Biosynthesis of mycobacterial arabinogalactan: identification of a novel (13)arabinofuranosyltransferase

The cell wall mycolyl-arabinogalactan-peptidoglycan complex is essential in mycobacterial species, such as Mycobacterium tuberculosis and is the target of several anti-tubercular drugs. For instance, ethambutol targets arabinogalactan biosynthesis through inhibition of the arabinofuranosyltransferases Mt-EmbA and Mt-EmbB. A bioinformatics approach identified putative integral membrane proteins, MSMEG2785 in Mycobacterium smegmatis, Rv2673 in Mycobacterium tuberculosis and NCgl1822 in Corynebacterium glutamicum, with 10 predicted transmembrane domains and a glycosyltransferase motif (DDX), features that are common to the GT-C superfamily of glycosyltransferases. Deletion of M. smegmatis MSMEG2785 resulted in altered growth and glycosyl linkage analysis revealed the absence of AG (13)-linked arabinofuranosyl (Araf) residues. Complementation of the M. smegmatis deletion mutant was fully restored to a wild type phenotype by MSMEG2785 and Rv2673, and as a result, we have now termed this previously uncharacterized open reading frame, arabinofuranosyltransferase C (aftC). Enzyme assays using the sugar donor -D-arabinofuranosyl-1-monophosphoryldecaprenol (DPA) and a newly synthesized linear (15)-linked Ara5 neoglycolipid acceptor together with chemical identification of products formed, clearly identified AftC as a branching (13) arabinofuranosyltransferase. This newly discovered glycosyltransferase sheds further light on the complexities of Mycobacterium cell wall biosynthesis, such as in M. tuberculosis and related species and represents a potential new drug target

Analysis of nucleosome repositioning by yeast ISWI and Chd1 chromatin remodeling complexes

ISWI proteins form the catalytic core of a subset of ATP-dependent chromatin remodelling activities in eukaryotes from yeast to man. Many of these complexes have been found to reposition nucleosomes, but with different directionalities. We find that the yeast Isw1a, Isw2 and Chd1 enzymes preferentially move nucleosomes towards more central locations on short DNA fragments whereas Isw1b does not. Importantly, the inherent positioning properties of the DNA play an important role in determining where nucleosomes are relocated to by all of these enzymes. However, a key difference is that the Isw1a, Isw2 and Chd1 enzymes are unable to move nucleosomes to positions closer than 15 bp from a DNA end whereas Isw1b can. We also find that there is a correlation between the inability of enzymes to move nucleosomes close to DNA ends and the preferential binding to nucleosomes bearing linker DNA. These observations suggest that the accessibility of linker DNA together with the positioning properties of the underlying DNA play important roles in determining the outcome of remodelling by these enzymes

Sensitive electrochemiluminescence (ECL) immunoassays for detecting lipoarabinomannan (LAM) and ESAT-6 in urine and serum from tuberculosis patients.

BackgroundTuberculosis (TB) infection was responsible for an estimated 1.3 million deaths in 2017. Better diagnostic tools are urgently needed. We sought to determine whether accurate TB antigen detection in blood or urine has the potential to meet the WHO target product profiles for detection of active TB.Materials and methodsWe developed Electrochemiluminescence (ECL) immunoassays for Lipoarabinomannan (LAM) and ESAT-6 detection with detection limits in the pg/ml range and used them to compare the concentrations of the two antigens in the urine and serum of 81 HIV-negative and -positive individuals with presumptive TB enrolled across diverse geographic sites.ResultsLAM and ESAT-6 overall sensitivities in urine were 93% and 65% respectively. LAM and ESAT-6 overall sensitivities in serum were 55% and 46% respectively. Overall specificity was ≥97% in all assays. Sensitivities were higher in HIV-positive compared to HIV-negative patients for both antigens and both sample types, with signals roughly 10-fold higher on average in urine than in serum. The two antigens showed similar concentration ranges within the same sample type and correlated.ConclusionsLAM and ESAT-6 can be detected in the urine and serum of TB patients, regardless of the HIV status and further gains in clinical sensitivity may be achievable through assay and reagent optimization. Accuracy in urine was higher with current methods and has the potential to meet the WHO accuracy target if the findings can be transferred to a point-of-care TB test

The Snf2 Homolog Fun30 acts as a homodimeric ATP-dependent chromatin-remodeling enzyme

The Saccharomyces cerevisiae Fun30 (Function unknown now 30) protein shares homology with an extended family of Snf2-related ATPases. Here we report the purification of Fun30 principally as a homodimer with a molecular mass of about 250 kDa. Biochemical characterization of this complex reveals that it has ATPase activity stimulated by both DNA and chromatin. Consistent with this, it also binds to both DNA and chromatin. The Fun30 complex also exhibits activity in ATP-dependent chromatin remodeling assays. Interestingly, its activity in histone dimer exchange is high relative to the ability to reposition nucleosomes. Fun30 also possesses a weakly conserved CUE motif suggesting that it may interact specifically with ubiquitinylated proteins. However, in vitro Fun30 was found to have no specificity in its interaction with ubiquitinylated histones

A Novel Sensitive Immunoassay Targeting the 5-Methylthio-d-Xylofuranose-Lipoarabinomannan Epitope Meets the WHO's Performance Target for Tuberculosis Diagnosis.

