dStrains of the Beijing genotype family of Mycobacterium tuberculosis are a cause of particular concern because of their increasing dissemination in the world and their association with drug resistance. Phylogenetically, this family includes distinct ancient and modern sublineages. The modern strains, contrary to the ancestral counterparts, demonstrated increasing prevalence in many world regions that suggest an enhanced bacterial pathogenicity. We therefore evaluated virulence of modern versus ancient Beijing strains with similar epidemiological and genotype characteristics. For this, we selected six strains that had very similar 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing profiles and belonged to the region of difference 181 (RD181) subgroup but differed using markers (mutT2 and mutT4 genes and NTF locus) that discriminate between modern and ancient Beijing sublineages. The strains were isolated from native patients in Brazil and Mozambique, countries with a low prevalence of Beijing strains. The virulence levels of these strains were determined in models of pulmonary infection in mice and in vitro macrophage infection and compared with that of a strain from Russia, part of the epidemic and hypervirulent Beijing clone B0/W148, and of the laboratory strain H37Rv. The results showed that two of the three modern Beijing strains were highly pathogenic, exhibiting levels of virulence comparable with that of the epidemic Russian strain. In contrast, all isolates of the ancient sublineage displayed intermediate or low virulence. The data obtained demonstrate that the strains of the modern Beijing sublineage are more likely to exhibit highly virulent phenotypes than ancient strains and suggest that genetic alterations characteristic of the modern Beijing sublineage favor selection of highly virulent bacteria. Despite extensive surveillance, tuberculosis (TB) remains a serious public health problem. In different parts of the world, there is concern about TB caused by the East Asian/Beijing lineage of Mycobacterium tuberculosis, demonstrating increasing prevalence in the global M. tuberculosis population (1). Clinical and epidemiological studies demonstrated that emergence of the Beijing strains could be associated with high levels of bacterial resistance to multiple drugs (2, 3) and enhanced pathogenicity of these strains, leading to increased transmissibility (4) and rapid progression from infection to active disease (5). However, the data on evaluation of the virulence of Beijing isolates were inconclusive, demonstrating a wide range of inflammatory and virulence phenotypes, as determined in animal models (6,7,8) and in vitro models of macrophage infection (9, 10).Such differences in the virulence of Beijing strains could be associated with genetic heterogeneity of the Beijing M. tuberculosis lineage. Indeed, bacterial genotyping and sequencing demonstrated that the Beijing lineage, having in common a characteristic spoligotype signature and lack of the region ...

The purinergic P2X7 receptor (P2X7R) is a sensor of extracellular ATP, a damage-associated molecule that is released from necrotic cells and that induces pro-inflammatory cytokine production and cell death. To investigate whether the innate immune response to damage signals could contribute to the development of pulmonary necrotic lesions in severe forms of tuberculosis, disease progression was examined in C57BL/6 and P2X7R−/− mice that were intratracheally infected with highly virulent mycobacterial strains (Mycobacterium tuberculosis strain 1471 of the Beijing genotype family and Mycobacterium bovis strain MP287/03). The low-dose infection of C57BL/6 mice with bacteria of these strains caused the rapid development of extensive granulomatous pneumonia with necrotic areas, intense bacillus dissemination and anticipated animal death. In contrast, in P2X7R−/− mice, the lung pathology presented with moderate infiltrates of mononuclear leukocytes without visible signs of necrosis; the disease attenuation was accompanied by a delay in mortality. In vitro, the hypervirulent mycobacteria grew rapidly inside macrophages and induced death by a P2X7R-dependent mechanism that facilitated the release of bacilli. Furthermore, these bacteria were resistant to the protective mechanisms elicited in macrophages following extracellular ATP stimulation. Based on this study, we propose that the rapid intracellular growth of hypervirulent mycobacteria results in massive macrophage damage. The ATP released by damaged cells engages P2X7R and accelerates the necrotic death of infected macrophages and the release of bacilli. This vicious cycle exacerbates pneumonia and lung necrosis by promoting widespread cell destruction and bacillus dissemination. These findings suggest the use of drugs that have been designed to inhibit the P2X7R as a new therapeutic approach to treat the aggressive forms of tuberculosis.

