Akaligenes eutrophus strain CH34, which was isolated as a bacterium resistant to cobalt, zinc, and cadmium ions, shares with A. eutrophus strain H16 the ability to grow lithoautotrophically on molecular hydrogen, to form a cytoplasmic NAD-reducing and a membrane-bound hydrogenase, and most metabolic attributes; however, it does not grow on fructose. Strain CH34 contains two plasmids, pMOL28 (163 kilobases) specifying nickel, mercury, and cobalt resistance and pMOL30 (238 kilobases) specifying zinc, cadmium, mercury, and cobalt resistance. The plasmids are self-transmissible in homologous matings, but at low frequencies. The transfer frequency was strongly increased with IncPl plasmids RP4 and pUZ8 as helper plasmids. The phenotypes of the wild type, cured strains, and transconjugants are characterized by the following MICs (Micromolar) in strains with the indicated phenotypes: Nic+, 2.5; Nic-, 0.6; Cob+A, 5.0; Cob+B, 20.0; Cob-, < 9.07; Zin+, 12.0; Zin-, 0.6; Cad', 2.5; and Cad-, 0.6. Plasmid-free cells of strain CH34 are still able to grow lithoautotrophicaily and to form both hydrogenases, indicating that the hydrogenase genes are located on the chromosome, in contrast to the Hox structural genes of strain H16, which are located on the megaplasmid pHG1 (450 kilobases).

BackgroundInterplays between Mycobacterium tuberculosis, the etiological agent of tuberculosis (TB) in human and host professional phagocytes, namely macrophages (Mφs) and dendritic cells (DCs), are central to immune protection against TB and to TB pathogenesis. We and others have recently shown that the C-type lectin dendritic cell–specific intercellular adhesion molecule-3 grabbing nonintegrin (DC-SIGN; CD209) mediates important interactions between mycobacteria and human monocyte-derived DCs (MoDCs) in vitro.Methods and FindingsIn order to explore the possible role of DC-SIGN in M. tuberculosis infection in vivo, we have analysed DC-SIGN expression in broncho-alveolar lavage (BAL) cells from patients with TB (n = 40) or with other non-mycobacterial lung pathologies, namely asthma (n = 14) and sarcoidosis (n = 11), as well as from control individuals (n = 9). We show that in patients with TB, up to 70% of alveolar Mφs express DC-SIGN. By contrast, the lectin is barely detected in alveolar Mφs from all other individuals. Flow cytometry, RT-PCR, and enzyme-linked immunosorbent assay analyses of BAL-derived fluids and cells indicated that M. tuberculosis infection induces DC-SIGN expression in alveolar Mφs by a mechanism that is independent of Toll-like receptor-4, interleukin (IL)-4, and IL-13. This mechanism most likely relies on the secretion of soluble host and/or mycobacterial factors that have yet to be identified, as both infected and uninfected bystander Mφs were found to express DC-SIGN in the presence of M. tuberculosis. Immunohistochemical examination of lung biopsy samples from patients with TB showed that the bacilli concentrate in pulmonary regions enriched in DC-SIGN-expressing alveolar Mφs in vivo. Ex vivo binding and inhibition of binding experiments further revealed that DC-SIGN–expressing alveolar Mφs constitute preferential target cells for M. tuberculosis, as compared to their DC-SIGN− counterparts. In contrast with what has been reported previously in MoDCs in vitro, ex vivo DC-SIGN ligation by mycobacterial products failed to induce IL-10 secretion by alveolar Mφs, and IL-10 was not detected in BALs from patients with TB.ConclusionAltogether, our results provide further evidence for an important role of DC-SIGN during TB in humans. DC-SIGN induction in alveolar Mφs may have important consequences on lung colonization by the tubercle bacillus, and on pulmonary inflammatory and immune responses in the infected host.

