Gabriel Waksman

The Institute of Structural and Molecular Biology, Londinium, England, United Kingdom

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Publications (164)1351.31 Total impact

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    ABSTRACT: Conjugation, the process by which plasmid DNA is transferred from one bacterium to another, is mediated by type IV secretion systems (T4SSs). T4SSs are versatile systems that can transport not only DNA, but also toxins and effector proteins. Conjugative T4SSs comprise 12 proteins named VirB1-11 and VirD4 that assemble into a large membrane-spanning exporting machine. Before being transported, the DNA substrate is first processed on the cytoplasmic side by a complex called the relaxosome. The substrate is then targeted to the T4SS for export into a recipient cell. In this review, we describe the recent progress made in the structural biology of both the relaxosome and the T4SS. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Trends in Microbiology 03/2015; 118. DOI:10.1016/j.tim.2015.02.012 · 9.81 Impact Factor
  • Andreas Busch, Gilles Phan, Gabriel Waksman
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    ABSTRACT: The formation of adhesive surface structures called pili or fimbriae ('bacterial hair') is an important contributor towards bacterial pathogenicity and persistence. To fight often chronic or recurrent bacterial infections such as urinary tract infections, it is necessary to understand the molecular mechanism of the nanomachines assembling such pili. Here, we focus on the so far best-known pilus assembly machinery: the chaperone-usher pathway producing the type 1 and P pili, and highlight the most recently acquired structural knowledge. First, we describe the subunits' structure and the molecular role of the periplasmic chaperone. Second, we focus on the outer-membrane usher structure and the catalytic mechanism of usher-mediated pilus biogenesis. Finally, we describe how the detailed understanding of the chaperone-usher pathway at a molecular level has paved the way for the design of a new generation of bacterial inhibitors called 'pilicides'. © 2015 The Author(s) Published by the Royal Society. All rights reserved.
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    ABSTRACT: In view of the relentless increase in antibiotic resistance in human pathogens, efforts are needed to safeguard our future therapeutic options against infectious diseases. In addition to regulatory changes in our antibiotic use, this will have to include the development of new therapeutic compounds. One area that has received growing attention in recent years is the possibility to treat or prevent infections by targeting the virulence mechanisms that render bacteria pathogenic. Antivirulence targets include bacterial adherence, secretion of toxic effector molecules, bacterial persistence through biofilm formation, quorum sensing and immune evasion. Effective small molecule compounds have already been identified that suppress such processes. In this review we discuss the susceptibility of such compounds to the development of resistance, by comparison with known resistance mechanisms observed for classical bacteriostatic or -lytic antibiotics, and by review of available experimental case studies. Unfortunately, appearance of resistance mechanisms has already been demonstrated for some, showing that the quest of new, lasting drugs remains complicated. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.
    Chemical Biology &amp Drug Design 01/2015; DOI:10.1111/cbdd.12517 · 2.51 Impact Factor
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    ABSTRACT: Pseudomonas aeruginosa is a Gram-negative opportunistic bacterium, synonymous with cystic fibrosis patients, which can cause chronic infection of the lungs. This pathogen is a model organism to study biofilms: a bacterial population embedded in an extracellular matrix that provide protection from environmental pressures and lead to persistence. A number of chaperone-usher pathways, namely CupA-CupE, play key roles in these processes by assembling adhesive pili on the bacterial surface. One of these, encoded by the cupB operon, is unique as it contains a non-chaperone-usher gene product, CupB5. Two-partner secretion (TPS) systems are comprised of a C-terminal integral membrane β-barrel pore with tandem N-terminal POTRA (polypeptide transport associated) domains located in the periplasm (TpsB) and a secreted substrate (TpsA). Using NMR 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 can translocate CupB5 across the P. aeruginosa outer membrane, which contrasts a previous observation that suggested the CupB3 P-usher secretes CupB5. In support of our findings we also demonstrate that tps4/cupB operons are co-regulated by the RocS1 sensor suggesting P. aeruginosa has developed synergy between these systems. Furthermore, we have determined the solution-structure of the TpsB4-POTRA1 domain and together with restraints from NMR chemical shift mapping and 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 residues for TpsA4/CupB5 recognition by TpsB4 in the periplasm and suggest distinct roles for each POTRA domain. This article is protected by copyright. All rights reserved.