The epidemiologically important Mycobacterium tuberculosis Beijing genotype strains, highly endemic in East Asia, have become an emerging infection in certain geographic areas, including Russia, because of its increasing prevalence and association with multidrug resistance (MDR). The aim was to verify whether MDR Beijing strains circulating in the emerging regions present some biological particularities that could contribute to their success in causing disease in comparison with the sporadic strains from locations with low prevalence of the Beijing genotype. We evaluated virulence-associated characteristics of the MDR Beijing strains isolated in Russia and compared them with those of the drug-resistant and susceptible Beijing strains from Brazil and reference H37Rv strain. We found that Russian MDR strains demonstrated an increased bacterial fitness and growth in THP-1 macrophage-like cells, as well as a higher capacity to induce non-protective cytokine synthesis and necrotic macrophage death. By contrast, the biological properties of the strains isolated in Brazil largely resembled those of the H37Rv strain, with the exception of the drug-resistant isolates that presented significantly reduced fitness. The data demonstrate that the emerging MDR strains of the Beijing genotype circulating in Russia do express a pattern of properties associated with the enhanced virulence favouring its clonal dissemination in this region.

The proinflammatory response of infected macrophages is an important early host defense mechanism against mycobacterial infection. Mycobacteria have been demonstrated to induce proinflammatory gene transcription through the Toll-like receptors, (TLR)2 and TLR 4, which initiate signaling cascades leading to nuclear factor (NF)-kappaB activation. The main transduction pathway responsible for NF-kappaB activation has been established and involves the MyD88, interleukin-1 receptor-associated kinase, tumor necrosis factor receptor-associated factor-6, NF-kappaB-inducing kinase, and inhibitor of kappaB kinase complex. The role of other kinase cascades triggered by mycobacteria in the NF-kappaB activation is less clear. We herein examine the role of the mitogen-activated protein kinases (MAPKs) and phosphatidylinositol 3-kinase (PI-3K) cascades in the expression of the bacillus Calmette-Guerin (BCG) mycobacteria-induced NF-kappaB-dependent genes, macrophage-inflammatory protein-2 (MIP-2) and inducible nitric oxide (NO) synthase. Specific pharmacological inhibition of the PI-3K, c-jun-N-terminal kinase (JNK), and to a smaller extent, p38 MAPK but not extracellular-regulated kinase (ERK), suppressed NF-kappaB-dependent reporter gene transcription and MIP-2 and NO secretion in BCG-induced RAW264.7 macrophages. A similar effect was obtained following molecular inhibition of JNK via JNK-interacting protein-1 overexpression. In addition, a kinase-dead mutant of MEK kinase-1, the up-stream regulator of JNK, also proved to be a potent inhibitor of NF-kappaB-reporter activity. The effect of inhibitors was mediated by the down-regulation of NF-kappaB transcription activity and without effecting its nuclear translocation. These data suggest an indirect mechanism of the NF-kappaB regulation by these kinases, probably through p65 phosphorylation and improved binding to the p300 transcription coactivator. The data obtained demonstrate that PI-3K, JNK, and p38 MAPK activation by mycobacteria enhance NF-kappaB-driven gene expression contributing to the proinflammatory macrophage response.

The success of Mycobacterium tuberculosis as a human pathogen has been attributed to the ability of the bacillus to proliferate inside macrophages and to induce cell death. This review describes how the sensors of the innate immune system modulate the cell death pathways in infected macrophages and, consequently, the pathogenesis of tuberculosis.

