Conflicting interests in the pathogen-host tug of war : fungal micronutrient scavenging versus mammalian nutritional immunity

Funding: The authors are supported by the European Research Council (STRIFE project funded on grant number ERC-2009-AdG-249793, http://erc.europa.eu). AJPB is also supported by the Wellcome Trust (grant numbers 080088, 097377, www.wellcome.ac.uk) and the UK Biotechnology and Biological Sciences Research Council (grant number BB/F00513X/1, www.bbsrc.ac.uk). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Platensimycin Activity against Mycobacterial β-Ketoacyl-ACP Synthases

Background - There is an urgent need for the discovery and development of new drugs against Mycobacterium tuberculosis, the causative agent of tuberculosis, especially due to the recent emergence of multi-drug and extensively-drug resistant strains. Herein, we have examined the susceptibility of mycobacteria to the natural product platensimycin. Methods and Findings - We have demonstrated that platensimycin has bacteriostatic activity against the fast growing Mycobacterium smegmatis (MIC = 14 µg/ml) and against Mycobacterium tuberculosis (MIC = 12 µg/ml). Growth in the presence of paltensimycin specifically inhibited the biosynthesis of mycolic acids suggesting that the antibiotic targeted the components of the mycolate biosynthesis complex. Given the inhibitory activity of platensimycin against β-ketoacyl-ACP synthases from Staphylococcus aureus, M. tuberculosis KasA, KasB or FabH were overexpressed in M. smegmatis to establish whether these mycobacterial KAS enzymes were targets of platensimycin. In M. smegmatis overexpression of kasA or kasB increased the MIC of the strains from 14 µg/ml, to 30 and 124 µg/ml respectively. However, overexpression of fabH on did not affect the MIC. Additionally, consistent with the overexpression data, in vitro assays using purified proteins demonstrated that platensimycin inhibited Mt-KasA and Mt-KasB, but not Mt-FabH. Significance - Our results have shown that platensimycin is active against mycobacterial KasA and KasB and is thus an exciting lead compound against M. tuberculosis and the development of new synthetic analogues

Engineering the indigoidine-synthesising enzyme BpsA for diverse applications in biotechnology

Blue pigment synthase A (BpsA) is a single module non-ribosomal peptide synthetase (NRPS) originally isolated from the bacterium Streptomyces lavendulae. It synthesises an easily detectible blue pigment called indigoidine from two molecules of L-glutamine in an ATP powered reaction. BpsA is readily purified and amenable to in vitro assays that have a variety of useful applications. By spectrophotometrically quantifying indigoidine levels it is possible to accurately measure the amount of L-glutamine in complex biological fluids including urine, blood plasma and cell culture media. This method has several advantages over existing methods for glutamine measurement, including that it directly reports on glutamine levels. Existing commercially available enzymatic kits first convert glutamine into glutamate and then measure the level of glutamate, which requires additional sample processing and introduces complexity if glutamate may also be present in the target sample. Additionally, we have shown that BpsA can also be used to measure ATP concentrations in a similar manner. We have further developed a BpsA based assay to detect inhibitors of 4’-phosphopantetheinyl transferases (PPTases). PPTases are enzymes that attach a phosphopantetheine arm to fatty acid synthases, NRPSs and polyketide synthases, thereby switching them from an inactive apo form to an active holo form. PPTases have been validated as promising drug targets in several pathogenic bacteria including P. aeruginosa and M. tuberculosis. In order to detect PPTase inhibition, we have shown that BpsA can be purified in its inactive apo form and mixed with the target PPTase as well as a candidate inhibitor in vitro. The level of PPTase inhibition can then be calculated by measuring the rate of indigoidine production. The assay has been optimised for high throughput screening and used to identify several compounds from chemical libraries that inhibit essential PPTases of P. aeruginosa and M. tuberculosis

Host-Imposed Copper Poisoning Impacts Fungal Micronutrient Acquisition during Systemic Candida albicans Infections

This work was supported by the European Research Council (http://erc.europa.eu/: STRIFE Advanced Grant ERC-2009-AdG-249793). A.J.P.B. was also supported by the UK Biotechnology and Biological Research Council (www.bbsrc.ac.uk: Research Grants BB/F00513X/1, BB/K017365/1), the UK Medical Research Council (www.mrc.ac.uk: Programme Grant MR/M026663/1; Centre Grant MR/ N006364/1), and the Wellcome Trust (www.wellcome.ac.uk: Strategic Award 097377)Peer reviewedPublisher PD

