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  • Journal article
    Lund-Palau H, Turnbull AR, Bush A, Bardin E, Cameron L, Soren O, Wierre-Gore N, Alton EW, Bundy JG, Connett G, Faust SN, Filloux A, Freemont P, Jones A, Khoo V, Morales S, Murphy R, Pabary R, Simbo A, Schelenz S, Takats Z, Webb J, Williams HD, Davies JCet al., 2016,

    Pseudomonas aeruginosa infection in cystic fibrosis: pathophysiological mechanisms and therapeutic approaches

    , Expert Review of Respiratory Medicine, Vol: 10, Pages: 685-697, ISSN: 1747-6348

    Pseudomonas aeruginosa is a remarkably versatile environmental bacterium with an extraordinary capacity to infect the cystic fibrosis (CF) lung. Infection with P. aeruginosa occurs early, and although eradication can be achieved following early detection, chronic infection occurs in over 60% of adults with CF. Chronic infection is associated with accelerated disease progression and increased mortality. Extensive research has revealed complex mechanisms by which P. aeruginosa adapts to and persists within the CF airway. Yet knowledge gaps remain, and prevention and treatment strategies are limited by the lack of sensitive detection methods and by a narrow armoury of antibiotics. Further developments in this field are urgently needed in order to improve morbidity and mortality in people with CF. Here, we summarize current knowledge of pathophysiological mechanisms underlying P. aeruginosa infection in CF. Established treatments are discussed, and an overview is offered of novel detection methods and therapeutic strategies in development.

  • Journal article
    Chambonnier G, Roux L, Redelberger D, Fadel F, Filloux A, Sivaneson M, de Bentzmann S, Bordi Cet al., 2016,

    The Hybrid Histidine Kinase LadS Forms a Multicomponent Signal Transduction System with the GacS/GacA Two-Component System in Pseudomonas aeruginosa.

    , PLOS Genetics, Vol: 12, ISSN: 1553-7390

    In response to environmental changes, Pseudomonas aeruginosa is able to switch from a planktonic (free swimming) to a sessile (biofilm) lifestyle. The two-component system (TCS) GacS/GacA activates the production of two small non-coding RNAs, RsmY and RsmZ, but four histidine kinases (HKs), RetS, GacS, LadS and PA1611, are instrumental in this process. RetS hybrid HK blocks GacS unorthodox HK autophosphorylation through the formation of a heterodimer. PA1611 hybrid HK, which is structurally related to GacS, interacts with RetS in P. aeruginosa in a very similar manner to GacS. LadS hybrid HK phenotypically antagonizes the function of RetS by a mechanism that has never been investigated. The four sensors are found in most Pseudomonas species but their characteristics and mode of signaling may differ from one species to another. Here, we demonstrated in P. aeruginosa that LadS controls both rsmY and rsmZ gene expression and that this regulation occurs through the GacS/GacA TCS. We additionally evidenced that in contrast to RetS, LadS signals through GacS/GacA without forming heterodimers, either with GacS or with RetS. Instead, we demonstrated that LadS is involved in a genuine phosphorelay, which requires both transmitter and receiver LadS domains. LadS signaling ultimately requires the alternative histidine-phosphotransfer domain of GacS, which is here used as an Hpt relay by the hybrid kinase. LadS HK thus forms, with the GacS/GacA TCS, a multicomponent signal transduction system with an original phosphorelay cascade, i.e. H1LadS→D1LadS→H2GacS→D2GacA. This highlights an original strategy in which a unique output, i.e. the modulation of sRNA levels, is controlled by a complex multi-sensing network to fine-tune an adapted biofilm and virulence response.

  • Journal article
    Armstrong-James DPH, Shah A, Kannambath S, Herbst S, Rogers A, Soresi S, Carby M, Reed A, Mostowy S, Fisher MC, Shaunak S, Armstrong-James DPet al., 2016,

    Calcineurin Orchestrates Lateral Transfer of Aspergillus fumigatus during Macrophage Cell Death

    , American Journal of Respiratory and Critical Care Medicine, Vol: 194, Pages: 1127-1139, ISSN: 1535-4970

    Rationale: Pulmonary aspergillosis is a lethal mold infection in the immunocompromised host. Understanding initial control of infection and how this is altered in the immunocompromised host are key goals for comprehension of the pathogenesis of pulmonary aspergillosis.Objectives: To characterize the outcome of human macrophage infection with Aspergillus fumigatus and how this is altered in transplant recipients on calcineurin inhibitor immunosuppressants.Methods: We defined the outcome of human macrophage infection with A. fumigatus, as well as the impact of calcineurin inhibitors, through a combination of single-cell fluorescence imaging, transcriptomics, proteomics, and in vivo studies.Measurements and Main Results: Macrophage phagocytosis of A. fumigatus enabled control of 90% of fungal germination. However, fungal germination in the late phagosome led to macrophage necrosis. During programmed necroptosis, we observed frequent cell–cell transfer of A. fumigatus between macrophages, which assists subsequent control of germination in recipient macrophages. Lateral transfer occurred through actin-dependent exocytosis of the late endosome in a vasodilator-stimulated phosphoprotein envelope. Its relevance to the control of fungal germination was also shown by direct visualization in our zebrafish aspergillosis model in vivo. The calcineurin inhibitor FK506 (tacrolimus) reduced cell death and lateral transfer in vitro by 50%. This resulted in uncontrolled fungal germination in macrophages and also resulted in hyphal escape.Conclusions: These observations identify programmed, necrosis-dependent lateral transfer of A. fumigatus between macrophages as an important host strategy for controlling fungal germination. This process is critically dependent on calcineurin. Our studies provide fundamental insights into the pathogenesis of pulmonary aspergillosis in the immunocompromised host.

