Complete genome sequences of four mammalian isolates of Chlamydophila psittaci

Chlamydia psittaci is an obligate intracellular zoonotic pathogen primarily of birds, but it is also known to infect a variety of mammalian species. Here we report the genomes of four strains isolated from sheep (C19/98), pigs (01DC11), cattle (02DC15), and humans (08DC60).     

The biofilm inhibitor carolacton disturbs membrane integrity and cell division of Streptococcus mutans through the serine/threonine protein kinase PknB

Carolacton, a secondary metabolite isolated from the myxobacterium Sorangium cellulosum, disturbs Streptococcus mutans biofilm viability at nanomolar concentrations. Here we show that carolacton causes leakage of cytoplasmic content (DNA and proteins) in growing cells at low pH and provide quantitative data on the membrane damage. Furthermore, we demonstrate that the biofilm-specific activity of carolacton is due to the strong acidification occurring during biofilm growth. The chemical conversion of the ketocarbonic function of the molecule to a carolacton methylester did not impact its activity, indicating that carolacton is not functionally activated at low pH by a change of its net charge. A comparative time series microarray analysis identified the VicKRX and ComDE two-component signal transduction systems and genes involved in cell wall metabolism as playing essential roles in the response to carolacton treatment. A sensitivity testing of mutants with deletions of all 13 viable histidine kinases and the serine/threonine protein kinase PknB of S. mutans identified only the ΔpknB deletion mutant as being insensitive to carolacton treatment. A strong overlap between the regulon of PknB in S. mutans and the genes affected by carolacton treatment was found. The data suggest that carolacton acts by interfering with PknB-mediated signaling in growing cells. The resulting altered cell wall morphology causes membrane damage and cell death at low pH.     

The E-loop is involved in hydrogen peroxide formation by the NADPH oxidase Nox4

In contrast to the NADPH oxidases Nox1 and Nox2, which generate superoxide (O(2)(·-)), Nox4 produces hydrogen peroxide (H(2)O(2)). We constructed chimeric proteins and mutants to address the protein region that specifies which reactive oxygen species is produced. Reactive oxygen species were measured with luminol/horseradish peroxidase and Amplex Red for H(2)O(2) versus L-012 and cytochrome c for O(2)(·-). The third extracytosolic loop (E-loop) of Nox4 is 28 amino acids longer than that of Nox1 or Nox2. Deletion of E-loop amino acids only present in Nox4 or exchange of the two cysteines in these stretches switched Nox4 from H(2)O(2) to O(2)(·-) generation while preserving expression and intracellular localization. In the presence of an NO donor, the O(2)()-producing Nox4 mutants, but not wild-type Nox4, generated peroxynitrite, excluding artifacts of the detection system as the apparent origin of O(2)(·-). In Cos7 cells, in which Nox4 partially localizes to the plasma membrane, an antibody directed against the E-loop decreased H(2)O(2) but increased O(2)(·-) formation by Nox4 without affecting Nox1-dependent O(2)(·-) formation. The E-loop of Nox4 but not Nox1 and Nox2 contains a highly conserved histidine that could serve as a source for protons to accelerate spontaneous dismutation of superoxide to form H(2)O(2). Mutation of this but not of four other conserved histidines also switched Nox4 from H(2)O(2) to O(2)(·-) formation. Thus, H(2)O(2) formation is an intrinsic property of Nox4 that involves its E-loop. The structure of the E-loop may hinder O(2)(·-) egress and/or provide a source for protons, allowing dismutation to form H(2)O(2).     

Plant D-2-hydroxyglutarate dehydrogenase participates in the catabolism of lysine especially during senescence

D-2-Hydroxyglutarate dehydrogenase (D-2HGDH) catalyzes the specific and efficient oxidation of D-2-hydroxyglutarate (D-2HG) to 2-oxoglutarate using FAD as a cofactor. In this work, we demonstrate that D-2HGDH localizes to plant mitochondria and that its expression increases gradually during developmental and dark-induced senescence in Arabidopsis thaliana, indicating an enhanced demand of respiration of alternative substrates through this enzymatic system under these conditions. Using loss-of-function mutants in D-2HGDH (d2hgdh1) and stable isotope dilution LC-MS/MS, we found that the D-isomer of 2HG accumulated in leaves of d2hgdh1 during both forms of carbon starvation. In addition to this, d2hgdh1 presented enhanced levels of most TCA cycle intermediates and free amino acids. In contrast to the deleterious effects caused by a deficiency in D-2HGDH in humans, d2hgdh1 and overexpressing lines of D-2HGDH showed normal developmental and senescence phenotypes, indicating a mild role of D-2HGDH in the tested conditions. Moreover, metabolic fingerprinting of leaves of plants grown in media supplemented with putative precursors indicated that D-2HG most probably originates during the catabolism of lysine. Finally, the L-isomer of 2HG was also detected in leaf extracts, indicating that both chiral forms of 2HG participate in plant metabolism.     

