Developmental transcriptome of resting cell formation in Mycobacterium smegmatis

Functional classification of gene numbers up- and down-regulated during shock starvation in PBS. (PDF 1796 kb

Defining the structural requirements for ribose 5-phosphate-binding and intersubunit cross-talk of the malarial pyridoxal 5-phosphate synthase

Most organisms synthesise the B(6) vitamer pyridoxal 5-phosphate (PLP) via the glutamine amidotransferase PLP synthase, a large enzyme complex of 12 Pdx1 synthase subunits with up to 12 Pdx2 glutaminase subunits attached. Deletion analysis revealed that the C-terminus has four distinct functionalities: assembly of the Pdx1 monomers, binding of the pentose substrate (ribose 5-phosphate), formation of the reaction intermediate I(320), and finally PLP synthesis. Deletions of distinct C-terminal regions distinguish between these individual functions. PLP formation is the only function that is conferred to the enzyme by the C-terminus acting in trans, explaining the cooperative nature of the complex

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

Primary tumor–derived systemic nANGPTL4 inhibits metastasis

Primary tumors and distant site metastases form a bidirectionally communicating system. Yet, the molecular mechanisms of this crosstalk are poorly understood. Here, we identified the proteolytically cleaved fragments of angiopoietin-like 4 (ANGPTL4) as contextually active protumorigenic and antitumorigenic contributors in this communication ecosystem. Preclinical studies in multiple tumor models revealed that the C-terminal fragment (cANGPTL4) promoted tumor growth and metastasis. In contrast, the N-terminal fragment of ANGPTL4 (nANGPTL4) inhibited metastasis and enhanced overall survival in a postsurgical metastasis model by inhibiting WNT signaling and reducing vascularity at the metastatic site. Tracing ANGPTL4 and its fragments in tumor patients detected full-length ANGPTL4 primarily in tumor tissues, whereas nANGPTL4 predominated in systemic circulation and correlated inversely with disease progression. The study highlights the spatial context of the proteolytic cleavage-dependent pro- and antitumorigenic functions of ANGPTL4 and identifies and validates nANGPTL4 as a novel biomarker of tumor progression and antimetastatic therapeutic agent

Novel Acetamide Indirectly Targets Mycobacterial Transporter MmpL3 by Proton Motive Force Disruption

To identify novel inhibitors of Mycobacterium tuberculosis cell envelope biosynthesis, we employed a two-step approach. First, we screened the diverse synthetic small molecule 71,544-compound Enamine library for growth inhibitors using the non-pathogenic surrogate Mycobacterium bovis BCG as screening strain and turbidity as readout. Second, 16 confirmed hits were tested for their ability to induce the cell envelope stress responsive promoter piniBAC controlling expression of red fluorescent protein in an M. bovis BCG reporter strain. Using a fluorescence readout, the acetamide E11 was identified. Resistant mutant selection and whole genome sequencing revealed the mycolic acid transporter Mmpl3 as a candidate target of E11. Biochemical analysis using mycobacterial spheroplasts and various membrane assays suggest that E11 indirectly inhibits MmpL3-facilitated translocation of trehalose monomycolates by proton motive force disruption. E11 showed potent bactericidal activity against growing and non-growing M. tuberculosis, low cytotoxic, and hemolytic activity and a dynamic structure activity relationship. In addition to activity against M. tuberculosis, E11 was active against the non-tuberculous mycobacterium M. abscessus, an emerging opportunistic pathogen. In conclusion, we identified a novel bactericidal anti-mycobacterial lead compound targeting MmpL3 providing an attractive starting point for optimization

Significant under expression of the DosR regulon in M. tuberculosis complex lineage 6 in sputum

YesMycobacterium africanum lineage (L) 6 is an important pathogen in West Africa, causing up to 40% of pulmonary tuberculosis (TB). The biology underlying the clinical differences between M. africanum and M. tuberculosis sensu stricto remains poorly understood. We performed ex vivo expression of 2179 genes of the most geographically dispersed cause of human TB, M. tuberculosis L4 and the geographically restricted, M. africanum L6 directly from sputa of 11 HIV-negative TB patients from The Gambia who had not started treatment. The DosR regulon was the most significantly decreased category in L6 relative to L4. Further, we identified nonsynonymous mutations in major DosR regulon genes of 44 L6 genomes of TB patients from The Gambia and Ghana. Using Lebek's test, we assessed differences in oxygen requirements for growth. L4 grew only at the aerobic surface while L6 grew throughout the medium. In the host, the DosR regulon is critical for M. tuberculosis in adaptation to oxygen limitation. However, M. africanum L6 appears to have adapted to growth under hypoxic conditions or to different biological niches. The observed under expression of DosR in L6 fits with the genomic changes in DosR genes, microaerobic growth and the association with extrapulmonary disease.European Research Council-INTERRUPTB starting grant nr.311725 (to BdJ, BO, FG, MA, CM)

Childhood tuberculosis is associated with decreased abundance of T cell gene transcripts and impaired T cell function

The WHO estimates around a million children contract tuberculosis (TB) annually with over 80 000 deaths from dissemination of infection outside of the lungs. The insidious onset and association with skin test anergy suggests failure of the immune system to both recognise and respond to infection. To understand the immune mechanisms, we studied genome-wide whole blood RNA expression in children with TB meningitis (TBM). Findings were validated in a second cohort of children with TBM and pulmonary TB (PTB), and functional T-cell responses studied in a third cohort of children with TBM, other extrapulmonary TB (EPTB) and PTB. The predominant RNA transcriptional response in children with TBM was decreased abundance of multiple genes, with 140/204 (68%) of all differentially regulated genes showing reduced abundance compared to healthy controls. Findings were validated in a second cohort with concordance of the direction of differential expression in both TBM (r2 = 0.78 p = 2x10-16) and PTB patients (r2 = 0.71 p = 2x10-16) when compared to a second group of healthy controls. Although the direction of expression of these significant genes was similar in the PTB patients, the magnitude of differential transcript abundance was less in PTB than in TBM. The majority of genes were involved in activation of leucocytes (p = 2.67E-11) and T-cell receptor signalling (p = 6.56E-07). Less abundant gene expression in immune cells was associated with a functional defect in T-cell proliferation that recovered after full TB treatment (p<0.0003). Multiple genes involved in T-cell activation show decreased abundance in children with acute TB, who also have impaired functional T-cell responses. Our data suggest that childhood TB is associated with an acquired immune defect, potentially resulting in failure to contain the pathogen. Elucidation of the mechanism causing the immune paresis may identify new treatment and prevention strategies