Adrian Robert Walmsley

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Produções bibliográficas

• JANGANAN, THAMARAI K. ; BAVRO, VASSILIY N. ; ZHANG, LI ; BORGES-WALMSLEY, MARIA INÊS ; WALMSLEY, ADRIAN R. . Tripartite efflux pumps: energy is required for dissociation, but not assembly or opening of the outer membrane channel of the pump. Molecular Microbiology (Print) , v. 88, p. 590-602, 2013.

• Janganan, T. K. ; Bavro, V. N. ; Zhang, L. ; Matak-Vinkovic, D. ; Barrera, N. P. ; Venien-Bryan, C. ; Robinson, C. V. ; Borges-Walmsley, M. I. ; Walmsley, A. R. ; WALMSLEY, A. . Evidence for the Assembly of a Bacterial Tripartite Multidrug Pump with a Stoichiometry of 3:6:3. The Journal of Biological Chemistry (Print) , v. 286, p. 26900-26912, 2011.

• Janganan, T. K. ; Zhang, L. ; Bavro, V. N. ; Matak-Vinkovic, D. ; Barrera, N. P. ; Burton, M. F. ; Steel, P. G. ; Robinson, C. V. ; Borges-Walmsley, M. I. ; Walmsley, A. R. ; WALMSLEY, A. . Opening of the Outer Membrane Protein Channel in Tripartite Efflux Pumps Is Induced by Interaction with the Membrane Fusion Partner. The Journal of Biological Chemistry (Print) , v. 286, p. 5484-5493, 2011.

• Lin HT ; Bavro, V. N. ; Barrera, N. P. ; Frankish HM ; Velamakanni S ; van Veen HW ; Robinson CV ; Borges-Walmsley, M. I. ; WALMSLEY, A. . MacB ABC transporter is a dimer whose ATPase activity and macrolide-binding capacity are regulated by the membrane fusion protein MacA.. The Journal of Biological Chemistry (Print) , v. 284, p. 1145-1154, 2009.

• Chen D ; Janganan, T. K. ; Chen G ; Marques ER ; Kress MR ; Goldman GH ; WALMSLEY, A. ; Borges-Walmsley, M. I. . The cAMP pathway is important for controlling the morphological switch to the pathogenic yeast form of Paracoccidioides brasiliensis.. Molecular Microbiology (Print) , v. 65, p. 761-769, 2007.

• Lin, Y.-F. ; Walmsley, A. R. ; Rosen, B. P. ; WALMSLEY, A. . An arsenic metallochaperone for an arsenic detoxification pump. Proceedings of the National Academy of Sciences of the United States of America , v. 103, p. 15617-15622, 2006.

• Borges-Walmsley, M. I. ; DU, D. ; McKeegan KS ; Sharples GJ ; WALMSLEY, A. . VceR regulates the vceCAB drug efflux pump operon of Vibrio cholerae by alternating between mutually exclusive conformations that bind either drugs or promoter DNA.. Journal of Molecular Biology , v. 349, p. 387-400, 2005.

• Federici L, ; DU, D. ; Walas F ; Matsumura H ; Fernandez-Recio J ; McKeegan KS ; Borges-Walmsley, M. I. ; Luisi BF ; WALMSLEY, A. . The crystal structure of the outer membrane protein VceC from the bacterial pathogen Vibrio cholerae at 1.8 A resolution.. The Journal of Biological Chemistry (Print) , v. 280, p. 15307-15314, 2005.

• Borges-Walmsley, M. I. ; WALMSLEY, A. . Identification of Oligomerization and Drug-binding Domains of the Membrane Fusion Protein EmrA. The Journal of Biological Chemistry (Print) , v. 278, p. 12903-12912, 2002.

• li s ; Rosen, B. P. ; Borges-Walmsley, M. I. ; WALMSLEY, A. . Evidence for cooperativity between the four binding sites of dimeric ArsD, an As(III)-responsive transcriptional regulator.. The Journal of Biological Chemistry (Print) , v. 277, p. 25992-25996, 2002.

• WALMSLEY, A. ; Zhou T ; Borges-Walmsley, M. I. ; Rosen, B. P. . A kinetic model for the action of a resistance efflux pump.. The Journal of Biological Chemistry (Print) , v. 276, p. 6378-6391, 2001.