Thinking strategically about assessment

Drawing upon the literature on strategy formulation in organisations, this paper argues for a focus on strategy as process. It relates this to the need to think strategically about assessment, a need engendered by resource pressures, developments in learning and the demands of external stakeholders. It is argued that in practice assessment strategies are often formed at the level of practice, but that this produces contradiction and confusion at higher levels. Such tensions cannot be managed away, but they can be reflected on and mitigated. The paper suggests a framework for the construction of assessment strategies at different levels of an institution. However, the main conclusion is that the process of constructing such strategies should be an opportunity for learning and reflection, rather than one of compliance

New Clox Systems for rapid and efficient gene disruption in Candida albicans

Acknowledgements: We are grateful to Janet Quinn, Lila Kastora, Joanna Potrykus, Michelle Leach, and others for sharing their experiences with the Clox cassettes. We thank Julia Kohler for her kind gift of the NAT1-flipper plasmid pJK863, Claudia Jacob for her advice with In-fusion cloning, and our colleagues in the Aberdeen Fungal Group for numerous stimulating discussions. Data Availability: The authors confirm that all data underlying the findings are fully available without restriction. The sequences of all Clox cassettes are available in GenBank: URA3-Clox (loxP-URA3-MET3p-cre-loxP): GenBank accession number KC999858. NAT1-Clox (loxP-NAT1-MET3p-cre-loxP): GenBank accession number KC999859. LAL (loxP-ARG4-loxP): GenBank accession number DQ015897. LHL (loxP-HIS1-loxP): GenBank accession number DQ015898. LUL (loxP-URA3-loxP): GenBank accession number DQ015899. Funding: This work was supported by the Wellcome Trust (www.wellcome.ac.uk): S.S., F.C.O., N.A.R.G., A.J.P.B. (080088); N.A.R.G., A.J.P.B. (097377). The authors also received support from the European Research Council [http://erc.europa.eu/]: DSC. ERB, AJPB (STRIFE Advanced Grant; C-2009-AdG-249793). The European Commission also provided funding [http://ec.europa.eu/research/fp7]: I.B., A.J.P.B. (FINSysB MC-ITN; PITN-GA-2008-214004). Also the UK Biotechnology and Biological Research Council provided support [www.bbsrc.ac.uk]: N.A.R.G., A.J.P.B. (Research Grant; BB/F00513X/1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Deletion of a mycobacterial gene encoding a reductase leads to an altered cell wall containing β-oxo-mycolic acid analogues, and the accumulation of longchain ketones related to mycolic acids.

Mycolic acids are essential components of the mycobacterial cell wall. In this study we show that a gene encoding a reductase involved in the final step of mycolic acid biosynthesis can be deleted in Mycobacterium smegmatis without affecting cell viability. Deletion of MSMEG4722 (ortholog of Mycobacterium tuberculosis Rv2509) altered culture characteristics and antibiotic sensitivity. The ΔMSMEG4722 strain synthesized α-alkyl, β-oxo intermediates of mycolic acids which were found esterified to cell wall-arabinogalactan. While the precursors could not be isolated directly due to their inherent instability during base-treatment, their presence was established by prior reduction of the β-oxo group by sodium borohydride. Interestingly, the mutant also accumulated unsaturated ketones, similar to tuberculenone from M. tuberculosis, which were shunt products derived from spontaneous decarboxylation of α-alkyl, β-oxo fatty acid precursors of mycolic acids

Redox Regulation, Rather than Stress-Induced Phosphorylation, of a Hog1 Mitogen-Activated Protein Kinase Modulates Its Nitrosative-Stress-Specific Outputs

