Tuberculosis (TB) is characterized by a tight interplay between Mycobacterium tuberculosis and host cells within granulomas. These cellular aggregates restrict bacterial spreading, but do not kill all the bacilli, which can persist for years. In-depth investigation of M. tuberculosis interactions with granuloma-specific cell populations are needed to gain insight into mycobacterial persistence, and to better understand the physiopathology of the disease. We have analyzed the formation of foamy macrophages (FMs), a granuloma-specific cell population characterized by its high lipid content, and studied their interaction with the tubercle bacillus. Within our in vitro human granuloma model, M. tuberculosis long chain fatty acids, namely oxygenated mycolic acids (MA), triggered the differentiation of human monocyte-derived macrophages into FMs. In these cells, mycobacteria no longer replicated and switched to a dormant non-replicative state. Electron microscopy observation of M. tuberculosis–infected FMs showed that the mycobacteria-containing phagosomes migrate towards host cell lipid bodies (LB), a process which culminates with the engulfment of the bacillus into the lipid droplets and with the accumulation of lipids within the microbe. Altogether, our results suggest that oxygenated mycolic acids from M. tuberculosis play a crucial role in the differentiation of macrophages into FMs. These cells might constitute a reservoir used by the tubercle bacillus for long-term persistence within its human host, and could provide a relevant model for the screening of new antimicrobials against non-replicating persistent mycobacteria.

SummaryMycobacterium tuberculosis thrives within macrophages by residing in phagosomes and preventing them from maturing and fusing with lysosomes. A parallel transcriptional survey of intracellular mycobacteria and their host macrophages revealed signatures of heavy metal poisoning. In particular, mycobacterial genes encoding heavy metal efflux P-type ATPases CtpC, CtpG, and CtpV, and host cell metallothioneins and zinc exporter ZnT1, were induced during infection. Consistent with this pattern of gene modulation, we observed a burst of free zinc inside macrophages, and intraphagosomal zinc accumulation within a few hours postinfection. Zinc exposure led to rapid CtpC induction, and ctpC deficiency caused zinc retention within the mycobacterial cytoplasm, leading to impaired intracellular growth of the bacilli. Thus, the use of P1-type ATPases represents a M. tuberculosis strategy to neutralize the toxic effects of zinc in macrophages. We propose that heavy metal toxicity and its counteraction might represent yet another chapter in the host-microbe arms race.

Receptors involved in the phagocytosis of microorganisms under nonopsonic conditions have been little studied in neutrophils. Complement receptor type 3 (CR3) is a pattern recognition receptor able to internalize zymosan and C3bi-coated particles. We report that Abs directed against CR3 strongly inhibited nonopsonic phagocytosis of Mycobacterium kansasii in human neutrophils. In these cells CR3 has been found associated with several GPI-anchored proteins localized in cholesterol-rich microdomains (rafts) of the plasma membrane. Cholesterol sequestration by nystatin, filipin, or β-cyclodextrin as well as treatment of neutrophils with phosphatidylinositol phospholipase C to remove GPI-anchored proteins from the cell surface markedly inhibited phagocytosis of M. kansasii, without affecting phagocytosis of zymosan or serum-opsonized M. kansasii. Abs directed against several GPI-anchored proteins inhibited phagocytosis of M. kansasii, but not of zymosan. N-acetyl-d-glucosamine, which is known to disrupt interactions between CR3 and GPI proteins, also strongly diminished phagocytosis of these mycobacteria. In conclusion, phagocytosis of M. kansasii involved CR3, GPI-anchored receptors, and cholesterol. In contrast, phagocytosis of zymosan or opsonized particles involved CR3, but not cholesterol or GPI proteins. We propose that CR3, when associated with a GPI protein, relocates in cholesterol-rich domains where M. kansasii are internalized. When CR3 is not associated with a GPI protein, it remains outside of these domains and mediates phagocytosis of zymosan and opsonized particles, but not of M. kansasii.

