A Rho GDP Dissociation Inhibitor Produced by Apoptotic T-Cells Inhibits Growth of Mycobacterium tuberculosis

In this study, we found that a subpopulation of CD4⁺CD25⁺ (85% Foxp3⁺) cells from persons with latent tuberculosis infection (LTBI) inhibits growth of M. tuberculosis (M. tb) in human monocyte-derived macrophages (MDMs). A soluble factor, Rho GDP dissociation inhibitor (D4GDI), produced by apoptotic CD4⁺CD25⁺ (85% Foxp3⁺) cells is responsible for this inhibition of M. tb growth in human macrophages and in mice. M. tb-expanded CD4⁺CD25⁺Foxp3⁺D4GDI⁺ cells do not produce IL-10, TGF-β and IFN-γ. D4GDI inhibited growth of M. tb in MDMs by enhancing production of IL-1β, TNF-α and ROS, and by increasing apoptosis of M. tb-infected MDMs. D4GDI was concentrated at the site of disease in tuberculosis patients, with higher levels detected in pleural fluid than in serum. However, in response to M. tb, PBMC from tuberculosis patients produced less D4GDI than PBMC from persons with LTBI. M. tb-expanded CD4⁺CD25⁺ (85% Foxp3⁺) cells and D4GDI induced intracellular M. tb to express the dormancy survival regulator DosR and DosR-dependent genes, suggesting that D4GDI induces a non-replicating state in the pathogen. Our study provides the first evidence that a subpopulation of CD4⁺CD25⁺ (85% Foxp3⁺) cells enhances immunity to M. tb, and that production of D4GDI by this subpopulation inhibits M. tb growth.     

Oxadiazolone derivatives,new promising multi-target inhibitors against M. tuberculosis

A set of 19 oxadiazolone (OX) derivatives have been investigated for their antimycobacterial activity against two pathogenic slow-growing mycobacteria, Mycobacterium marinum and Mycobacterium bovis BCG, and the avirulent Mycobacterium tuberculosis (M. tb) mc²6230. The encouraging minimal inhibitory concentrations (MIC) values obtained prompted us to test them against virulent M. tb H37Rv growth either in broth medium or inside macrophages. The OX compounds displayed a diversity of action and were found to act either on extracellular M. tb growth only with moderated MIC₅₀, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth. Of interest, all OX derivatives exhibited very low toxicity towards host macrophages. Among the six potential OXs identified, HPOX, a selective inhibitor of extracellular M. tb growth, was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP, in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 18 potential candidates, all being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA, TesA, KasA and MetA have been reported as essential for in vitro growth of M. tb and/or its survival and persistence inside macrophages. Overall, our findings support the assumption that OX derivatives may represent a novel class of multi-target inhibitors leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes involved in various important physiological processes.     

Differential recruitment of CD63 and Rab7‐interacting‐lysosomal‐protein to phagosomes containing Mycobacterium tuberculosis in macrophages

M.tb is an intracellular pathogen which survives within the phagosomes of host macrophages by inhibiting their fusion with lysosomes. Here, it has been demonstrated that a lysosomal glycoprotein, CD63, is recruited to the majority of M.tb phagosomes, while RILP shows limited localization. This is consistent with the author's findings that CD63, but not RILP, is recruited to the phagosomes in macrophages expressing the dominant negative form of Rab7. These results suggest that M.tb phagosomes selectively fuse with endosomes and lysosomes to escape killing activity while acquiring nutrients.     

Interaction of alveolar epithelial cells with CFP21, a mycobacterial cutinase-like enzyme

Mycobacterium tuberculosis (M. tb), an intracellular pathogen, has the ability to infect alveolar epithelial cells (AEC) also in addition to alveolar macrophages. The virulence of M. tb is attributed to proteins encoded by genomic regions of deletion (RD) and till date 16 such regions (RD1-RD16) have been identified. Culture filtrate protein 21 (CFP21), a RD2 secretory protein, is a cutinase-like enzyme that possesses esterase and lipolytic activity. It is hypothesized that CFP21 could be playing a role in M. tb virulence by disrupting the host cell integrity. In this study, recombinant CFP21 was expressed and purified. The in vitro exposure of type I (WI26) and type II (A549) AEC to CFP21 resulted in a significant decline in their cellular viability by inducing cell apoptosis. However, the cytotoxic effects were more pronounced in WI26 cells than in A549 cells. The analysis of immune responses in CFP21-treated AEC exhibited significant production of reactive oxygen species and anti-inflammatory cytokine TGF-β which indicated oxidative stress-mediated cell death. These results show that CFP21 could play an important role in M. tb pathogenesis by disrupting the host alveolar barrier and thereby facilitating mycobacterial dissemination.     

