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  • Journal article
    Schrader SM, Vaubourgeix J, Nathan C, 2020,

    Biology of antimicrobial resistance and approaches to combat it

    , SCIENCE TRANSLATIONAL MEDICINE, Vol: 12, ISSN: 1946-6234
  • Journal article
    Sarah Wettstadt S, Lai E-M, Filloux AAM, 2020,

    Solving the puzzle: connecting a heterologous Agrobacterium tumefaciens T6SS effector to a Pseudomonas aeruginosa spike complex

    , Frontiers in Cellular and Infection Microbiology, Vol: 10, ISSN: 2235-2988

    The type VI secretion system (T6SS) is a contractile injection apparatus that translocates a spike loaded with various effectors directly into eukaryotic and prokaryotic target cells. Such T6SS spike consists of a needle-shaped trimer of VgrG proteins topped by a conical and sharp PAAR protein that facilitates puncturing of the target membrane. T6SS-delivered effector proteins can be either fused to one of the two spike proteins or interact with either in a highly specific manner. In Agrobacterium tumefaciens the T6SS effector Tde1 is targeted to its cognate VgrG1 protein. Here, we attempted to use a VgrG shuttle to deliver a heterologous T6SS effector by directing Tde1 onto a T6SS spike in Pseudomonas aeruginosa. For this, we designed chimeras between VgrG1 from A. tumefaciens and VgrG1a from P. aeruginosa and showed that modification of the spike protein hampered T6SS functionality in the presence of the Tde1 effector complex. We provide evidence suggesting that Tde1 specifically binds to the VgrG spike in the heterologous environment and propose that there are additional requirements to allow proper effector delivery and translocation. Our work sheds light on complex aspects of the molecular mechanisms of T6SS delivery and highlights some limitations on how effectors can be translocated using this nanomachine.
  • Journal article
    Stelzl LS, Mavridou DA, Saridakis E, Gonzalez D, Baldwin AJ, Ferguson SJ, Sansom MS, Redfield Cet al., 2020,

    Local frustration determines loop opening during the catalytic cycle of an oxidoreductase.

    , eLife, Vol: 9, Pages: 1-27, ISSN: 2050-084X

    Local structural frustration, the existence of mutually exclusive competing interactions, may explain why some proteins are dynamic while others are rigid. Frustration is thought to underpin biomolecular recognition and the flexibility of protein-binding sites. Here, we show how a small chemical modification, the oxidation of two cysteine thiols to a disulfide bond, during the catalytic cycle of the N-terminal domain of the key bacterial oxidoreductase DsbD (nDsbD), introduces frustration ultimately influencing protein function. In oxidized nDsbD, local frustration disrupts the packing of the protective cap-loop region against the active site allowing loop opening. By contrast, in reduced nDsbD the cap loop is rigid, always protecting the active-site thiols from the oxidizing environment of the periplasm. Our results point toward an intricate coupling between the dynamics of the active-site cysteines and of the cap loop which modulates the association reactions of nDsbD with its partners resulting in optimized protein function.
  • Journal article
    Letertre MPM, Munjoma NC, Wolfer K, Pechlivanis A, McDonald J, Hardwick RN, Cherrington NJ, Coen M, Nicholson J, Hoyles L, Swann J, Wilson Iet al., 2020,

    A two-way interaction between methotrexate and the gut microbiota of male Sprague Dawley rats

    , Journal of Proteome Research, Vol: 19, Pages: 3326-3339, ISSN: 1535-3893

    Methotrexate (MTX) is a chemotherapeutic agent that cancause a range of toxic side effects including gastrointestinal damage,hepatotoxicity, myelosuppression, and nephrotoxicity and has potentiallycomplex interactions with the gut microbiome. Following untargeted UPLCqtof-MS analysis of urine and fecal samples from male Sprague−Dawley ratsadministered at either 0, 10, 40, or 100 mg/kg of MTX, dose-dependentchanges in the endogenous metabolite profiles were detected. Semiquantitativetargeted UPLC-MS detected MTX excreted in urine as well as MTX and twometabolites, 2,4-diamino-N-10-methylpteroic acid (DAMPA) and 7-hydroxyMTX, in the feces. DAMPA is produced by the bacterial enzymecarboxypeptidase glutamate 2 (CPDG2) in the gut. Microbiota profiling(16S rRNA gene amplicon sequencing) of fecal samples showed an increase inthe relative abundance of Firmicutes over the Bacteroidetes at low doses ofMTX but the reverse at high doses. Firmicutes relative abundance was positively correlated with DAMPA excretion in feces at 48 h,which were both lower at 100 mg/kg compared to that seen at 40 mg/kg. Overall, chronic exposure to MTX appears to inducecommunity and functionality changes in the intestinal microbiota, inducing downstream perturbations in CPDG2 activity, and thusmay delay MTX detoxication to DAMPA. This reduction in metabolic clearance might be associated with increased gastrointestinaltoxicity.
  • Journal article
    Asai M, Li Y, Spiropoulos J, Cooley W, Everest D, Robertson BD, Langford PR, Newton SMet al., 2020,

    A novel biosafety level 2 compliant tuberculosis infection model using a ΔleuDΔpanCD double auxotroph of Mycobacterium tuberculosis H37Rv and Galleria mellonella