SummaryAfter internalization into macrophages non-pathogenic mycobacteria are killed within phagosomes. Pathogenic mycobacteria can block phagosome maturation and grow inside phagosomes but under some conditions can also be killed by macrophages. Killing mechanisms are poorly understood, although phagolysosome fusion and nitric oxide (NO) production are implicated. We initiated a systematic analysis addressing how macrophages kill 'non-pathogenic' Mycobacterium smegmatis . This system was dynamic, involving periods of initial killing, then bacterial multiplication, followed by two additional killing stages. NO synthesis represented the earliest killing factor but its synthesis stopped during the first killing period. Phagosome actin assembly and fusion with late endocytic organelles coincided with the first and last killing phase, while recycling of phagosome content and membrane coincided with bacterial growth. Phagosome acidification and acquisition of the vacuolar (V) ATPase followed a different pattern coincident with later killing phases. Moreover, V-ATPase localized to vesicles distinct from classical late endosomes and lysosomes. Map kinase p38 is a crucial regulator of all processes investigated, except NO synthesis, that facilitated the host for some functions while being usurped by live bacteria for others. A mathematical model argues that periodic high and low cellular killing activity is more effective than is a continuous process.

RNase E, which is the central component of the multienzyme RNA degradosome, serves as a scaffold for interaction with other enzymes involved in mRNA degradation including the DEAD-box RNA helicase RhlB. Epifluorescence microscopy under live cell conditions shows that RNase E and RhlB are membrane associated, but neither protein forms cytoskeletal-like structures as reported earlier by Taghbalout and Rothfield. We show that association of RhlB with the membrane depends on a direct protein interaction with RNase E, which is anchored to the inner cytoplasmic membrane through an MTS (Membrane Targeting Sequence). Molecular dynamics simulations show that the MTS interacts with the phospholipid bilayer by forming a stabilized amphipathic α-helix with the helical axis oriented parallel to the plane of the bilayer and hydrophobic side chains buried deep in the acyl core of the membrane. Based on the molecular dynamics simulations, we propose that the MTS freely diffuses in the membrane by a novel mechanism in which a large number of weak contacts are rapidly broken and reformed. TIRFm (Total Internal Reflection microscopy) shows that RNase E in live cells rapidly diffuses over the entire inner membrane forming short-lived foci. Diffusion could be part of a scanning mechanism facilitating substrate recognition and cooperativity. Remarkably, RNase E foci disappear and the rate of RNase E diffusion increases with rifampicin treatment. Control experiments show that the effect of rifampicin is specific to RNase E and that the effect is not a secondary consequence of the shut off of E. coli transcription. We therefore interpret the effect of rifampicin as being due to the depletion of RNA substrates for degradation. We propose a model in which formation of foci and constraints on diffusion arise from the transient clustering of RNase E into cooperative degradation bodies.

Actin is implicated in membrane fusion, but the precise mechanisms remain unclear. We showed earlier that membrane organelles catalyze the de novo assembly of F-actin that then facilitates the fusion between latex bead phagosomes and a mixture of early and late endocytic organelles. Here, we correlated the polymerization and organization of F-actin with phagosome and endocytic organelle fusion processes in vitro by using biochemistry and light and electron microscopy. When membrane organelles and cytosol were incubated at 37°C with ATP, cytosolic actin polymerized rapidly and became organized into bundles and networks adjacent to membrane organelles. By 30-min incubation, a gel-like state was formed with little further polymerization of actin thereafter. Also during this time, the bulk of in vitro fusion events occurred between phagosomes/endocytic organelles. The fusion between latex bead phagosomes and late endocytic organelles, or between late endocytic organelles themselves was facilitated by actin, but we failed to detect any effect of perturbing F-actin polymerization on early endosome fusion. Consistent with this, late endosomes, like phagosomes, could nucleate F-actin, whereas early endosomes could not. We propose that actin assembled by phagosomes or late endocytic organelles can provide tracks for fusion-partner organelles to move vectorially toward them, via membranebound myosins, to facilitate fusion. INTRODUCTIONActin is essential for many cellular processes and dynamic polymerization/depolymerization of actin filaments is a critical property of all eukaryotic cells (Mitchison and Cramer, 1996;Carlier, 1998;Machesky and Insall, 1999;Small et al., 1999;Amann and Pollard, 2000). Much of the F-actin assembled in cells is intimately associated with membranes, especially the plasma membrane (Tilney, 1976;Hoglund et al., 1980;Lindberg et al., 1981;Carraway and Carraway, 1989;Dickinson and Purich, 2002). The first links between F-actin and membranes were reported by Tilney and Cardell (1970) who demonstrated the role of plasma membranes in actin nucleation, and Orci et al. (1972) who showed that cytochalasin can either stimulate or inhibit exocytic fusion, depending on the conditions. In addition to exocytosis (Bernstein et al., 1998;Lang et al., 2000;Bader et al., 2002), actin is now known to be involved in many other trafficking events, including phagocytosis (Swanson et al., 1999; Desjardin and Griffiths, 2003) and transcytosis (Durrbach et al., 2000). In the endocytic pathway, actin and myosins are essential for two different transport steps: clathrin-dependent internalization from the plasma membrane and transport to lysosomes (Durrbach et al., 1996;Riezman et al., 1996;Buss et al., 2001). More recently, actin has also been shown to be directly involved in homotypic fusion between yeast vacuoles (see DISCUSSION).Our interest in actin emerged from our analysis of an in vitro phagosome-endocytic organelle fusion assay that used latex bead phagosomes (LBP) and early endosomes (EE) and late endocytic org...