Role of Tissue Factor in Mycobacterium tuberculosis-Induced Inflammation and Disease Pathogenesis

Tuberculosis (TB) is a chronic lung infectious disease characterized by severe inflammation and lung granulomatous lesion formation. Clinical manifestations of TB include hypercoagulable states and thrombotic complications. We previously showed that Mycobacterium tuberculosis (M.tb) infection induces tissue factor (TF) expression in macrophages in vitro. TF plays a key role in coagulation and inflammation. In the present study, we investigated the role of TF in M.tb-induced inflammatory responses, mycobacterial growth in the lung and dissemination to other organs. Wild-type C57BL/6 and transgenic mice expressing human TF, either very low levels (low TF) or near to the level of wild-type (HTF), in place of murine TF were infected with M.tb via aerosol exposure. Levels of TF expression, proinflammatory cytokines and thrombin-antithrombin complexes were measured post M.tb infection and mycobacterial burden in the tissue homogenates were evaluated. Our results showed that M.tb infection did not increase the overall TF expression in lungs. However, macrophages in the granulomatous lung lesions in all M.tb-infected mice, including low TF mice, showed increased levels of TF expression. Conspicuous fibrin deposition in the granuloma was detected in wild-type and HTF mice but not in low TF mice. M.tb infection significantly increased expression levels of cytokines IFN-γ, TNF-α, IL-6 and IL-1ß in lung tissues. However, no significant differences were found in proinflammatory cytokines among the three experimental groups. Mycobacterial burden in lungs and dissemination into spleen and liver were essentially similar in all three genotypes. Our data indicate, in contrast to that observed in acute bacterial infections, that TF-mediated coagulation and/or signaling does not appear to contribute to the host-defense in experimental tuberculosis.     

Mycobacterium tuberculosis Subverts the TLR-2 - MyD88 Pathway to Facilitate Its Translocation into the Cytosol

Mycobacterium tuberculosis (M.tb) has evolved mechanisms to evade its destruction in phagolysosomes, where it successfully survives and replicates within phagocytes. Recent studies have shown that virulent strains of M.tb can translocate from the phagosome into the cytosol of dendritic cells (DC). The molecular mechanisms by which virulent M.tb strains can escape the phagosome remain unknown. Here we show that the virulent M.tb strain H37Rv, but not the vaccine strain Bacille Calmette-Guérin (BCG), escapes from the phagolysosome and enters the cytosol by interfering with the TLR-2-MyD88 signaling pathway. Using H37Rv mutants, we further demonstrate that the region of difference-1 (RD-1) locus and ESAT-6, a gene within the RD-1 locus, play an important role in the capacity of M.tb to migrate from the phagosome to the cytosol of macrophages. H37Rv, BCG, H37RvΔRD1, and H37RvΔESAT6 were able to translocate to the cytosol in macrophages derived from TLR-2- and MyD88-deficient animals, whereas only virulent H37Rv was able to enter the cytosol in macrophages from wild type mice. Therefore, signaling through the TLR-2–MyD88 pathway in macrophages plays an important role in confining M.tb within phagolysomes. Virulent strains of M.tb have evolved mechanisms to subvert this pathway, thus facilitating their translocation to the cytosol and to escape the toxic microenvironment of the phagosome or phagolysosome.     

Exosomes released from M. tuberculosis infected cells can suppress IFN-γ mediated activation of naïve macrophages

Background

Macrophages infected with Mycobacterium tuberculosis (M.tb) are known to be refractory to IFN-γ stimulation. Previous studies have shown that M.tb express components such as the 19-kDa lipoprotein and peptidoglycan that can bind to macrophage receptors including the Toll-like receptor 2 resulting in the loss in IFN-γresponsiveness. However, it is unclear whether this effect is limited to infected macrophages. We have previously shown that M.tb-infected macrophages release exosomes which are 30–100 nm membrane bound vesicles of endosomal origin that function in intercellular communication. These exosomes contain mycobacterial components including the 19-kDa lipoprotein and therefore we hypothesized that macrophages exposed to exosomes may show limited response to IFN-γ stimulation.