    Protein Science 01/2015; DOI:10.1002/pro.2640 · 2.86 Impact Factor
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    ABSTRACT: eLife digest Escherichia coli is a bacterium that commonly lives in the intestines of mammals, including humans, where it is usually harmless and can even be beneficial to its host. However, some types of E. coli produce hair-like filaments called P pili that allow the bacteria to attach to the human urinary tract and cause disease. To pass through the outer membrane of the E. coli cell, the filaments have to travel through a protein in the membrane called PapC usher. The PapC usher protein—which is also involved in the assembly of the P pili filaments—contains a tube-like part called a β-barrel that is usually blocked by another part of the protein called the ‘plug domain’. For the P pili to pass through the β-barrel, the plug domain has to move. This movement is controlled by two parts of the PapC protein, known as the α-helix and the β-hairpin, but it is not clear how. To address this question, Farabella et al. made computer models of the normal PapC protein and versions that lacked the α-helix and/or the β-hairpin. Looking at these structural models and analyzing the evolution of PapC proteins helped to predict that certain regions of the β-barrel may be involved in controlling the movement of the plug domain, and this was then confirmed experimentally. Farabella et al. propose that these regions—together with the α-helix and β-hairpin—control the opening and closing of the β-barrel. Further work is needed to investigate how other parts of the PapC protein are involved in P pili formation. These new insights could prove useful in the development of alternative treatments to fight bacterial infection. DOI: http://dx.doi.org/10.7554/eLife.03532.002
    eLife Sciences 10/2014; 3. DOI:10.7554/eLife.03532 · 8.52 Impact Factor
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    ABSTRACT: A novel series of 8-amino imidazo[1,2-a] pyrazine derivatives has been developed as inhibitors of the VirB11 ATPase HP0525, a key component of the bacterial type IV secretion system. A flexible synthetic route to both 2- and 3-aryl substituted regioisomers has been developed. The resulting series of imidazo[1,2-a] pyrazines has been used to probe the structure-activity relationships of these inhibitors, which show potential as antibacterial agents. (C) 2014 The Authors. Published by Elsevier Ltd.
    Bioorganic & Medicinal Chemistry 09/2014; 22(22). DOI:10.1016/j.bmc.2014.09.036 · 2.95 Impact Factor
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    Tamir Gonen, Gabriel Waksman
    Current Opinion in Structural Biology 09/2014; 27. DOI:10.1016/j.sbi.2014.09.002 · 8.75 Impact Factor
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    David Steadman, Alvin Lo, Gabriel Waksman, Han Remaut
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    ABSTRACT: The rise of multidrug resistant bacteria is a major worldwide health concern. There is currently an unmet need for the development of new and selective antibacterial drugs. Therapies that target and disarm the crucial virulence factors of pathogenic bacteria, while not actually killing the cells themselves, could prove to be vital for the treatment of numerous diseases. This article discusses the main surface architectures of pathogenic Gram-negative bacteria and the small molecules that have been discovered, which target their specific biogenesis pathways and/or actively block their virulence. The future perspective for the use of antivirulence compounds is also assessed.
    Future Microbiology 07/2014; 9(7):887-900. DOI:10.2217/fmb.14.46 · 3.82 Impact Factor
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    ABSTRACT: Uropathogenic Escherichia coli (UPEC) cause urinary tract infections (UTIs) in approximately 50% of women. These bacteria use type 1 and P pili for host recognition and attachment. These pili are assembled by the chaperone-usher pathway of pilus biogenesis. The review examines the biogenesis and adhesion of the UPEC type 1 and P pili. Particular emphasis is drawn to the role of the outer membrane usher protein. The structural properties of the complete pilus are also examined to highlight the strength and functionality of the final assembly. The usher orchestrates the sequential addition of pilus subunits in a defined order. This process follows a subunit-incorporation cycle which consists of four steps: recruitment at the usher N-terminal domain, donor-strand exchange with the previously assembled subunit, transfer to the usher C-terminal domains and translocation of the nascent pilus. Adhesion by the type 1 and P pili is strengthened by the quaternary structure of their rod sections. The rod is endowed with spring-like properties which provide mechanical resistance against urine flow. The distal adhesins operate differently from one another, targeting receptors in a specific manner. The biogenesis and adhesion of type 1 and P pili are being therapeutically targeted, and efforts to prevent pilus growth or adherence are described. The combination of structural and biochemical study has led to the detailed mechanistic understanding of this membrane spanning nano-machine. This can now be exploited to design novel drugs able to inhibit virulence. This is vital in the present era of resurgent antibiotics resistance. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins.
    Biochimica et Biophysica Acta 05/2014; 1850(3). DOI:10.1016/j.bbagen.2014.04.021 · 4.66 Impact Factor
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    ABSTRACT: Bacteria use type IV secretion (T4S) systems to deliver DNA and protein substrates to a diverse range of prokaryotic and eukaryotic target cells. T4S systems have great impact on human health, as they are a major source of antibiotic resistance spread among bacteria and are central to infection processes of many pathogens. Therefore, deciphering the structure and underlying translocation mechanism of T4S systems is crucial to facilitate development of new drugs. The last five years have witnessed considerable progress in unraveling the structure of T4S system subassemblies, notably that of the T4S system core complex, a large 1MegaDalton (MDa) structure embedded in the double membrane of Gram-negative bacteria and made of 3 of the 12 T4S system components. However, the recent determination of the structure of ∼3MDa assembly of 8 of these components has revolutionized our views of T4S system architecture and opened up new avenues of research, which are discussed in this review.