Projetos de pesquisa

• 2014 - Atual

  Engineering Zymomonas mobilis for enhanced bioethanol production and the integrated bioprocessing of lignocellulose., Descrição: The major objective of our program is to elucidate the AI-2 signalling pathway that activates ethanol production in Z. mobilis, and to identify additional genes that are regulated by this pathway, through a combined mutagenesis, functional, transcriptomic and proteomic approach. It is important to stress that Z. mobilis does not possess the genes generally associated with sensing AI-2 in other organisms; and, consequently, most likely utilizes a new and novel quorum-sensing system that awaits elucidation. These studies will provide information of fundamental importance in optimizing strains for ethanol production and tolerance. Furthermore, by exploiting our finding that AI-2 not only activates enhanced bioethanol production but also protein extrusion, our studies could be a milestone towards the holy grail of producing a microbial strain in which the processes of cellulose degradation and sugar fermentation are integrated. For example; Z. mobilis could be used to express cellulases that have been tagged at their N-terminus with the signal sequence for protein export; and by placing the genes for these cellulases under the control of an AI-2 responsive promoter, the expression and secretion of these enzymes could be co-ordinated with the fermentation process. Considering that the market for cellulosic ethanol has been estimated at US$ 75 billion per year, our research has considerable potential for commercial exploitation. Considering the potential exploitation of our discovery, we have been granted a patent for this technology in the US (No. 12/446,139), Europe (EP2092070) and a patent is pending in Brazil.. , Situação: Em andamento; Natureza: Pesquisa. , Alunos envolvidos: Doutorado: (2) . , Integrantes: Adrian Robert Walmsley - Coordenador / Lidia Pepe de Moares - Integrante / Marciano Regis Rubini - Integrante / Vanessa Rodrigues Viera - Integrante / Tiago Benoliel - Integrante., Financiador(es): Conselho Nacional de Desenvolvimento Científico e Tecnológico - Cooperação / Durham University - Auxílio financeiro.

• 2012 - Atual

  TARGIT - Transcriptional Activators of Resistance Genes as Inhibitor Targets, Descrição: Many pathogenic bacteria have a pair of genes that encode a regulator and an enzyme, a beta-lactamase, which destroys beta-lactam drugs, such as penicillin. This regulator senses changes in the cell wall, the target of the beta-lactam drugs. Since the cell wall is continuously being remodeled, the inhibition of its synthesis leads to a build-up of degradation products that bind to the regulator protein, which then activates the expression of the beta-lactamase. Regulatory proteins also control the expression of the multi-drug resistance pumps but these regulators act as cytosolic drug-sensors that frequently bind the same drugs as the pumps. In the pathogen Vibrio cholerae we have identified a novel cluster of genes in which a regulatory protein controls the expression of both a beta-lactamase and the components of a drug pump. In this novel arrangement the beta-lactamase is a type, a metallo-beta-lactamase, which can destroy the newer carbapenems, which are among the few useful antibiotics against multidrug resistant gram-negative bacteria and are often referred to as drugs of 'last resort'. Bacteria that produce these 'carbapenemases', which destroy carbapenems, are often called "superbugs" because infections caused by them are difficult to treat. Worryingly, the production of this carbapenemase is linked with that of a drug pump. In some bacteria the same regulator can control the expression of many genes, including those for virulence factors. It is imperative that we understand why these complex regulatory systems have arisen and how they operate to control different aspects of bacterial resistance and virulence before they spread more widely to other bacteria. We propose a detailed characterization of the new system we have identified in V. cholerae, (which has the potential to be transferred to other pathogenic bacteria, such as Campylobacter or Salmonella), and to incorporate the regulatory protein into a 'biosensor' that we can use to screen for small molecule ligands that could attenuate the expression of the resistance genes. Of course these small-molecules will represent valuable leads for a new class of antibiotics - "resistance suppressors". Notably, since these 'suppressors' do not kill or inhibit bacterial growth, they represent a new paradigm of antibacterial treatment.. , Situação: Em andamento; Natureza: Pesquisa. , Alunos envolvidos: Doutorado: (2) . , Integrantes: Adrian Robert Walmsley - Coordenador / Paul Millner - Integrante.

• 2008 - 2012

  Elucidation of the assembly of a tripartite multidrug pump, Descrição: The proposed programme of work will build upon our current structural and functional studies of the MtrCDE multi-drug pump from Neisseria gonorrhoeae. As part of these studies we have overproduced MtrC, D and E; and established, via pull-down assays and chemical cross-linking, that MtrD and MtrE can interact with MtrC and with one another independently of MtrC. However, microcalorimetry revealed that MtrE and MtrD have a relatively strong interaction with MtrC, but the interaction of these proteins is weak in the absence of MtrC. In assays in which one protein component is immobilised, the other two components can be pulled-down, indicative of the formation of a tripartite complex in vitro. Our aims are to map the sites of interaction of MtrC with MtrE and D by proteolytic fragmentation and mass spectrometry fingerprinting of the cross-linked proteins. This will enable us to model the docking of the proteins as a guide to the insertion of cysteines for cross-linking that would enable us to stabilise the ternary complex for imaging by AFM. Ultimately, our aim is to build an atomic resolution model of the assembly by docking the high-resolution structures of the individual proteins into the AFM topographs. Towards this end, we have already obtained crystals of each protein that diffract to around 7-8 , which will be further refined to generate crystals that give high-resolution diffraction. However, a major effort will be to focus of the determination of the structures of the binary complexes because currently there is no structural data on the protein interactions. Importantly, we have expressed the hairpin of MtrC, which retains its alpha-helical structure and interacts with MtrE, with which it can be used immediate for co-crystallisation. We will utilise our docking models and structures to identifying residues that can stabilize the ternary complex. We will mutate these residues and test for pump assembly and function.. , Situação: Em andamento; Natureza: Pesquisa. , Alunos envolvidos: Graduação: (2) / Doutorado: (2) . , Integrantes: Adrian Robert Walmsley - Coordenador., Financiador(es): Wellcome Trust - Auxílio financeiro., Número de produções C, T & A: 2 / Número de orientações: 3