Methodology/Principal Findings

Exosomes were isolated from resting as well as M.tb-infected RAW264.7 macrophages. Mouse bone marrow-derived macrophages (BMMØ) were treated with exosomes +/− IFN-γ. Cells were harvested and analyzed for suppression of IFN-γ responsive genes by flow cytometry and real time PCR. We found that exosomes derived from M.tb H37Rv-infected but not from uninfected macrophages inhibited IFN-γ induced MHC class II and CD64 expression on BMMØ. This inhibition was only partially dependent on the presence of lipoproteins but completely dependent on TLR2 and MyD88. The exosomes isolated from infected cells did not inhibit STAT1 Tyrosine phosphorylation but down-regulated IFN-γ induced expression of the class II major histocompatibity complex transactivator; a key regulator of class II MHC expression. Microarray studies showed that subsets of genes induced by IFN-γ were inhibited by exosomes from H37Rv-infeced cells including genes involved in antigen presentation. Moreover, this set of genes partially overlapped with the IFN-γ-induced genes inhibited by H37Rv infection.

Conclusions

Our study suggests that exosomes, as carriers of M.tb pathogen associated molecular patterns (PAMPs), may provide a mechanism by which M.tb may exert its suppression of a host immune response beyond the infected cell.     

Protective immunity to Mycobacterium tuberculosis infection by chemokine and cytokine conditioned CFP-10 differentiated dendritic cells

Background

Dendritic cells (DCs) play major roles in mediating immune responses to mycobacteria. A crucial aspect of this is the priming of T cells via chemokines and cytokines. In this study we investigated the roles of chemokines RANTES and IP-10 in regulating protective responses from Mycobacterium tuberculosis (M. tb) 10 kDa Culture Filtrate Protein-10 (CFP-10) differentiated DCs (CFP10-DCs).

Methods and Findings

Infection of CFP10-DCs with mycobacteria down-modulated RANTES and IP-10 levels. Pathway specific microarray analyses showed that in addition to RANTES and IP-10, mycobacteria infected CFP10-DCs showed reduced expression of many Th1 promoting chemokines and chemokine receptors. Importantly, T cells co-cultured with RANTES and IP-10 conditioned CFP10-DCs mediated killing of mycobacteria from infected macrophages. Similarly, T cells recruited by RANTES and IP-10 conditioned CFP10-DCs mediated significant killing of mycobacteria from infected macrophages. IFN-gamma treatment of CFP10-DCs restored RANTES and IP-10 levels and T cells activated by these DCs mediated significant killing of virulent M. tb inside macrophages. Adoptive transfer of either RANTES and IP-10 or IL-12 and IFN-gamma conditioned CFP10-DCs cleared an established M. tb infection in mice. The extent of clearance was similar to that obtained with drug treatment.

Conclusions

These results indicate that chemokine and cytokine secretion by DCs differentiated by M. tb antigens such as CFP-10 play major roles in regulating protective immune responses at sites of infection.     

The Role of Selection in Shaping Diversity of Natural M. tuberculosis Populations

Mycobacterium tuberculosis (M.tb), the cause of tuberculosis (TB), is estimated to infect a new host every second. While analyses of genetic data from natural populations of M.tb have emphasized the role of genetic drift in shaping patterns of diversity, the influence of natural selection on this successful pathogen is less well understood. We investigated the effects of natural selection on patterns of diversity in 63 globally extant genomes of M.tb and related pathogenic mycobacteria. We found evidence of strong purifying selection, with an estimated genome-wide selection coefficient equal to −9.5×10⁻⁴ (95% CI −1.1×10⁻³ to −6.8×10⁻⁴); this is several orders of magnitude higher than recent estimates for eukaryotic and prokaryotic organisms. We also identified different patterns of variation across categories of gene function. Genes involved in transport and metabolism of inorganic ions exhibited very low levels of non-synonymous polymorphism, equivalent to categories under strong purifying selection (essential and translation-associated genes). The highest levels of non-synonymous variation were seen in a group of transporter genes, likely due to either diversifying selection or local selective sweeps. In addition to selection, we identified other important influences on M.tb genetic diversity, such as a 25-fold expansion of global M.tb populations coincident with explosive growth in human populations (estimated timing 1684 C.E., 95% CI 1620–1713 C.E.). These results emphasize the parallel demographic histories of this obligate pathogen and its human host, and suggest that the dominant effect of selection on M.tb is removal of novel variants, with exceptions in an interesting group of genes involved in transportation and defense. We speculate that the hostile environment within a host imposes strict demands on M.tb physiology, and thus a substantial fitness cost for most new mutations. In this respect, obligate bacterial pathogens may differ from other host-associated microbes such as symbionts.     

