High-level expression, high-throughput screening and direct recovery of nitroreductase enzymes from metagenome libraries

We have developed generally applicable library generation methods to maximize expression of cloned environmental genes, enabling screening for weak phenotypes in metagenome libraries. Our method also permits direct recovery of the encoded enzymes, providing rapid access to an almost unlimited diversity of previously unexplored biocatalysts. We have exemplified this for nitroreductases, members of a diverse family of oxidoreductase enzymes that can catalyze the bioreductive activation of nitroaromatic prodrugs such as metronidazole. These capabilities have diverse applications in medicine and research, including anti-cancer gene therapy and targeted ablation of nitroreductase-expressing tissues in transgenic animal models. However, research in these fields has largely been focused on the canonical nitroreductase NfsB from Escherichia coli, which exhibits sub-optimal levels of metronidazole activity. In previous work we have investigated alternative nitroreductase enzymes, sourced from genome-sequenced bacteria. To complement this work we have now turned to the discovery of novel nitroreductases from metagenomic DNA fragments, derived from the uncultivable bacteria present in New Zealand soil and lichen species

Characterization of pyoverdine and achromobactin in Pseudomonas syringae pv. phaseolicola 1448a

<p>Abstract</p> <p>Background</p> <p><it>Pseudomonas syringae </it>pv. phaseolicola 1448a (<it>P. syringae </it>1448a), the causative agent of bean halo blight, is a bacterium capable of occupying diverse biological niches. Under conditions of iron starvation <it>P. syringae </it>1448a secretes siderophores for active uptake of iron. The primary siderophore of <it>P. syringae </it>1448a is pyoverdine, a fluorescent molecule that is assembled from amino acid precursors by non-ribosomal peptide synthetase (NRPS) enzymes. Whereas other species of <it>Pseudomonas </it>often exhibit structural variations in the pyoverdine produced by different strains, all <it>P. syringae </it>pathovars previously tested have been found to make an identical pyoverdine molecule. <it>P. syringae </it>1448a also appears to have the genetic potential to make two secondary siderophores, achromobactin and yersiniabactin, each of which has previously been detected in different <it>P. syringae </it>pathovars.</p> <p>Results</p> <p>Five putative pyoverdine NRPS genes in <it>P. syringae </it>1448a were characterized <it>in-silico </it>and their role in pyoverdine biosynthesis was confirmed by gene knockout. Pyoverdine was purified from <it>P. syringae </it>1448a and analyzed by MALDI-TOF and MS/MS spectroscopy. Peaks were detected corresponding to the expected sizes for the pyoverdine structure previously found in other <it>P. syringae </it>pathovars, but surprisingly <it>P. syringae </it>1448a appears to also produce a variant pyoverdine species that has an additional 71 Da monomer incorporated into the peptide side chain. Creation of pyoverdine null mutants of <it>P. syringae </it>1448a revealed that this strain also produces achromobactin as a temperature-regulated secondary siderophore, but does not appear to make yersiniabactin. Pyoverdine and achromobactin null mutants were characterized in regard to siderophore production, iron uptake, virulence and growth in iron limited conditions.</p> <p>Conclusions</p> <p>This study provides the first evidence of a <it>P. syringae </it>pathovar producing a side chain variant form of pyoverdine. We also describe novel IC₅₀and liquid CAS assays to quantify the contribution of different siderophores across a range of iron starvation conditions, and show that although achromobactin has potential to contribute to fitness its contribution is masked by the presence of pyoverdine, which is a significantly more effective siderophore. Neither pyoverdine nor achromobactin appear to be required for <it>P. syringae </it>1448a to cause bean halo blight, indicating that these siderophores are not promising targets for crop protection strategies.</p

Discovering and engineering novel prodrug activating and detoxifying enzymes to improve targeted cell ablation

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Discovery and evolution of primordial antibiotic resistance genes from soil microbes

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Accessing bacterial dark matter for improved enzyme discovery and engineering

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Restoring tumour selectivity of the bioreductive prodrug pr-104 by developing an analogue resistant to aerobic metabolism by human aldo-keto reductase 1c3

