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Journal articleHerbst S, Shah A, Moya MM, et al., 2015,
Phagocytosis-dependent activation of a TLR9-BTK-calcineurin-NFAT pathway co-ordinates innate immunity to Aspergillus fumigatus
, EMBO Molecular Medicine, Vol: 7, Pages: 240-258, ISSN: 1757-4676Transplant recipients on calcineurin inhibitors are at high risk of invasive fungal infection. Understanding how calcineurin inhibitors impair fungal immunity is a key priority for defining risk of infection. Here, we show that the calcineurin inhibitor tacrolimus impairs clearance of the major mould pathogen Aspergillus fumigatus from the airway, by inhibiting macrophage inflammatory responses. This leads to defective early neutrophil recruitment and fungal clearance. We confirm these findings in zebrafish, showing an evolutionarily conserved role for calcineurin signalling in neutrophil recruitment during inflammation. We find that calcineurin–NFAT activation is phagocytosis dependent and collaborates with NF‐κB for TNF‐α production. For yeast zymosan particles, activation of macrophage calcineurin–NFAT occurs via the phagocytic Dectin‐1–spleen tyrosine kinase pathway, but for A. fumigatus, activation occurs via a phagosomal TLR9‐dependent and Bruton's tyrosine kinase‐dependent signalling pathway that is independent of MyD88. We confirm the collaboration between NFAT and NF‐κB for TNF‐α production in primary alveolar macrophages. These observations identify inhibition of a newly discovered macrophage TLR9–BTK–calcineurin–NFAT signalling pathway as a key immune defect that leads to organ transplant‐related invasive aspergillosis.
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Journal articleCorrigan RM, Bowman L, Willis AR, et al., 2015,
Cross-talk between Two Nucleotide-signaling Pathways in Staphylococcus aureus
, Journal of Biological Chemistry, Vol: 290, Pages: 5826-5839, ISSN: 0021-9258Nucleotide-signaling pathways are found in all kingdoms oflife and are utilized to coordinate a rapid response to externalstimuli. The stringent response alarmones guanosine tetra(ppGpp)and pentaphosphate (pppGpp) control a globalresponse allowing cells to adapt to starvation conditions such asamino acid depletion. One more recently discovered signalingnucleotide is the secondary messenger cyclic diadenosinemonophosphate (c-di-AMP). Here, we demonstrate that thissignaling nucleotide is essential for the growth of Staphylococcusaureus, and its increased production during late growthphases indicates that c-di-AMP controls processes that areimportant for the survival of cells in stationary phase. By examiningthe transcriptional profile of cells with high levels of c-diAMP,we reveal a significant overlap with a stringent responsetranscription signature. Examination of the intracellular nucleotidelevels under stress conditions provides further evidencethat high levels of c-di-AMP lead to an activation of the stringentresponse through a RelA/SpoT homologue (RSH) enzymedependentincrease in the (p)ppGpp levels. This activation isshown to be indirect as c-di-AMP does not interact directly withthe RSH protein. Our data extend this interconnection furtherby showing that the S. aureus c-di-AMP phosphodiesteraseenzyme GdpP is inhibited in a dose-dependent manner byppGpp, which itself is not a substrate for this enzyme. Altogether,these findings add a new layer of complexity to ourunderstanding of nucleotide signaling in bacteria as they highlightintricate interconnections between different nucleotidesignalingnetworks.
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Journal articleGarnett JA, Muhl D, Douse CH, et al., 2015,
Structure-function analysis reveals that the Pseudomonas aeruginosa Tps4 two-partner secretion system is involved in CupB5 translocation
, Protein Science, Vol: 24, Pages: 670-687, ISSN: 1469-896XPseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous withcystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a modelorganism to study biofilms: a bacterial population embedded in an extracellular matrix that provideprotection from environmental pressures and lead to persistence. A number of Chaperone-UsherPathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili onthe bacterial surface. One of these, encoded by the cupB operon, is unique as it contains anonchaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised ofa C-terminal integral membrane b-barrel pore with tandem N-terminal POTRA (POlypeptide TRansportAssociated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). UsingNMR we show that TpsB4 (LepB) interacts with CupB5 and its predicted cognate partner TpsA4(LepA), an extracellular protease. Moreover, using cellular studies we confirm that TpsB4 cantranslocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observationthat suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstratethat tps4/cupB operons are coregulated by the RocS1 sensor suggesting P. aeruginosa hasdeveloped synergy between these systems. Furthermore, we have determined the solutionstructureof the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mappingand in vivo mutational analysis we have calculated models for the entire TpsB4 periplasmic region in complex with both TpsA4 and CupB5 secretion motifs. The data highlight specific residuesfor TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for eachPOTRA domain.
