Mycobacterium tuberculosis Responds to Chloride and pH as Synergistic Cues to the Immune Status of its Host Cell

PubMed ID: 23592993This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited

Probing host pathogen cross-talk by transcriptional profiling of both Mycobacterium tuberculosis and infected human dendritic cells and macrophages

This study provides the proof of principle that probing the host and the microbe transcriptomes simultaneously is a valuable means to accessing unique information on host pathogen interactions. Our results also underline the extraordinary plasticity of host cell and pathogen responses to infection, and provide a solid framework to further understand the complex mechanisms involved in immunity to M. tuberculosis and in mycobacterial adaptation to different intracellular environments

Monosodium urate crystals promote innate anti-mycobacterial immunity and improve BCG efficacy as a vaccine against tuberculosis

A safer and more effective anti-Tuberculosis vaccine is still an urgent need. We probed the effects of monosodium urate crystals (MSU) on innate immunity to improve the Bacille Calmette-Guerin (BCG) vaccination. Results showed that in vitro MSU cause an enduring macrophage stimulation of the anti-mycobacterial response, measured as intracellular killing, ROS production and phagolysosome maturation. The contribution of MSU to anti-mycobacterial activity was also shown in vivo. Mice vaccinated in the presence of MSU showed a lower number of BCG in lymph nodes draining the vaccine inoculation site, in comparison to mice vaccinated without MSU. Lastly, we showed that MSU improved the efficacy of BCG vaccination in mice infected with Mycobacterium tuberculosis (MTB), measured in terms of lung and spleen MTB burden. These results demonstrate that the use of MSU as adjuvant may represent a novel strategy to enhance the efficacy of BCG vaccination

Pathogenic mycobacteria achieve cellular persistence by inhibiting the Niemann-Pick Type C disease cellular pathway

Background. Tuberculosis remains a major global health concern. The ability to prevent phagosome-lysosome fusion is a key mechanism by which intracellular mycobacteria, including Mycobacterium tuberculosis, achieve long-term persistence within host cells. The mechanisms underpinning this key intracellular pro-survival strategy remain incompletely understood. Host macrophages infected with intracellular mycobacteria share phenotypic similarities with cells taken from patients suffering from Niemann-Pick Disease Type C (NPC), a rare lysosomal storage disease in which endocytic trafficking defects and lipid accumulation within the lysosome lead to cell dysfunction and cell death. We investigated whether these shared phenotypes reflected an underlying mechanistic connection between mycobacterial intracellular persistence and the host cell pathway dysfunctional in NPC.  Methods. The induction of NPC phenotypes in macrophages from wild-type mice or obtained from healthy human donors was assessed via infection with mycobacteria and subsequent measurement of lipid levels and intracellular calcium homeostasis. The effect of NPC therapeutics on intracellular mycobacterial load was also assessed.  Results. Macrophages infected with intracellular mycobacteria phenocopied NPC cells, exhibiting accumulation of multiple lipid types, reduced lysosomal Ca 2+ levels, and defects in intracellular trafficking. These NPC phenotypes could also be induced using only lipids/glycomycolates from the mycobacterial cell wall. These data suggest that intracellular mycobacteria inhibit the NPC pathway, likely via inhibition of the NPC1 protein, and subsequently induce altered acidic store Ca 2+ homeostasis. Reduced lysosomal calcium levels may provide a mechanistic explanation for the reduced levels of phagosome-lysosome fusion in mycobacterial infection. Treatments capable of correcting defects in NPC mutant cells via modulation of host cell calcium were of benefit in promoting clearance of mycobacteria from infected host cells.  Conclusion. These findings provide a novel mechanistic explanation for mycobacterial intracellular persistence, and suggest that targeting interactions between the mycobacteria and host cell pathways may provide a novel avenue for development of anti-TB therapies

Inhibition of Host Vacuolar H+-ATPase Activity by a Legionella pneumophila Effector

Legionella pneumophila is an intracellular pathogen responsible for Legionnaires' disease. This bacterium uses the Dot/Icm type IV secretion system to inject a large number of bacterial proteins into host cells to facilitate the biogenesis of a phagosome permissive for its intracellular growth. Like many highly adapted intravacuolar pathogens, L. pneumophila is able to maintain a neutral pH in the lumen of its phagosome, particularly in the early phase of infection. However, in all cases, the molecular mechanisms underlying this observation remain unknown. In this report, we describe the identification and characterization of a Legionella protein termed SidK that specifically targets host v-ATPase, the multi-subunit machinery primarily responsible for organelle acidification in eukaryotic cells. Our results indicate that after being injected into infected cells by the Dot/Icm secretion system, SidK interacts with VatA, a key component of the proton pump. Such binding leads to the inhibition of ATP hydrolysis and proton translocation. When delivered into macrophages, SidK inhibits vacuole acidification and impairs the ability of the cells to digest non-pathogenic E. coli. We also show that a domain located in the N-terminal portion of SidK is responsible for its interactions with VatA. Furthermore, expression of sidK is highly induced when bacteria begin to enter new growth cycle, correlating well with the potential temporal requirement of its activity during infection. Our results indicate that direct targeting of v-ATPase by secreted proteins constitutes a virulence strategy for L. pneumophila, a vacuolar pathogen of macrophages and amoebae