Abstract:Two new chamigrane sesquiterpenes 1-2 and three known compounds 3-5 were isolated from a lipophilic extract of the red alga Laurencia dendroidea collected from the OPEN ACCESSMolecules 2014, 19 3182 Southeastern Brazilian coast. Dendroidone (1) and dendroidiol (2) were isolated from samples collected at Biscaia Inlet, Angra dos Reis, Rio de Janeiro and at Manguinhos Beach, Serra, Espírito Santo, respectively. Debromoelatol (3), obtusane (4) and (1S*,2S*,3S*,5S*,8S*,9S*)-2,3,5,9-tetramethyltricyclo[6.3.0.0 1.5 ]undecan-2-ol (5) were obtained from specimens collected at Vermelha Beach, Parati, Rio de Janeiro. The structures of new compounds were elucidated by extensive NMR ( 1 H-, 13 C-, COSY, HSQC, HMBC and NOESY) and high resolution mass spectrometry analysis. Additionally, the absolute configuration of compound 2 was assigned by X-ray analysis. Full spectroscopic data is described for the first time for compound 3. Anti-inflammatory and antimycobacterial activities of compounds 2-5 were evaluated. Compounds 3-5 inhibited the release of inflammatory mediator NO while TNF-α levels were only affected by 3. All compounds tested displayed moderate antimycobacterial action.

Macrophage migration and adhesion are important for the control of mycobacterial infection and are critically dependent on the reorganization of the cytoskeleton. Mycobacteria elicit rapid morphological changes, such as cell spreading, a process relevant to in vivo changes of macrophage shape during extravasation and migration. In this study, we investigated the BCG mycobacteria-induced signaling events leading to macrophage cytoskeletal rearrangements employing specific pharmacological inhibitors to suppress distinct kinase pathways known to be elicited by infection. Viable or lysed mycobacteria, as well as purified cell wall lipoprotein p19, TLR2 agonist, induced RAW264.7 cells to extend actin-rich pseudopods, which impart radial spreading within 3 h, leading later to persistent cell polarization. BCG induced rapid activation of phosphatidylinositol 3-kinase, PI3K, activation that was recruited to the activated TLR2 receptor. TLR2- neutralizing antibody inhibited macrophage spreading and PI3K activation induced by p19. Additionally, BCG induced spreading and polarization of bone marrow-derived macrophages from TLR2- expressing mice in contrast to their TLR2-knockout counterparts. Neither MEK1/ERK, p38 MAPK, nor NF-kappaB activation were important for the early cytoskeletal rearrangements observed, although suppression of these pathways is known to inhibit chemokine secretion by activated macrophages. Beta2-integrins blockade with a corresponding antibody inhibited macrophage spreading and polarization but had no effect on pseudopodia protrusions demonstrating the downstream position of integrin-mediated adhesion in PI3K- dependent signaling pathway leading to the motility phenotype. The obtained data demonstrate that the direct effect of mycobacteria on macrophage shape might be mediated through TLR2-dependent PI3K activation.

Tuberculosis (TB) remains a serious public health problem aggravated by the emergence of M. tuberculosis (Mtb) strains resistant to multiple drugs (MDR). Delay in TB OPEN ACCESSMolecules 2015, 20 8073 treatment, common in the MDR-TB cases, can lead to deleterious life-threatening inflammation in susceptible hyper-reactive individuals, encouraging the discovery of new anti-Mtb drugs and the use of adjunctive therapy based on anti-inflammatory interventions. In this study, a series of forty synthetic chalcones was evaluated in vitro for their anti-inflammatory and antimycobacterial properties and in silico for pharmacokinetic parameters. Seven compounds strongly inhibited NO and PGE2 production by LPS-stimulated macrophages through the specific inhibition of iNOS and COX-2 expression, respectively, with compounds 4 and 5 standing out in this respect. Four of the seven most active compounds were able to inhibit production of TNF-α and IL-1β. Chalcones that were not toxic to cultured macrophages were tested for antimycobacterial activity. Eight compounds were able to inhibit growth of the M. bovis BCG and Mtb H37Rv strains in bacterial cultures and in infected macrophages. Four of them, including compounds 4 and 5, were active against a hypervirulent clinical Mtb isolate as well. In silico analysis of ADMET properties showed that the evaluated chalcones displayed satisfactory pharmacokinetic parameters. In conclusion, the obtained data demonstrate that at least two of the studied chalcones, compounds 4 and 5, are promising antimycobacterial and anti-inflammatory agents, especially focusing on an anti-tuberculosis dual treatment approach.