AhR sensing of bacterial pigments regulates antibacterial defence.

The aryl hydrocarbon receptor (AhR) is a highly conserved ligand-dependent transcription factor that senses environmental toxins and endogenous ligands, thereby inducing detoxifying enzymes and modulating immune cell differentiation and responses. We hypothesized that AhR evolved to sense not only environmental pollutants but also microbial insults. We characterized bacterial pigmented virulence factors, namely the phenazines from Pseudomonas aeruginosa and the naphthoquinone phthiocol from Mycobacterium tuberculosis, as ligands of AhR. Upon ligand binding, AhR activation leads to virulence factor degradation and regulated cytokine and chemokine production. The relevance of AhR to host defence is underlined by heightened susceptibility of AhR-deficient mice to both P. aeruginosa and M. tuberculosis. Thus, we demonstrate that AhR senses distinct bacterial virulence factors and controls antibacterial responses, supporting a previously unidentified role for AhR as an intracellular pattern recognition receptor, and identify bacterial pigments as a new class of pathogen-associated molecular patterns.

Identification of RNA targets for the nuclear multidomain cyclophilin atCyp59 and their effect on PPIase activity.

AtCyp59 is a multidomain cyclophilin containing a peptidyl-prolyl cis/trans isomerase (PPIase) domain and an evolutionarily highly conserved RRM domain. Deregulation of this class of cyclophilins has been shown to affect transcription and to influence phosphorylation of the C-terminal repeat domain of the largest subunit of the RNA polymerase II. We used a genomic SELEX method for identifying RNA targets of AtCyp59. Analysis of the selected RNAs revealed an RNA-binding motif (G[U/C]N[G/A]CC[A/G]) and we show that it is evolutionarily conserved. Binding to this motif was verified by gel shift assays in vitro and by RNA immunopreciptation assays of AtCyp59 in vivo. Most importantly, we show that binding also occurs on unprocessed transcripts in vivo and that binding of specific RNAs inhibits the PPIase activity of AtCyp59 in vitro. Surprisingly, genome-wide analysis showed that the RNA motif is present in about 70% of the annotated transcripts preferentially in exons. Taken together, the available data suggest that these cyclophilins might have an important function in transcription regulation.

Humanized Mouse Model Mimicking Pathology of Human Tuberculosis for in vivo Evaluation of Drug Regimens.

Human immune system mice are highly valuable for in vivo dissection of human immune responses. Although they were employed for analyzing tuberculosis (TB) disease, there is little data on the spatial organization and cellular composition of human immune cells in TB granuloma pathology in this model. We demonstrate that human immune system mice, generated by transplanted human fetal liver derived hematopoietic stem cells develop a continuum of pulmonary lesions upon Mycobacterium tuberculosis aerosol infection. In particular, caseous necrotic granulomas, which contribute to prolonged TB treatment time, developed, and had cellular phenotypic spatial-organization similar to TB patients. By comparing two recommended drug regimens, we confirmed observations made in clinical settings: Adding Moxifloxacin to a classical chemotherapy regimen had no beneficial effects on bacterial eradication. We consider this model instrumental for deeper understanding of human specific features of TB pathogenesis and of particular value for the pre-clinical drug development pipeline.

Heterogeneity and timing of translocation and membrane-mediated assembly of different annexins.

Many cell types, including neurons and epithelial cells, express a variety of annexins. Although the overall function has only been partially unravelled, a dominant feature is the formation of two-dimensional assemblies under the plasma membrane in a calcium-dependent manner. Here we show that fluorescently tagged annexins A1, A2, A4, A5, and A6 translocate and assemble at the plasma membrane and the nuclear envelope, except annexin A2, which only attaches to the plasma membrane. All annexins have different response times to elevated calcium levels as was shown by the translocation of co-expressed proteins. Fluorescence recovery after photobleaching revealed the static nature of all annexin assemblies. Analysis of the assemblies by Foerster resonance energy transfer (FRET) using acceptor bleaching demonstrated mostly annexin-specific self-assembly. Heterogeneous assembly formation was shown between annexins A5 and A1, and A5 and A2. The formation of homo- and heterogeneous annexin assemblies may play an important role when high increases in calcium occur, such as after disruption of the plasma membrane.