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Journal articleDembek M, Barquist L, Boinett CJ, et al., 2015,
High-Throughput Analysis of Gene Essentiality and Sporulation in Clostridium difficile
, mBio, Vol: 6, ISSN: 2161-2129Clostridium difficile is the most common cause of antibiotic-associated intestinal infections and a significant cause of morbidity and mortality. Infection with C. difficile requires disruption of the intestinal microbiota, most commonly by antibiotic usage. Therapeutic intervention largely relies on a small number of broad-spectrum antibiotics, which further exacerbate intestinal dysbiosis and leave the patient acutely sensitive to reinfection. Development of novel targeted therapeutic interventions will require a detailed knowledge of essential cellular processes, which represent attractive targets, and species-specific processes, such as bacterial sporulation. Our knowledge of the genetic basis of C. difficile infection has been hampered by a lack of genetic tools, although recent developments have made some headway in addressing this limitation. Here we describe the development of a method for rapidly generating large numbers of transposon mutants in clinically important strains of C. difficile. We validated our transposon mutagenesis approach in a model strain of C. difficile and then generated a comprehensive transposon library in the highly virulent epidemic strain R20291 (027/BI/NAP1) containing more than 70,000 unique mutants. Using transposon-directed insertion site sequencing (TraDIS), we have identified a core set of 404 essential genes, required for growth in vitro. We then applied this technique to the process of sporulation, an absolute requirement for C. difficile transmission and pathogenesis, identifying 798 genes that are likely to impact spore production. The data generated in this study will form a valuable resource for the community and inform future research on this important human pathogen.
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Journal articlePainter KL, Strange E, Parkhill J, et al., 2015,
Staphylococcus aureus adapts to oxidative stress by producing H2O2-resistant small colony variants via the SOS response
, Infection and Immunity, ISSN: 1098-5522 -
Journal articleEvans ML, Chorell E, Taylor JD, et al., 2015,
The Bacterial Curli System Possesses a Potent and Selective Inhibitor of Amyloid Formation
, MOLECULAR CELL, Vol: 57, Pages: 445-455, ISSN: 1097-2765- Author Web Link
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- Citations: 138
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Journal articleTorres M, Garcia-Garcia L, Cruz-Hervert P, et al., 2015,
Effect of isoniazid on antigen-specific interferon-γ secretion in latent tuberculosis
, EUROPEAN RESPIRATORY JOURNAL, Vol: 45, Pages: 473-482, ISSN: 0903-1936- Author Web Link
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- Citations: 10
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Journal articleEldridge MJG, Shenoy AR, 2015,
Antimicrobial inflammasomes: unified signalling against diverse bacterial pathogens
, CURRENT OPINION IN MICROBIOLOGY, Vol: 23, Pages: 32-41, ISSN: 1369-5274- Author Web Link
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- Citations: 30
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Journal articleHolden DW, Philpott DJ, 2015,
Editorial overview: Host-microbe interactions: bacteria
, CURRENT OPINION IN MICROBIOLOGY, Vol: 23, Pages: V-VIII, ISSN: 1369-5274 -
Journal articleCampeotto I, Zhang Y, Mladenov MG, et al., 2015,
Complex Structure and Biochemical Characterization of the Staphylococcus aureus Cyclic Diadenylate Monophosphate (c-di-AMP)-binding Protein PstA, the Founding Member of a New Signal Transduction Protein Family
, Journal of Biological Chemistry, Vol: 290, Pages: 2888-2901, ISSN: 1083-351XSignaling nucleotides are integral parts of signal transductionsystems allowing bacteria to cope with and rapidly respond tochanges in the environment. The Staphylococcus aureus PII-likesignal transduction protein PstA was recently identified as acyclic diadenylate monophosphate (c-di-AMP)-binding protein.Here, we present the crystal structures of the apo- and c-diAMP-boundPstA protein, which is trimeric in solution as wellas in the crystals. The structures combined with detailed bioinformaticsanalysis revealed that the protein belongs to a newfamily of proteins with a similar core fold but with distinct featuresto classical PII proteins, which usually function in nitrogenmetabolism pathways in bacteria. The complex structurerevealed three identical c-di-AMP-binding sites per trimer witheach binding site at a monomer-monomer interface. Althoughdistinctly different from other cyclic-di-nucleotide-bindingsites, as the half-binding sites are not symmetrical, the complexstructure also highlighted common features for c-di-AMPbindingsites. A comparison between the apo and complexstructures revealed a series of conformational changes thatresult in the ordering of two anti-parallel !-strands that protrudefrom each monomer and allowed us to propose a mechanismon how the PstA protein functions as a signaling transductionprotein.
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