Mycobacterium tuberculosis triggers host type I IFN signaling to regulate IL-1β production in human macrophages

Mycobacterium tuberculosis is a virulent intracellular pathogen that survives in macrophages even in the presence of an intact adaptive immune response. Type I IFNs have been shown to exacerbate tuberculosis in mice and to be associated with disease progression in infected humans. Nevertheless, the mechanisms by which type I IFNs regulate the host response to M. tuberculosis infection are poorly understood. In this study, we show that M. tuberculosis induces an IFN-related gene expression signature in infected primary human macrophages, which is dependent on host type I IFN signaling as well as the mycobacterial virulence factor, region of difference-1. We further demonstrate that type I IFNs selectively limit the production of IL-1β, a critical mediator of immunity to M. tuberculosis. This regulation occurs at the level of IL1B mRNA expression, rather than caspase-1 activation or autocrine IL-1 amplification and appears to be preferentially used by virulent mycobacteria since avirulent M. bovis bacillus Calmette-Guérin (BCG) fails to trigger significant expression of type I IFNs or release of mature IL-1β protein. The latter property is associated with decreased caspase-1-dependent IL-1β maturation in the BCG-infected macrophages. Interestingly, human monocytes in contrast to macrophages produce comparable levels of IL-1β in response to either M. tuberculosis or BCG. Taken together, these findings demonstrate that virulent and avirulent mycobacteria employ distinct pathways for regulating IL-1β production in human macrophages and reveal that in the case of M. tuberculosis infection the induction of type I IFNs is a major mechanism used for this purpose.     

IL-1β and TGF-β act antagonistically in induction and differentially in propagation of human proinflammatory precursor CD4+ T cells

Cytokines are critical messengers that control the differentiation of Th cells. To evaluate their impact on the fate of human naive CD4(+) T cells from cord and adult blood, early T cell differentiation was monitored after T cell activation in the presence of pro- and anti-inflammatory cytokines. Interestingly, the analysis of Th cell lineage-specific molecules revealed that IL-1β on its own mediates differentiation of Th cells that secrete a wide range of proinflammatory cytokines and stably express CD69, STAT1, IFN-γ, and IL-17. Notably, our data suggest that IL-1β induces Th17 cells independent of RORC upregulation. In contrast, TGF-β that triggers RORC prevents Th17 cell development. This suppressive function of TGF-β is characterized by inhibition of STAT1, STAT3, and CD69. However, after repeated anti-CD3 and anti-CD28 stimulation, we observe that TGF-β provokes an increase in Th17 cells that presumably relies on reactivation of a default pathway by preferential inhibition of IFN-γ. Hence, our data extend the view that the principal cytokines for determining Th cell fate are IL-12 for the Th1 lineage, IL-4 for the Th2 lineage, and TGF-β in conjunction with IL-6 for the Th17 lineage. We propose that IL-1β induces a general proinflammatory Th cell precursor that, in the presence of the lineage-specifying cytokines, further differentiates into one of the specific Th cell subpopulations.     

Multispan mitochondrial outer membrane protein Ugo1 follows a unique Mim1-dependent import pathway

The mitochondrial outer membrane (MOM) harbors several multispan proteins that execute various functions. Despite their importance, the mechanisms by which these proteins are recognized and inserted into the outer membrane remain largely unclear. In this paper, we address this issue using yeast mitochondria and the multispan protein Ugo1. Using a specific insertion assay and analysis by native gel electrophoresis, we show that the import receptor Tom70, but not its partner Tom20, is involved in the initial recognition of the Ugo1 precursor. Surprisingly, the import pore formed by the translocase of the outer membrane complex appears not to be required for the insertion process. Conversely, the multifunctional outer membrane protein mitochondrial import 1 (Mim1) plays a central role in mediating the insertion of Ugo1. Collectively, these results suggest that Ugo1 is inserted into the MOM by a novel pathway in which Tom70 and Mim1 contribute to the efficiency and selectivity of the process.