    , Virulence, Vol: 11, Pages: 811-824, ISSN: 2150-5594

    Mammalian infection models have contributed significantly to our understanding of the host-mycobacterial interaction, revealing potential mechanisms and targets for novel antimycobacterial therapeutics. However, the use of conventional mammalian models such as mice, are typically expensive, high maintenance, require specialised animal housing, and are ethically regulated. Furthermore, research using Mycobacterium tuberculosis (MTB), is inherently difficult as work needs to be carried out at biosafety level 3 (BSL3). The insect larvae of Galleria mellonella (greater wax moth), have become increasingly popular as an infection model, and we previously demonstrated its potential as a mycobacterial infection model using Mycobacterium bovis BCG. Here we present a novel BSL2 complaint MTB infection model using G. mellonella in combination with a bioluminescent ΔleuDΔpanCD double auxotrophic mutant of MTB H37Rv (SAMTB lux) which offers safety and practical advantages over working with wild type MTB. Our results show a SAMTB lux dose dependent survival of G. mellonella larvae and demonstrate proliferation and persistence of SAMTB lux bioluminescence over a 1 week infection time course. Histopathological analysis of G. mellonella, highlight the formation of early granuloma-like structures which matured over time. We additionally demonstrate the drug efficacy of first (isoniazid, rifampicin, and ethambutol) and second line (moxifloxacin) antimycobacterial drugs. Our findings demonstrate the broad potential of this insect model to study MTB infection under BSL2 conditions. We anticipate that the successful adaptation and implementation of this model will remove the inherent limitations of MTB research at BSL3 and increase tuberculosis research output.
  • Journal article
    Kiga K, Tan X-E, Ibarra-Chávez R, Watanabe S, Aiba Y, Sato'o Y, Li F-Y, Sasahara T, Cui B, Kawauchi M, Boonsiri T, Thitiananpakorn K, Taki Y, Azam AH, Suzuki M, Penadés JR, Cui Let al., 2020,

    Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

    , Nature Communications, Vol: 11, ISSN: 2041-1723

    The emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. Here we report the development of a series of CRISPR-Cas13a-based antibacterial nucleocapsids, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus by recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs are generated by packaging programmed CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibit strong bacterial killing activities upon recognizing target genes regardless of their location. Moreover, we also demonstrate that the CapsidCas13a(s) can be applied to detect bacterial genes through gene-specific depletion of bacteria without employing nucleic acid manipulation and optical visualization devices. Our data underscore the potential of CapsidCas13a(s) as both therapeutic agents against antimicrobial-resistant bacteria and nonchemical agents for detection of bacterial genes.
  • Journal article
    Larrouy-Maumus G, Dortet L, Filloux A, bonnin, le hello, bonnet, kostrzewaet al., 2020,

    Detection of colistin resistance in Salmonella enterica using MALDIxin test on the routine MALDI Biotyper Sirius mass spectrometer

    , Frontiers in Microbiology, Vol: 11, Pages: 1-6, ISSN: 1664-302X

    Resistance to polymyxins in most Gram-negative bacteria arises from chemical modifications to the lipid A portion of their lipopolysaccharide (LPS) mediated by chromosomally-encoded mutations or the recently discovered plasmid-encoded mcr genes that have further complicated the landscape of colistin resistance. Currently, minimal inhibitory concentration (MIC) determination by broth microdilution, the gold standard for the detection of polymyxin resistance, is time consuming (24 hours) and challenging to perform in clinical and veterinatryveterinary laboratories. Here we present the use of the MALDIxin to detect colistin resistant Salmonella enterica using the MALDxin test on the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system.
  • Journal article
    Zeden MS, Kviatkovski I, Schuster CF, Thomas VC, Fey PD, Grundling Aet al., 2020,

    Identification of the main glutamine and glutamate transporters in Staphylococcus aureus and their impact on c-di-AMP production

    , Molecular Microbiology, Vol: 113, Pages: 1085-1100, ISSN: 0950-382X

    A Staphylococcus aureus strain deleted for the c‐di‐AMP cyclase gene dacA is unable to survive in rich medium unless it acquires compensatory mutations. Previously identified mutations were in opuD, encoding the main glycine‐betaine transporter, and alsT, encoding a predicted amino acid transporter. Here, we show that inactivation of OpuD restores the cell size of a dacA mutant to near wild‐type (WT) size, while inactivation of AlsT does not. AlsT was identified as an efficient glutamine transporter, indicating that preventing glutamine uptake in rich medium rescues the growth of the S. aureus dacA mutant. In addition, GltS was identified as a glutamate transporter. By performing growth curves with WT, alsT and gltS mutant strains in defined medium supplemented with ammonium, glutamine or glutamate, we revealed that ammonium and glutamine, but not glutamate promote the growth of S. aureus. This suggests that besides ammonium also glutamine can serve as a nitrogen source under these conditions. Ammonium and uptake of glutamine via AlsT and hence likely a higher intracellular glutamine concentration inhibited c‐di‐AMP production, while glutamate uptake had no effect. These findings provide, besides the previously reported link between potassium and osmolyte uptake, a connection between nitrogen metabolism and c‐di‐AMP signalling in S. aureus.
  • Journal article
    Mathioudakis AG, Janssens W, Sivapalan P, Singanayagam A, Dransfield MT, Jensen J-US, Vestbo Jet al., 2020,

    Acute exacerbations of chronic obstructive pulmonary disease: in search of diagnostic biomarkers and treatable traits

    , THORAX, Vol: 75, Pages: 520-527, ISSN: 0040-6376
  • Journal article
    Kumar K, Hinks TSC, Singanayagam A, 2020,

    Treatment of COVID-19-exacerbated asthma: should systemic corticosteroids be used?

    , AMERICAN JOURNAL OF PHYSIOLOGY-LUNG CELLULAR AND MOLECULAR PHYSIOLOGY, Vol: 318, Pages: L1244-L1247, ISSN: 1040-0605

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