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Arabinofuranosyltransferase enzymes, such as EmbA, EmbB, and AftA, play pivotal roles in the biosynthesis of arabinogalactan, and the anti-tuberculosis agent ethambutol (EMB) targets arabinogalactan biosynthesis through inhibition of Mt-EmbA and Mt-EmbB. Herein, we describe the identification and characterization of a novel arabinofuranosyltransferase, now termed AftB (Rv3805c), which is essential in Mycobacterium tuberculosis. Deletion of its orthologue NCgl2780 in the closely related species Corynebacterium glutamicum resulted in a viable mutant. Analysis of the cell wall-associated lipids from the deletion mutant revealed a decreased abundance of cell wall-bound mycolic acids, consistent with a partial loss olmycolylation sites. Subsequent glycosyl linkage analysis of arabinogalactan also revealed the complete absence of terminal β(1 → 2)-linked arabinofuranosyl residues. The deletion mutant biochemical phenotype was fully complemented by either Mt-AftB or Cg-AftB, but not with muteins of Mt-AftB, where the two adjacent aspartic acid residues, which have been suggested to be involved in glycosyltransferase activity, were replaced by alanine. In addition, the use of C. glutamicum and C. glutamicumΔaftB in an in vitro assay utilizing the sugar donor β-D-arabinofuranosyl-1-monophosphoryl-decaprenol together with the neoglycolipid acceptor α-D-Araf-(1 → 5)-α-D-Araf-O-C8 is a substrate con- firmed AftB as a terminal β(1 → 2) arabinofuranosyltransferase, which was also insensitive to EMB. Altogether, these studies have shed further light on the complexities of Corynebacterianeae cell wall biosynthesis, and Mt-AftB represents a potential new drug target. [ABSTRACT FROM AUTHOR]

The cell wall of Mycobacterium tuberculosis has a complex ultrastructure that consists of mycolic acids connected to peptidoglycan via arabinogalactan (AG) and abbreviated as the mAGP complex. The mAGP complex is crucial for the survival and pathogenicity of M. tuberculosis and is the target of several anti-tubercular agents. Apart from sharing a similar mAGP and the availability of the complete genome sequence, Corynebacterium glutamicum has proven useful in the study of orthologous M. tuberculosis genes essential for viability. Here we examined the effects of particular genes involved in AG polymerization by gene deletion in C. glutamicum. The anti-tuberculosis drug ethambutol is thought to target a set of arabinofuranosyltransferases (Emb) that are involved in arabinan polymerization. Deletion of emb in C. glutamicum results in a slow growing mutant with profound morphological changes. Chemical analysis revealed a dramatic reduction of arabinose resulting in a novel truncated AG structure possessing only terminal arabinofuranoside (t-Araf) residues with a corresponding loss of cell wall bound mycolic acids. Treatment of wild-type C. glutamicum with ethambutol and subsequent cell wall analyses resulted in an identical phenotype comparable to the C. glutamicum emb deletion mutant. Additionally, disruption of ubiA in C. glutamicum, the first enzyme involved in the biosynthesis of the sugar donor decaprenol phosphoarabinose (DPA), resulted in a complete loss of cell wall arabinan. Herein, we establish for the first time, (i) that in contrast to M. tuberculosis embA and embB mutants, deletion of C. glutamicum emb leads to a highly truncated AG possessing t-Araf residues, (ii) the exact site of attachment of arabinan chains in AG, and (iii) DPA is the only Araf sugar donor in AG biosynthesis suggesting the presence of a novel enzyme responsible for ‘priming’ the galactan domain for further elaboration by Emb, resulting in the final maturation of the native AG polysaccharide. [ABSTRACT FROM AUTHOR]

Background: Active decision support systems implementing goal directed therapy may be an approach to reduce disparities in outcome between different health care providers. We assessed feasibility of and adherence to an active decision support system (ADSS) comprising fluids, vasopressors, and dobutamine to optimize hemodynamics during high-risk vascular surgery., Methods: In this prospective observational trial a closed-loop goal-directed therapy protocol, employing the minimally-invasive LiDCOrapid device, was used to actively provide advice to the anesthesiologist during surgery. All given suggestions and all interventions were recorded. Every intervention without or against the given advice had to be justified. The primary outcome parameters were the number of interventions done according to the ADSS and its duration of use. Reasons for non-compliance served to describe its limitations., Results: The active decision support system was employed in 32 patients for 137 hours. Median (IQR) use of the ADSS as percentage of surgery time was 100% (94-100%) with 743 interventions being executed. 634 interventions were done according to ADSS proposals. Reasons to act against or without advice were: hemodynamic instability (6%), foreseeing a surgical event (2%), medical reasons (2%), awaiting hemodynamic improvement (1%) and orders by senior physician or surgeons (1%). In five patients the anesthesiologist decided to modify intervention thresholds of the underlying protocol., Conclusions: High rates of compliance underline clinical acceptability and feasibility of this ADSS during vascular surgery. It may therefore facilitate the work of anesthesiologists and reduce disparities in patient outcomes due to different healthcare providers. Particularly, rapidly developing hemodynamic perturbances as well as co-factors the ADSS as of now does not anticipate are current limitations. These findings may serve to further improve this stand-alone real-time ADSS.

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. Following a detailed bioinformatics analysis of genes surrounding the conserved emb locus, we present the identification and characterization of a novel arabinofuranosyltransferase AftA (Rv3792). The enzyme catalyzes the addition of the first key arabinofuranosyl residue from the sugar donor beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to the galactan domain of the cell wall, thus "priming" the galactan for further elaboration by the arabinofuranosyltransferases. Because aftA is an essential gene in M. tuberculosis, we deleted its orthologue in Corynebacterium glutamicum to produce a slow growing but viable mutant. Analysis of its cell wall revealed the complete absence of arabinose resulting in a truncated cell wall structure possessing only a galactan core with a concomitant loss of cell wall-bound mycolates. Complementation of the mutant was fully restored to the wild type phenotype by Cg-aftA. In addition, by developing an in vitro assay using recombinant Escherichia coli expressing Mt-aftA and use of cell wall galactan as an acceptor, we demonstrated the transfer of arabinose from beta-D-arabinofuranosyl-1-monophosphoryldecaprenol to galactan, and unlike the Mt-Emb proteins, Mt-AftA was not inhibited by ethambutol. This newly discovered glycosyltransferase represents an attractive drug target for further exploitation by chemotherapeutic intervention.