T o evaluate the potential contribution of extracellular enzymes to the pathogenicity of mycobacteria, the presence of selected enzyme activities was investigated in the culture filtrates of the obligate human pathogen Mycobacterium tuberculosis, M. bovis BCG, the opportunistic pathogens M. kansasii and M. fortuitum, and the non-pathogenic species M. phlei and M. smegmatis. For M. tuberculosis and M. bovis, 22 enzyme activities were detected in the culture filtrates and/or cell surfaces, of which eight were absent from the culture fluids of non-pathogens: alanine dehydrogenase, glutamine synthetase, nicotinamidase, isonicotinamidase, superoxide dismutase, catalase, peroxidase and alcohol dehydrogenase. These activities, which correspond to secreted enzymes, formed a significant part (up to 92 O/ O) of the total enzyme activities of the bacteria and were absent from the culture fluids and the cell surfaces of the non-pathogenic species M. smegmatis and M. phlei. The extracellular location of superoxide dismutase and glutamine synthetase seemed to be restricted to the obligate pathogens examined. The difference in the enzyme profiles was not attributable to the growth rates of the two groups of bacteria. The presence of the eight enzyme activities in the outermost compartments of obligate pathogens and their absence in those of non-pathogens provides further evidence that these enzymes may be involved in the pathogenicity of mycobacteria. In addition, the eight enzyme activities were demonstrated in the cell extract of M. smegmatis. Stepwise erosion of the cell surface of M. smegmatis to expose internal capsular constituents showed that the various enzyme activities, with the possible exception of superoxide dismutase, were located more deeply in the cell envelope of this bacterium. This suggests that the molecular architecture of the mycobacterial envelopes may play an important role in the pathogenicity of these organisms.

BackgroundAs a species, Mycobacterium tuberculosis is more diverse than previously thought. In particular, the Beijing family of M. tuberculosis strains is spreading and evoluating throughout the world and this is giving rise to public health concerns. Genetic diversity within this family has recently been delineated further and a specific genotype, called Bmyc10, has been shown to represent over 60% of all Beijing clinical isolates in several parts of the world. How the host immune system senses and responds to various M. tuberculosis strains may profoundly influence clinical outcome and the relative epidemiological success of the different mycobacterial lineages. We hypothesised that the success of the Bmyc10 group may, at least in part, rely upon its ability to alter innate immune responses and the secretion of cytokines and chemokines by host phagocytes.Methodology/Principal FindingsWe infected human macrophages and dendritic cells with a collection of genetically well-defined M. tuberculosis clinical isolates belonging to various mycobacterial families, including Beijing. We analyzed cytokine and chemokine secretion on a semi-global level using antibody arrays allowing the detection of sixty-five immunity-related soluble molecules. Our data indicate that Beijing strains induce significantly less interleukin (IL)-6, tumor necrosis factor (TNF), IL-10 and GRO-α than the H37Rv reference strain, a feature that is variously shared by other modern and ancient M. tuberculosis families and which constitutes a signature of the Beijing family as a whole. However, Beijing strains did not differ relative to each other in their ability to modulate cytokine secretion.Conclusions/SignificanceOur results confirm and expand upon previous reports showing that M. tuberculosis Beijing strains in general are poor in vitro cytokine inducers in human phagocytes. The results suggest that the epidemiological success of the Beijing Bmyc10 is unlikely to rely upon any specific ability of this group of strains to impair anti-mycobacterial innate immunity.