The mechanisms by which the airborne pathogen Mycobacterium tuberculosis spreads within the lung and leaves its primary niche to colonize other organs, thus inducing extrapulmonary forms of tuberculosis (TB) in humans, remains poorly understood. Herein, we used a transcriptomic approach to investigate the host cell gene expression profile in M. tuberculosis–infected human macrophages (ΜΦ). We identified 33 genes, encoding proteins involved in angiogenesis, for which the expression was significantly modified during infection, and we show that the potent angiogenic factor VEGF is secreted by M. tuberculosis-infected ΜΦ, in an RD1-dependent manner. In vivo these factors promote the formation of blood vessels in murine models of the disease. Inhibiting angiogenesis, via VEGF inactivation, abolished mycobacterial spread from the infection site. In accordance with our in vitro and in vivo results, we show that the level of VEGF in TB patients is elevated and that endothelial progenitor cells are mobilized from the bone marrow. These results strongly strengthen the most recent data suggesting that mycobacteria take advantage of the formation of new blood vessels to disseminate.

Mycobacteria produce two unusual polymethylated polysaccharides (PMPS), 3 the 3-O-methylmannose polysaccharides (MMP) (1-2) and the 6-O-methylglucosyl-containing lipopolysaccharides (MGLP) (3, 4). Both polysaccharides localize to the cytoplasm, where they have been postulated to regulate fatty acid synthesis by FAS-I as a consequence of their ability to form stable 1:1 complexes with long-chain fatty acids and acylcoenzyme A derivatives (5-9) (for a review, see Ref. 10). PMPS were also proposed to protect fatty acid products from degradation and to serve as general lipid carriers facilitating the synthesis of the very large and insoluble mycolic acid esters while at the same time increasing the tolerance of mycobacteria to high cytoplasmic concentrations of long-chain acyl-CoA derivatives (10 -12). PMPS were first isolated from Mycobacterium phlei, Mycobacterium smegmatis, and Mycobacterium tuberculosis in the 1960s (2-4), and much of the information we have about the structure, biosynthesis, and biological activities of these molecules comes from this early work. Others then revised the structure of MGLP and extended the analysis of these molecules to other mycobacterial species (13-15). The structures of MGLP and MMP are shown in Fig. 1. MMP have been found in multiple nonpathogenic fast growing species of mycobacteria (16) and in Streptomyces griseus (17), whereas MGLP have been isolated from several Nocardia species as well as M. phlei, M. smegmatis, Mycobacterium bovis BCG, M. tuberculosis, M. leprae,[13][14][15]18,19).Ballou and co-workers (2, 16, 20, 21) isolated precursors of MMP and characterized an ␣-(134)-mannosyltransferase and a 3-O-methyltransferase from cell-free extracts of M. smegmatis. These studies led to a biosynthetic model in which MMP is elongated by a linear alternating process of mannosylation followed by O-methylation, in which GDP-Man serves as the sugar donor for the mannosyltransferase and S-adenosylmethionine serves as the source of methyl groups. Termination of the elongation reaction occurs when the length of the chain is sufficient to confer on the polysaccharide fatty acid-binding properties (11-13 3-O-methylmannoses). At this stage, the chain is terminated with an unmethylated mannose because the acylCoA-bound oligosaccharides are no longer available as acceptors for the 3-O-methyltransferase.Knowledge of the initiation, elongation, and termination reactions involved in the biosynthesis of MGLP is more limited. A membrane-associated acyltransferase activity responsible for the transfer of acetyl, propionyl, isobutyryl, octanoyl, and succinyl groups from their respective acyl-CoA derivatives onto *

Abstractp-Hydroxybenzoic acid derivatives (p-HBADs) are glycoconjugates secreted by all Mycobacterium tuberculosis isolates whose contribution to pathogenicity remains to be determined. The pathogenicity of three transposon mutants of M. tuberculosis deficient in the biosynthesis of some or all forms of p-HBADs was studied. Whilst the mutants grew similarly to the wild-type strain in macrophages and C57BL/6 mice, two of the mutants induced a more severe and diffuse inflammation in the lungs. The lack of production of some or all forms of p-HBADs in these two mutants also correlated with an increased secretion of the pro-inflammatory cytokines tumour-necrosis factor α, interleukin 6 and interleukin 12 in vivo. We propose that the loss of production of p-HBADs by tubercle bacilli results in their diminished ability to suppress the proinflammatory response to infection and that this ultimately provokes extensive pulmonary lesions in the C57BL/6 model of tuberculosis infection.