Interactions between Naïve and Infected Macrophages Reduce Mycobacterium tuberculosis Viability

A high intracellular bacillary load of Mycobacterium tuberculosis in macrophages induces an atypical lysosomal cell death with early features of apoptosis that progress to necrosis within hours. Unlike classical apoptosis, this cell death mode does not appear to diminish M. tuberculosis viability. We previously reported that culturing heavily infected macrophages with naïve macrophages produced an antimicrobial effect, but only if naïve macrophages were added during the pre-necrotic phase of M. tuberculosis-induced cell death. In the present study we investigated the mechanism of antimicrobial activity in co-cultures, anticipating that efferocytosis of bacilli in apoptotic bodies would be required. Confocal microscopy revealed frustrated phagocytosis of M. tuberculosis-infected macrophages with no evidence that significant numbers of bacilli were transferred to the naïve macrophages. The antimicrobial effect of naïve macrophages was retained when they were separated from infected macrophages in transwells, and conditioned co-culture supernatants transferred antimicrobial activity to cultures of infected macrophages alone. Antimicrobial activity in macrophage co-cultures was abrogated when the naïve population was deficient in IL-1 receptor or when the infected population was deficient in inducible nitric oxide synthase. The participation of nitric oxide suggested a conventional antimicrobial mechanism requiring delivery of bacilli to a late endosomal compartment. Using macrophages expressing GFP-LC3 we observed the induction of autophagy specifically by a high intracellular load of M. tuberculosis. Bacilli were identified in LC3-positive compartments and LC3-positive compartments were confirmed to be acidified and LAMP1 positive. Thus, the antimicrobial effect of naïve macrophages acting on M. tuberculosis in heavily-infected macrophages is contact-independent. Interleukin-1 provides an afferent signal that induces an as yet unidentified small molecule which promotes nitric oxide-dependent antimicrobial activity against bacilli in autolysosomes of heavily infected macrophages. This cooperative, innate antimicrobial interaction may limit the maximal growth rate of M. tuberculosis prior to the expression of adaptive immunity in pulmonary tuberculosis

A two-component regulator induces the transmission phenotype of stationary-phase Legionella pneumophila

Pathogenic Legionella pneumophila evolved as a parasite of aquatic amoebae. To persist in the environment, the microbe must be proficient at both replication and transmission. In laboratory cultures, as nutrients become scarce a stringent response-like pathway coordinates exit from the exponential growth phase with induction of traits correlated with virulence, including motility. A screen for mutants that express the flagellin gene poorly identified five activators of virulence: LetA/LetS, a two-component regulator homologous to GacA/GacS of Pseudomonas and SirA/BarA of Salmonella ; the stationary-phase sigma factor RpoS; the flagellar sigma factor FliA; and a new locus, letE . Unlike wild type, post-exponential-phase letA and letS mutants were not motile, cytotoxic, sodium sensitive or proficient at infecting macrophages. L. pneumophila also required fliA to become motile, cytotoxic and to infect macrophages efficiently and letE to express sodium sensitivity and maximal motility and cytotoxicity. When induced to express RelA, all of the strains exited the exponential phase, but only wild type converted to the fully virulent form. In contrast, intracellular replication was independent of letA, letS, letE or fliA . Together, the data indicate that, as the nutrient supply wanes, ppGpp triggers a regulatory cascade mediated by LetA/ LetS, RpoS, FliA and letE that coordinates differentiation of replicating L. pneumophila to a transmissible form.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75531/1/j.1365-2958.2002.02884.x.pd

Identification of a Novel Gene Product That Promotes Survival of Mycobacterium smegmatis in Macrophages

BACKGROUND: Bacteria of the suborder Corynebacterineae include significant human pathogens such as Mycobacterium tuberculosis and M. leprae. Drug resistance in mycobacteria is increasingly common making identification of new antimicrobials a priority. Mycobacteria replicate intracellularly, most commonly within the phagosomes of macrophages, and bacterial proteins essential for intracellular survival and persistence are particularly attractive targets for intervention with new generations of anti-mycobacterial drugs. METHODOLOGY/PRINCIPAL FINDINGS: We have identified a novel gene that, when inactivated, leads to accelerated death of M. smegmatis within a macrophage cell line in the first eight hours following infection. Complementation of the mutant with an intact copy of the gene restored survival to near wild type levels. Gene disruption did not affect growth compared to wild type M. smegmatis in axenic culture or in the presence of low pH or reactive oxygen intermediates, suggesting the growth defect is not related to increased susceptibility to these stresses. The disrupted gene, MSMEG_5817, is conserved in all mycobacteria for which genome sequence information is available, and designated Rv0807 in M. tuberculosis. Although homology searches suggest that MSMEG_5817 is similar to the serine:pyruvate aminotransferase of Brevibacterium linens suggesting a possible role in glyoxylate metabolism, enzymatic assays comparing activity in wild type and mutant strains demonstrated no differences in the capacity to metabolize glyoxylate. CONCLUSIONS/SIGNIFICANCE: MSMEG_5817 is a previously uncharacterized gene that facilitates intracellular survival of mycobacteria. Interference with the function of MSMEG_5817 may provide a novel therapeutic approach for control of mycobacterial pathogens by assisting the host immune system in clearance of persistent intracellular bacteria