The only currently commercialized point-of-care assay for tuberculosis (TB) that measures lipoarabinomannan (LAM) in urine (Alere LF-LAM) has insufficient sensitivity. We evaluated the potential of 100 novel monoclonal antibody pairs targeting a variety of LAM epitopes on a sensitive electrochemiluminescence platform to improve the diagnostic accuracy. In the screening, many antibody pairs showed high reactivity to purified LAM but performed poorly at detecting urinary LAM in clinical samples, suggesting differences in antigen structure and immunoreactivity of the different LAM sources. The 12 best antibody pairs from the screening were tested in a retrospective case-control study with urine samples from 75 adults with presumptive TB. The best antibody pair reached femtomolar analytical sensitivity for LAM detection and an overall clinical sensitivity of 93% (confidence interval [CI], 80% to 97%) and specificity of 97% (CI, 85% to 100%). Importantly, in HIV-negative subjects positive for TB by sputum smear microscopy, the test achieved a sensitivity of 80% (CI, 55% to 93%). This compares to an overall sensitivity of 33% (CI, 20% to 48%) of the Alere LF-LAM and a sensitivity of 13% (CI, 4% to 38%) in HIV-negative subjects in the same sample set. The capture antibody targets a unique 5-methylthio-d-xylofuranose (MTX)-dependent epitope in LAM that is specific to the Mycobacterium tuberculosis complex and shows no cross-reactivity with fast-growing mycobacteria or other bacteria. The present study provides evidence that improved assay methods and reagents lead to increased diagnostic accuracy. The results of this work have informed the development of a sensitive and specific novel LAM point-of-care assay with the aim to meet the WHO's performance target for TB diagnosis

Sequence Dependence of Transcription Factor-Mediated DNA Looping

DNA is subject to large deformations in a wide range of biological processes. Two key examples illustrate how such deformations influence the readout of the genetic information: the sequestering of eukaryotic genes by nucleosomes, and DNA looping in transcriptional regulation in both prokaryotes and eukaryotes. These kinds of regulatory problems are now becoming amenable to systematic quantitative dissection with a powerful dialogue between theory and experiment. Here we use a single-molecule experiment in conjunction with a statistical mechanical model to test quantitative predictions for the behavior of DNA looping at short length scales, and to determine how DNA sequence affects looping at these lengths. We calculate and measure how such looping depends upon four key biological parameters: the strength of the transcription factor binding sites, the concentration of the transcription factor, and the length and sequence of the DNA loop. Our studies lead to the surprising insight that sequences that are thought to be especially favorable for nucleosome formation because of high flexibility lead to no systematically detectable effect of sequence on looping, and begin to provide a picture of the distinctions between the short length scale mechanics of nucleosome formation and looping.Comment: Nucleic Acids Research (2012); Published version available at http://nar.oxfordjournals.org/cgi/content/abstract/gks473? ijkey=6m5pPVJgsmNmbof&keytype=re

Chromatin: a tunable spring at work inside chromosomes

This paper focuses on mechanical aspects of chromatin biological functioning. Within a basic geometric modeling of the chromatin assembly, we give for the first time the complete set of elastic constants (twist and bend persistence lengths, stretch modulus and twist-stretch coupling constant) of the so-called 30-nm chromatin fiber, in terms of DNA elastic properties and geometric properties of the fiber assembly. The computation naturally embeds the fiber within a current analytical model known as the ``extensible worm-like rope'', allowing a straightforward prediction of the force-extension curves. We show that these elastic constants are strongly sensitive to the linker length, up to 1 bp, or equivalently to its twist, and might locally reach very low values, yielding a highly flexible and extensible domain in the fiber. In particular, the twist-stretch coupling constant, reflecting the chirality of the chromatin fiber, exhibits steep variations and sign changes when the linker length is varied. We argue that this tunable elasticity might be a key feature for chromatin function, for instance in the initiation and regulation of transcription.Comment: 38 pages 15 figure

RNA Binding to CBP Stimulates Histone Acetylation and Transcription

CBP/p300 are transcription co-activators whose binding is a signature of enhancers, cis-regulatory elements that control patterns of gene expression in multicellular organisms. Active enhancers produce bi-directional enhancer RNAs (eRNAs) and display CBP/p300-dependent histone acetylation. Here, we demonstrate that CBP binds directly to RNAs in vivo and in vitro. RNAs bound to CBP in vivo include a large number of eRNAs. Using steady-state histone acetyltransferase (HAT) assays, we show that an RNA binding region in the HAT domain of CBP—a regulatory motif unique to CBP/p300—allows RNA to stimulate CBP’s HAT activity. At enhancers where CBP interacts with eRNAs, stimulation manifests in RNA-dependent changes in the histone acetylation mediated by CBP, such as H3K27ac, and by corresponding changes in gene expression. By interacting directly with CBP, eRNAs contribute to the unique chromatin structure at active enhancers, which, in turn, is required for regulation of target genes

Synthesis of the highly branched hexasaccharide core of chlorella virus N-linked glycans

Chlorella viruses produce N-linked glycoproteins with carbohydrate moieties that differ in structure from all other N- linked glycans. In addition, unlike most viruses, these organisms do not hijack the biosynthetic machinery of the host to make glycocoproteins; instead, they produce their own carbohydrate- processing enzymes. A better understanding of the function and assembly of these fascinating and structurally-unprecedented glycans requires access to probe molecules. We describe here the first synthesis of a chlorella virus N-linked glycan, a highly branched hexasaccharide that contains the pentasaccharide present in all of the >15 structures reported to date. The target molecule includes a glucosyl-asparagine linkage and a ‘hyperbranched’ fucose residue in which all of the hydroxyl groups are glycosylated. Both convergent and linear approaches were investigated with the latter being successful in providing the target in 16 steps and 13% overall yield

An oxidation–amidation approach for the synthesis of glycuronamides

A route for the synthesis of glycuronamides via the intermediacy of 6-S-tolyl-glycosides and uronic acid thioesters, is reported. The route, which is compatible with a variety of carbohydrate residues and protecting groups, was used to synthesize the repeating unit of the E. coli K54 capsular polysaccharide