treatment trials of Mycobacterium avium complex (MAC) lung disease were assessed by pulsed-field gel electrophoresis (PFGE). Having >or=2 cultures positive for MAC after completion of therapy was documented 23 times; of 20 episodes studied by PFGE, 17 (85%) represented new genotypes (i.e., new infections), and 87% occurred in patients with nodular bronchiectasis. With >or=2 positive cultures after therapy was stopped prematurely, 6 (86%) of 7 episodes were relapses. Single positive cultures after completion of therapy occurred 16 times; only 1 (6%) was predictive of a subsequent relapse. No late isolates were macrolide resistant. Thus, relapses of MAC lung disease with these macrolide regimens are unusual, and most infections after completing therapy resulted from new strains in patients with nodular bronchiectasis.