  • Journal article
    Khara JS, Priestman M, Uhia I, Hamilton MS, Krishnan N, Wang Y, Yang YY, Langford PR, Newton SM, Robertson BD, Ee PLRet al., 2016,

    Unnatural amino acid analogues of membrane-active helical peptides with anti-mycobacterial activity and improved stability

    , Journal of Antimicrobial Chemotherapy, Vol: 71, Pages: 2181-2191, ISSN: 1460-2091

    Objectives The emergence of MDR-TB, coupled with shrinking antibiotic pipelines, has increased demands for new antimicrobials with novel mechanisms of action. Antimicrobial peptides have increasingly been explored as promising alternatives to antibiotics, but their inherent poor in vivo stability remains an impediment to their clinical utility. We therefore systematically evaluated unnatural amino acid-modified peptides to design analogues with enhanced anti-mycobacterial activities.Methods Anti-mycobacterial activities were evaluated in vitro and intracellularly against drug-susceptible and MDR isolates of Mycobacterium tuberculosis using MIC, killing efficacy and intracellular growth inhibition studies. Toxicity profiles were assessed against mammalian cells to verify cell selectivity. Anti-mycobacterial mechanisms were investigated using microfluidic live-cell imaging with time-lapse fluorescence microscopy and confocal laser-scanning microscopy.Results Unnatural amino acid incorporation was well tolerated without an appreciable effect on toxicity profiles and secondary conformations of the synthetic peptides. The modified peptides also withstood proteolytic digestion by trypsin. The all D-amino acid peptide, i(llkk)2i (II-D), displayed superior activity against all six mycobacterial strains tested, with a 4-fold increase in selectivity index as compared with the unmodified L-amino acid peptide in broth. II-D effectively reduced the intracellular bacterial burden of both drug-susceptible and MDR clinical isolates of M. tuberculosis after 4 days of treatment. Live-cell imaging studies demonstrated that II-D permeabilizes the mycobacterial membrane, while confocal microscopy revealed that II-D not only permeates the cell membrane, but also accumulates within the cytoplasm.Conclusions Unnatural amino acid modifications not only decreased the susceptibility of peptides to proteases, but also enhanced mycobacterial selectivity.

  • Journal article
    Valentini M, Filloux A, 2016,

    Biofilms and cyclic di-GMP (c-di-GMP) signaling: lessons from Pseudomonas aeruginosa and other bacteria

    , Journal of Biological Chemistry, Vol: 291, Pages: 12547-12555, ISSN: 1083-351X

    The cyclic-di-GMP (c-di-GMP) second messenger represents a signaling system that regulates many bacterial behaviors and is of key importance for driving the lifestyle switch between motile loner cells and biofilm formers. This review provides an up-to-date compendium of c-di-GMP pathways connected to biofilm formation, biofilm-associated motilities and other functionalities in the ubiquitous and opportunistic human pathogen Pseudomonas aeruginosa. This bacterium is frequently adopted as model organism to study bacterial biofilm formation. Importantly, its versatility and adaptation capabilities are linked with a broad range of complex regulatory networks, including a large set of genes involved in c-di-GMP biosynthesis, degradation and transmission.

  • Journal article
    Taylor JD, Hawthorne WJ, Lo J, Dear A, Jain N, Meisl G, Andreasen M, Fletcher C, Koch M, Darvill N, Scull N, Escalera-Maurer A, Sefer L, Wenman R, Lambert S, Jean J, Xu Y, Turner B, Kazarian SG, Chapman MR, Bubeck D, de Simone A, Knowles TPJ, Matthews SJet al., 2016,

    Electrostatically-guided inhibition of Curli amyloid nucleation by the CsgC-like family of chaperones

    , Scientific Reports, Vol: 6, ISSN: 2045-2322

    Polypeptide aggregation into amyloid is linked with several debilitating human diseases.Despite the inherent risk of aggregation-induced cytotoxicity, bacteria control the export ofamyloid-prone subunits and assemble adhesive amyloid fibres during biofilm formation. AnEscherichia protein, CsgC potently inhibits amyloid formation of curli amyloid proteins.Here we unlock its mechanism of action, and show that CsgC strongly inhibits primarynucleation via electrostatically-guided molecular encounters, which expands theconformational distribution of disordered curli subunits. This delays the formation of higherorder intermediates and maintains amyloidogenic subunits in a secretion-competent form.New structural insight also reveal that CsgC is part of diverse family of bacterial amyloidinhibitors. Curli assembly is therefore not only arrested in the periplasm, but the preservationof conformational flexibility also enables efficient secretion to the cellsurface. Understanding how bacteria safely handle amyloidogenic polypeptides contributetowards efforts to control aggregation in disease-causing amyloids and amyloid-based biotechnological applications.