M.tuberculosis Mutants Lacking Oxygenated Mycolates Show Increased Immunogenicity and Protective Efficacy as Compared to M. bovis BCG Vaccine in an Experimental Mouse Model

The existing vaccine against tuberculosis (M. bovis BCG) exerts some protection against the extrapulmonary forms of the disease, particularly in young children, but is not very effective against the pulmonary form of TB, which often results from the reactivation of a latent M. tuberculosis (M.tb)infection. Among the new approaches in TB vaccine development, live attenuated M.tb mutants are a promising new avenue. Here we report on the vaccine potential of two highly attenuated M.tb mutants, MGM1991 and M.tbhma::hyg (HMA), lacking all oxygenated mycolates in their cell wall. In C57BL/6 mice, stronger Th1 (IFN-γ, IL-2 and TNF-α) and IL-17 responses could be induced following subcutaneous vaccination with either of the two mutants, than following vaccination with M. bovis BCG. Significantly more mycobacteria specific IFN-γ producing CD4⁺ and particularly CD8⁺ T cells could be detected by intracellular cytokine staining in mice vaccinated with the M.tb mutants. Finally, vaccination with either of the two mutants conferred stronger protection against intratracheal M.tb challenge than vaccination with BCG, as indicated by reduced bacterial replication in lungs at 4 to 12 weeks after challenge. Protection against M. tb dissemination, as indicated by reduced bacterial numbers in spleen, was comparable for both mutants to protection conferred by BCG.     

Guanine nucleotide exchange factor -H1 promotes inflammatory cytokine production and intracellular mycobacterial elimination in macrophages

Mycobacterium tuberculosis (M.tb), which causes tuberculosis, is a host-adapted intracellular pathogen that can live within macrophages owning to its ability to arrest phagolysosome biogenesis. The guanine nucleotide exchange factor H1 (GEF-H1) may contribute to the phagocytosis of bacteria by macrophages through mediating the crosstalk between microtubules and the actin cytoskeleton. Its role in Shigella infection has been determined but little is known about the role of GEF-H1 in mycobacterial infection. In the present study, we demonstrated that GEF-H1 functioned as a key regulator of the macrophage-mediated anti-mycobacterial response. We found that both mRNA and protein expression levels of GEF-H1 were significantly upregulated in macrophage during mycobacterial infection. Moreover, silencing of GEF-H1 with specific siRNAs reduced the phosphorylation of p38 mitogen-activated protein kinase and TANK binding kinase 1 as well as the expression of interleukin-1β (IL-1β), IL-6, and interferon-β (IFN-β), without affecting nitric oxide production or autophagy. Importantly, GEF-H1 depletion attenuated macrophages-mediated mycobacterial phagocytosis and elimination. Taken together, our data supported that GEF-H1 was a novel regulator of inflammatory cytokine production and mycobacterial elimination, and may serve as a novel potential target for clinical treatment of tuberculosis.     

Anti-tuberculosis lead molecules from natural products targeting <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> ClpC1

Tuberculosis (TB) is a serious and potentially fatal disease caused by Mycobacterium tuberculosis (M. tb). The occurrence of multidrug-resistant (MDR) and extensively drug-resistant (XDR) M. tb is a significant public health concern because most of the anti-TB drugs that have been in use for over 40 years are no longer effective for the treatment of these infections. Recently, new anti-TB lead compounds such as cyclomarin A, lassomycin, and ecumicin, which are cyclic peptides from actinomycetes, have shown potent anti-TB activity against MDR and XDR M. tb as well as drug-susceptible M. tb in vitro. The target molecule of these antibiotics is ClpC1, a protein that is essential for the growth of M. tb. In this review, we introduce the three anti-TB lead compounds as potential anti-TB therapeutic agents targeting ClpC1 and compare them with the existing anti-TB drugs approved by the US Food and Drug Administration.     

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.     

Identification of a small molecule SIRT2 inhibitor with selective tumor cytotoxicity

As a member of the class III histone deacetylases, Sirtuin-2 (SIRT2) is critical in cell cycle regulation which makes it a potential target for cancer therapeutics. In this study, we identified a novel SIRT2 inhibitor, AC-93253, with IC₅₀ of 6 μM in vitro. The compound is selective, inhibiting SIRT2 7.5- and 4-fold more potently than the closely related SIRT1 and SIRT3, respectively. AC-93253 significantly enhanced acetylation of tubulin, p53, and histone H4, confirming SIRT2 and SIRT1 as its cellular targets. AC-93253 as a single agent exhibited submicromolar selective cytotoxicity towards all four tumor cell lines tested with a therapeutic window up to 200-fold, comparing to any of the three normal cell types tested. Results from high content analysis suggested that AC-93253 significantly triggered apoptosis. Taken together, SIRT2 selective inhibitor AC-93253 may serve as a novel chemical scaffold for structure-activity relationship study and future lead development.