• 2005 - 2007

  Elucidation of the molecular mechanism of drug-responsive transcriptional repressors, Descrição: Our objective is to determine the structure and mechanism of the VceR and VarR transcriptional-regulators from V.cholerae. VceR is a drug-responsive TetR-type repressor of the vceABC-operon that encodes a tripartite multidrug-pump. We have crystallized the VceR/DNA-complex and obtained a 4 X-ray-diffraction data-set that we can build upon to determine the structure; whilst crystals with bound drug-substrates will be refined to elucidate the mechanism of drug-induced de-repression. VarR is a LysR-type regulator of the varR-varGABCDE-regulon that encodes a metallo--lactamase VarG and the components of a tripartite antimicrobial-resistance-pump. This consists of an inner-membrane ABC-transporter, composed of an ATPase VarF and two membrane-translocases VarD and VarE, a membrane-fusion-protein VarA and an outer-membrane-protein VarC. VarR binds to the varR-G, varG-A and varB-CDEF intergenic-regions; we will characterize its role as a repressor/activator of the upstream-genes. Importantly, varR-varG resembles the organization of ampR-ampC, in which AmpR responds to muropeptides released from peptidoglycan to activate expression of the AmpC serine--lactamase. We aim to test if VarR is responsive to both muropeptides and antimicrobials. Ultimately, our aim is to determine the structure of VarR to understand its mechanism of operation. Although LysR-regulators tend to precipitate at high-concentrations; we have developed procedures to concentrate VarR, enabling us to generate protein crystals, which we will refine for structure determination.. , Situação: Concluído; Natureza: Pesquisa. , Alunos envolvidos: Graduação: (2) / Doutorado: (1) . , Integrantes: Adrian Robert Walmsley - Integrante / Borges-Walmsley, M. I. - Coordenador., Financiador(es): Wellcome Trust - Auxílio financeiro., Número de produções C, T & A: 2 / Número de orientações: 2

• 2000 - 2005

  Cell signalling in the human pathogenic fungus Paracoccidioides brasiliensis, Descrição: Paracoccidioides brasiliensis (Pb) is one of a group of six phylogenetically related ascomycete fungi, from more than a hundred-thousand environmental fungi, that have adapted for survival in mammalian hosts. These six fungi are dimorphic, undergoing extensive changes that allow them to switch from a nonpathogenic filamentous mycelium, usually found in soil, to pathogenic single-cellular yeast that every year causes infections in millions of people across the globe. Infection is the result of hypha-fragments or spores released from mycelium, which are inhaled by the host, exposing them to an elevated temperature that triggers the morphological switch. The pathogenicity of these fungi is intimately linked to the morphological change since strains that are unable to transform from mycelium to yeast are often avirulent. Our knowledge of the mechanisms that these fungi use to sense and respond to the temperature change to switch morphology is still rudimentary. We established that the cAMP-signalling pathway is important in controlling the morphological transition from mycelium to yeast. Importantly, we found that the Gbeta protein Gpb1 interacts with adenylate-cyclase (AC) and there is a change in its expression during the morphological transition; this suggested a novel signaling mechanism in which the activity of adenylate-cyclase is differentially modulated by Gpb1 and its cognate Galpha protein Gpa1, such that the transient increase in Gpb1 during the morphological transition is used to attenuate cAMP-levels. Most recently we have established that Gpb1 and the co-repressor TupA from P. brasiliensis both bind to the PKA protein Tpk2; through which they act as molecular switches of the cell morphology, with TupA and Gpb1 inducing and repressing filamentous growth, respectively. The controlled differential expression of these genes during the morphological transition, as we have observed for both GPB1 and TUPA in P. brasiliensis, would be expected to induce the mo. , Situação: Concluído; Natureza: Pesquisa. , Alunos envolvidos: Doutorado: (3) . , Integrantes: Adrian Robert Walmsley - Integrante / Borges-Walmsley, M. I. - Coordenador., Número de orientações: 2