Data availability. The RNA sequencing dataset is available at EBI (www.ebi.ac.uk/arrayexpress/) under accession number E-MTAB-5990. Other data that support the findings of this study are available from the corresponding author upon reasonable request. ACKNOWLEDGMENTS We thank Debbie Smith for constructing the strains JC41 and JC310, Arnab Pradhan for help with DHE control experiments, and our colleagues in the Aberdeen Fungal Group and Newcastle Yeast Group for insightful discussions. We are also grateful to Mike Gustin for his advice. We are grateful to the Centre for Genome Enabled Biology and Medicine, Aberdeen Proteomics, the Iain Fraser Cytometry Centre, the Microscopy and Histology Facility, and the qPCR facility at the University of Aberdeen for their help, advice, and support. This work was funded by the UK Biotechnology and Biological Research Council (http://www.bbsrc.ac.uk) (grants BB/K017365/1 and BB/F00513X/1 to A.J.P.B. and grant BB/K016393/1 to J.Q.). This work was also supported by the European Research Council (http://erc.europa.eu/) (STRIFE advanced grant C-2009-AdG-249793 to A.J.P.B.), the UK Medical Research Council (http://www.mrc.ac.uk) (grant MR/M026663/1 to A.J.P.B. and grant MR/M000923/1 to P.S.S.), the Wellcome Trust (https://wellcome.ac.uk) (grant 097377 to A.J.P.B. and J.Q.), the MRC Centre for Medical Mycology and the University of Aberdeen (grant MR/M026663/1 to A.J.P.B.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Crystal structure of Mycobacterium tuberculosis FadB2 implicated in mycobacterial β-oxidation

The intracellular pathogen Mycobacterium tuberculosis is the causative agent of tuberculosis, which is a leading cause of mortality worldwide. The survival of M. tuberculosis in host macrophages through long-lasting periods of persistence depends, in part, on breaking down host cell lipids as a carbon source. The critical role of fatty-acid catabolism in this organism is underscored by the extensive redundancy of the genes implicated in β-oxidation (∼100 genes). In a previous study, the enzymology of the M. tuberculosisl-3-hydroxyacyl-CoA dehydrogenase FadB2 was characterized. Here, the crystal structure of this enzyme in a ligand-free form is reported at 2.1 Å resolution. FadB2 crystallized as a dimer with three unique dimer copies per asymmetric unit. The structure of the monomer reveals a dual Rossmann-fold motif in the N-terminal domain, while the helical C-terminal domain mediates dimer formation. Comparison with the CoA- and NAD + -bound human orthologue mitochondrial hydroxyacyl-CoA dehydrogenase shows extensive conservation of the residues that mediate substrate and cofactor binding. Superposition with the multi-catalytic homologue M. tuberculosis FadB, which forms a trifunctional complex with the thiolase FadA, indicates that FadB has developed structural features that prevent its self-association as a dimer. Conversely, FadB2 is unable to substitute for FadB in the tetrameric FadA–FadB complex as it lacks the N-terminal hydratase domain of FadB. Instead, FadB2 may functionally (or physically) associate with the enoyl-CoA hydratase EchA8 and the thiolases FadA2, FadA3, FadA4 or FadA6 as suggested by interrogation of the STRING protein-network database

New \u3ci\u3eClox\u3c/i\u3e Systems for Rapid and Efficient Gene Disruption in \u3ci\u3eCandida albicans\u3c/i\u3e

Precise genome modification is essential for the molecular dissection of Candida albicans, and is yielding invaluable information about the roles of specific gene functions in this major fungal pathogen of humans. C. albicans is naturally diploid, unable to undergo meiosis, and utilizes a non-canonical genetic code. Hence, specialized tools have had to be developed for gene disruption in C. albicans that permit the deletion of both target alleles, and in some cases, the recycling of the Candida-specific selectable markers. Previously, we developed a tool based on the Cre recombinase, which recycles markers in C. albicans with 90–100% efficiency via site-specific recombination between loxP sites. Ironically, the utility of this system was hampered by the extreme efficiency of Cre, which prevented the construction in Escherichia coli of stable disruption cassettes carrying a methionine-regulatable CaMET3p-cre gene flanked by loxP sites. Therefore, we have significantly enhanced this system by engineering new Clox cassettes that carry a synthetic, intron-containing cre gene. The Clox kit facilitates efficient transformation and marker recycling, thereby simplifying and accelerating the process of gene disruption in C. albicans. Indeed, homozygous mutants can be generated and their markers resolved within two weeks. The Clox kit facilitates strategies involving single marker recycling or multi-marker gene disruption. Furthermore, it includes the dominant NAT1 marker, as well as URA3, HIS1 and ARG4 cassettes, thereby permitting the manipulation of clinical isolates as well as genetically marked strains of C. albicans. The accelerated gene disruption strategies afforded by this new Clox system are likely to have a profound impact on the speed with which C. albicans pathobiology can be dissected