Mycobacterium tuberculosis interacts with macrophages and epithelial cells in the alveolar space of the lung, where it is able to invade and replicate in both cell types. M. tuberculosis -associated cytotoxicity to these cells has been well documented, but the mechanisms of host cell death are not well understood. We examined the induction of apoptosis and necrosis of human macrophages (U937) and type II alveolar epithelial cells (A549) by virulent (H37Rv) and attenuated (H37Ra) M. tuberculosis strains. Apoptosis was determined by both enzyme-linked immunosorbent assay (ELISA) and TdT-mediated dUTP nick end labelling (TUNEL) assay, whereas necrosis was evaluated by the release of lactate dehydrogenase (LDH). Both virulent and attenuated M. tuberculosis induced apoptosis in macrophages; however, the attenuated strain resulted in significantly more apoptosis than the virulent strain after 5 days of infection. In contrast, cytotoxicity

Mycobacterium avium is widely distributed in the environment, and it is chiefly found in water and soil. M. avium, as well as Mycobacterium smegmatis, has been recognized to produce a biofilm or biofilm-like structure. We screened an M. avium green fluorescent protein (GFP) promoter library in M. smegmatis for genes involved in biofilm formation on polyvinyl chloride (PVC) plates. Clones associated with increased GFP expression >2.0-fold over the baseline were sequenced. Seventeen genes, most encoding proteins of the tricarboxylic acid (TCA) cycle and GDP-mannose and fatty acid biosynthesis, were identified. Their regulation in M. avium was confirmed by examining the expression of a set of genes by real-time PCR after incubation on PVC plates. In addition, screening of 2,000 clones of a transposon mutant bank constructed using M. avium strain A5, a mycobacterial strain with the ability to produce large amounts of biofilm, revealed four mutants with an impaired ability to form biofilm. Genes interrupted by transposons were homologues of M. tuberculosis 6-oxodehydrogenase (sucA), enzymes of the TCA cycle, protein synthetase (pstB), enzymes of glycopeptidolipid (GPL) synthesis, and Rv1565c (a hypothetical membrane protein). In conclusion, it appears that GPL biosynthesis, including the GDP-mannose biosynthesis pathway, is the most important pathway involved in the production of M. avium biofilm.Mycobacterium avium complex is widely distributed in the environment, such as in water and soil, and is a chief component of many natural aquatic biofilms (8). M. avium is also known to cause chronic pulmonary infection in patients with predisposing lung disease, such as previous tuberculosis and chronic obstructive pulmonary disease (28). Urban water systems contain organisms of the M. avium complex in biofilm or a biofilm-like structure, and individuals can potentially be exposed to the bacterium, either by inhalation of aerosol particles or ingestion of contaminated water. Studies have established an association between M. avium in urban water and the development of disseminated disease in individuals with AIDS (36).Mycobacterium smegmatis, as well as M. avium, has been shown to produce a biofilm or a biofilm-like structure (6, 19). The outermost layers of the M. smegmatis and M. avium cell walls contain glycopeptidolipid (GPL), whereas the outermost layer of M. tuberculosis is made of phenolic glycolipids, dimycocerosate, and lipo-oligosaccharides (24). Recent studies suggest that the M. smegmatis biofilm is associated with a GPL present on the cell wall, and indirect evidence indicates a similar role in M. avium (6). Aspects of biofilm formation have begun to be examined with M. smegmatis. Transposon inactivation of the GPL gene clusters in M. smegmatis decreased the production of biofilm, and the deletion of the genes tmtp and mps revealed their involvement in biofilm formation upon seeding of the bacterium on polyvinyl chloride (PVC) plates (19,26). The tmtp gene is highly conserved between M. smegmatis and M. avium, ...

SummaryOrganisms of the Mycobacterium avium complex (MAC) are widely distributed in the environment, form biofilms in water pipes and potable water tanks, and cause chronic lung infections in patients with chronic obstructive pulmonary disease and cystic fibrosis. Pathological studies in patients with pulmonary MAC infection revealed granulomatous inflammation around bronchi and bronchioles. BEAS-2B human bronchial epithelial cell line was used to study MAC invasion. MAC strain A5 entered polarized BEAS-2B cells with an efficiency of 0.1 ± ± ± ± 0.03% in 2 h and 11.3 ± ± ± ± 4.0% in 24 h. In contrast, biofilm-deficient transposon mutants 5G4, 6H9 and 9B5 showed impaired invasion. Bacteria exposed to BEAS-2B cells for 24 h had greater ability to invade BEAS-2B cells compared with bacteria incubated in broth. M. avium had no impact on the monolayer transmembrane resistance. Scanning electron microscopy showed that MAC A5 forms aggregates on the surface of BEAS-2B cell monolayers, and transmission electron microscopy evidenced MAC within vacuoles in BEAS-2B cells. Cells infected with the 5G4 mutant, however, showed significantly fewer bacteria and no aggregates on the cell surface. Mutants had impaired ability to cause infection in mice, as well. The ability to form biofilm appeared to be associated with the invasiveness of MAC A5.

BackgroundInhibition of macrophage apoptosis by Mycobacterium tuberculosis has been proposed as one of the virulence mechanisms whereby the pathogen avoids the host defense. The mechanisms by which M. tuberculosis H37Rv strain suppress apoptosis and escapes human macrophage killing was investigated.Methodology/Principal FindingsThe screening of a transposon mutant bank identified several mutants, which, in contrast to the wild-type bacterium, had impaired ability to inhibit apoptosis of macrophages. Among the identified genes, Rv3659c (31G12 mutant) belongs to an operon reminiscent of type IV pili. The Rv3654c and Rv3655c putative proteins in a seven-gene operon are secreted into the macrophage cytoplasm and suppress apoptosis by blocking the extrinsic pathway. The operon is highly expressed when the bacterium is within macrophages, compared to the expression level in the extracellular environment. Rv3654c recognizes the polypyrimidine tract binding Protein-associated Splicing Factor (PSF) and cleaves it, diminishing the availability of caspase-8. While M. tuberculosis inhibits apoptosis by the extrinsic pathway, the pathogen does not appear to affect the intrinsic pathway. Inactivation of the intrinsic pathway by pharmacologic agents afftects M. tuberculosis and induces cell necrosis. Likewise, inactivation of PSF by siRNA significantly decreased the level of caspase-8 in macrophages.ConclusionWhile M. tuberculosis inhibits the extrinsic pathway of apoptosis, it appears to activate the intrinsic pathway leading to macrophage necrosis as a potential exit strategy.