  • Journal article
    Miliara X, Matthews S, 2016,

    Structural comparison of yeast and human intra-mitochondrial lipid transport systems

    , Biochemical Society Transactions, Vol: 44, Pages: 479-485, ISSN: 1470-8752

    Mitochondria depend on a tightly regulated supply of phospholipids. The protein of relevant evolutionary and lymphoid interest (PRELI)/Ups1 family together with its mitochondrial chaperones [TP53-regulated inhibitor of apoptosis 1 (TRIAP1)/Mdm35] represents a unique heterodimeric lipid-transfer system that is evolutionary conserved from yeast to man. Recent X-ray crystal structures of the human and yeast systems are compared and discuss here and shed new insight into the mechanism of the PRELI/Ups1 system.

  • Journal article
    O'Neill A, Thurston T, Holden D, 2016,

    Cytosolic Replication of Group A Streptococcus in Human Macrophages

    , mBio, Vol: 7, ISSN: 2161-2129

    As key components of innate immune defense, macrophages are essential in controlling bacterial pathogens, includinggroup A Streptococcus (GAS). Despite this, only a limited number of studies have analyzed the recovery of GAS from withinhuman neutrophils and macrophages. Here, we determined the intracellular fate of GAS in human macrophages by using severalquantitative approaches. In both U937 and primary human macrophages, the appearance over time of long GAS chains revealedthat despite GAS-mediated cytotoxicity, replication occurred in viable, propidium iodide-negative macrophages. Whereas themajor virulence factor M1 did not contribute to bacterial growth, a GAS mutant strain deficient in streptolysin O (SLO) was impairedfor intracellular replication. SLO promoted bacterial escape from the GAS-containing vacuole (GCV) into the macrophagecytosol. Up to half of the cytosolic GAS colocalized with ubiquitin and p62, suggesting that the bacteria were targeted bythe autophagy machinery. Despite this, live imaging of U937 macrophages revealed proficient replication of GAS after GCV rupture,indicating that escape from the GCV is important for growth of GAS in macrophages. Our results reveal that GAS can replicatewithin viable human macrophages, with SLO promoting GCV escape and cytosolic growth, despite the recruitment of autophagyreceptors to bacteria.

  • Journal article
    Lobato-Márquez D, Mostowy S, 2016,

    Septins recognize micron-scale membrane curvature

    , Journal of Cell Biology, Vol: 213, Pages: 5-6, ISSN: 1540-8140

    How cells recognize membrane curvature is not fully understood. In this issue, Bridges et al. (2016.J. Cell Biol.http://dx.doi.org/10.1083/jcb.201512029) discover that septins, a component of the cytoskeleton, recognize membrane curvature at the micron scale, a common morphological hallmark of eukaryotic cellular processes.

  • Journal article
    Helaine S, holden DW, sampson SL, Mouton JMet al., 2016,

    Elucidating population-wide mycobacterial replication dynamics at the single-cell level

    , Microbiology, Vol: 162, Pages: 966-978, ISSN: 1350-0872

    Mycobacterium tuberculosis infections result in a spectrum of clinical outcomes, and frequently the infection persists in a latent, clinically asymptomatic state. The within-host bacterial population is likely to be heterogeneous, and it is thought that persistent mycobacteria arise from a small population of viable, but non-replicating (VBNR) cells. These are likely to be antibiotic tolerant and necessitate prolonged treatment. Little is known about these persistent mycobacteria, since they are very difficult to isolate. To address this, we have successfully developed a replication reporter system for use in M. tuberculosis. This approach, termed fluorescence dilution, exploits 2 fluorescent reporters; a constitutive reporter allows the tracking of bacteria, while an inducible reporter enables the measurement of bacterial replication. The application of fluorescent single-cell analysis to characterise intracellular M. tuberculosis identified a distinct subpopulation of non-growing mycobacteria in murine macrophages. The presence of VBNR and actively replicating mycobacteria was observed within the same macrophage after 48 hours of infection. Furthermore, our results suggest that macrophage uptake resulted in enrichment of non- or slowly replicating bacteria (as revealed by DCS treatment); this population is likely to be highly enriched for persisters, based on its drug tolerant phenotype. These results demonstrate the successful application of the novel dual fluorescent reporter system both in vitro and in macrophage infection models to provide a window into mycobacterial population heterogeneity.

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