PR-104 is a phosphate ester pre-prodrug that is converted in vivo to its cognate alcohol, PR-104A, a latent alkylator which forms potent cytotoxins upon bioreduction. Hypoxia selectivity results from one-electron nitro reduction of PR-104A, in which cytochrome P450 oxidoreductase (POR) plays an important role. However, PR-104A also undergoes ‘off-target’ two-electron reduction by human aldo-keto reductase 1C3 (AKR1C3), resulting in activation in oxygenated tissues. AKR1C3 expression in human myeloid progenitor cells probably accounts for the dose-limiting myelotoxicity of PR-104 documented in clinical trials, resulting in human PR-104A plasma exposure levels 3.4- to 9.6-fold lower than can be achieved in murine models. Structure-based design to eliminate AKR1C3 activation thus represents a strategy for restoring the therapeutic window of this class of agent in humans. Here, we identified SN29176, a PR-104A analogue resistant to human AKR1C3 activation. SN29176 retains hypoxia selectivity in vitro with aerobic/hypoxic IC(50) ratios of 9 to 145, remains a substrate for POR and triggers γH2AX induction and cell cycle arrest in a comparable manner to PR-104A. SN35141, the soluble phosphate pre-prodrug of SN29176, exhibited superior hypoxic tumour log cell kill (>4.0) to PR-104 (2.5–3.7) in vivo at doses predicted to be achievable in humans. Orthologues of human AKR1C3 from mouse, rat and dog were incapable of reducing PR-104A, thus identifying an underlying cause for the discrepancy in PR-104 tolerance in pre-clinical models versus humans. In contrast, the macaque AKR1C3 gene orthologue was able to metabolise PR-104A, indicating that this species may be suitable for evaluating the toxicokinetics of PR-104 analogues for clinical development. We confirmed that SN29176 was not a substrate for AKR1C3 orthologues across all four pre-clinical species, demonstrating that this prodrug analogue class is suitable for further development. Based on these findings, a prodrug candidate was subsequently identified for clinical trials

Psychophysical Investigations into the Role of Low-Threshold C Fibres in Non-Painful Affective Processing and Pain Modulation

We recently showed that C low-threshold mechanoreceptors (CLTMRs) contribute to touch-evoked pain (allodynia) during experimental muscle pain. Conversely, in absence of ongoing pain, the activation of CLTMRs has been shown to correlate with a diffuse sensation of pleasant touch. In this study, we evaluated (1) the primary afferent fibre types contributing to positive (pleasant) and negative (unpleasant) affective touch and (2) the effects of tactile stimuli on tonic muscle pain by varying affective attributes and frequency parameters. Psychophysical observations were made in 10 healthy participants. Two types of test stimuli were applied: stroking stimulus using velvet or sandpaper at speeds of 0.1, 1.0 and 10.0 cm/s; focal vibrotactile stimulus at low (20 Hz) or high (200 Hz) frequency. These stimuli were applied in the normal condition (i.e. no experimental pain) and following the induction of muscle pain by infusing hypertonic saline (5%) into the tibialis anterior muscle. These observations were repeated following the conduction block of myelinated fibres by compression of sciatic nerve. In absence of muscle pain, all participants reliably linked velvet-stroking to pleasantness and sandpaper-stroking to unpleasantness (no pain). Likewise, low-frequency vibration was linked to pleasantness and high-frequency vibration to unpleasantness. During muscle pain, the application of previously pleasant stimuli resulted in overall pain relief, whereas the application of previously unpleasant stimuli resulted in overall pain intensification. These effects were significant, reproducible and persisted following the blockade of myelinated fibres. Taken together, these findings suggest the role of low-threshold C fibres in affective and pain processing. Furthermore, these observations suggest that temporal coding need not be limited to discriminative aspects of tactile processing, but may contribute to affective attributes, which in turn predispose individual responses towards excitatory or inhibitory modulation of pain

A922 Sequential measurement of 1 hour creatinine clearance (1-CRCL) in critically ill patients at risk of acute kidney injury (AKI)

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