    Current Opinion in Structural Biology 04/2014; 27C:16-23. DOI:10.1016/j.sbi.2014.02.006 · 8.75 Impact Factor
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    ABSTRACT: Bacterial type IV secretion systems translocate virulence factors into eukaryotic cells, distribute genetic material between bacteria and have shown potential as a tool for the genetic modification of human cells. Given the complex choreography of the substrate through the secretion apparatus, the molecular mechanism of the type IV secretion system has proved difficult to dissect in the absence of structural data for the entire machinery. Here we use electron microscopy to reconstruct the type IV secretion system encoded by the Escherichia coli R388 conjugative plasmid. We show that eight proteins assemble in an intricate stoichiometric relationship to form an approximately 3 megadalton nanomachine that spans the entire cell envelope. The structure comprises an outer membrane-associated core complex connected by a central stalk to a substantial inner membrane complex that is dominated by a battery of 12 VirB4 ATPase subunits organized as side-by-side hexameric barrels. Our results show a secretion system with markedly different architecture, and consequently mechanism, to other known bacterial secretion systems.
    Nature 03/2014; 508(7497). DOI:10.1038/nature13081 · 42.35 Impact Factor
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    Gabriel Waksman, Elena V Orlova
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    ABSTRACT: Type IV secretion (T4S) systems are large dynamic nanomachines that transport DNAs and/or proteins through the membranes of bacteria. Because of their complexity and multi-protein organisation, T4S systems have been extremely challenging to study structurally. However in the past five years significant milestones have been achieved by X-ray crystallography and cryo-electron microscopy. This review describes some of the more recent advances: the structures of some of the protein components of the T4S systems and the complete core complex structure that was determined using electron microscopy.
    Current opinion in microbiology 02/2014; 17C:24-31. DOI:10.1016/j.mib.2013.11.001 · 7.22 Impact Factor
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    ABSTRACT: Bacteria use type IV secretion (T4S) systems to deliver DNA and protein substrates to a diverse range of prokaryotic and eukaryotic target cells. T4S systems have great impact on human health, as they are a major source of antibiotic resistance spread among bacteria and are central to infection processes of many pathogens. Therefore, deciphering the structure and underlying translocation mechanism of T4S systems is crucial to facilitate development of new drugs. The last five years have witnessed considerable progress in unraveling the structure of T4S system subassemblies, notably that of the T4S system core complex, a large 1 MegaDalton (MDa) structure embedded in the double membrane of Gram-negative bacteria and made of 3 of the 12 T4S system components. However, the recent determination of the structure of ∼3 MDa assembly of 8 of these components has revolutionized our views of T4S system architecture and opened up new avenues of research, which are discussed in this review.
    Current Opinion in Structural Biology 01/2014; 27:16–23. · 8.75 Impact Factor
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    ABSTRACT: To identify and to characterize small-molecule inhibitors that target the subunit polymerization of the type 1 pilus assembly in uropathogenic Escherichia coli (UPEC). Using an SDS-PAGE-based assay, in silico pre-filtered small-molecule compounds were screened for specific inhibitory activity against the critical subunit polymerization step of the chaperone-usher pathway during pilus biogenesis. The biological activity of one of the compounds was validated in assays monitoring UPEC type 1 pilus biogenesis, type 1 pilus-dependent biofilm formation and adherence to human bladder epithelial cells. The time dependence of the in vivo inhibitory activity and the overall effect of the compound on UPEC growth were determined. N-(4-chloro-phenyl)-2-{5-[4-(pyrrolidine-1-sulfonyl)-phenyl]-[1,3,4]oxadiazol-2-yl sulfanyl}-acetamide (AL1) inhibited in vitro pilus subunit polymerization. In bacterial cultures, AL1 disrupted UPEC type 1 pilus biogenesis and pilus-dependent biofilm formation, and resulted in the reduction of bacterial adherence to human bladder epithelial cells, without affecting bacterial cell growth. Bacterial exposure to the inhibitor led to an almost instantaneous loss of type 1 pili. We have identified and characterized a small molecule that interferes with the assembly of type 1 pili. The molecule targets the polymerization step during the subunit incorporation cycle of the chaperone-usher pathway. Our discovery provides new insight into the design and development of novel anti-virulence therapies targeting key virulence factors of bacterial pathogens.
    Journal of Antimicrobial Chemotherapy 12/2013; 69(4). DOI:10.1093/jac/dkt467 · 5.44 Impact Factor
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    ABSTRACT: Extracellular fibers called chaperone-usher pathway pili are critical virulence factors in a wide range of Gram-negative pathogenic bacteria that facilitate binding and invasion into host tissues and mediate biofilm formation. Chaperone-usher pathway ushers, which catalyze pilus assembly, contain five functional domains: a 24-stranded transmembrane β-barrel translocation domain (TD), a β-sandwich plug domain (PLUG), an N-terminal periplasmic domain, and two C-terminal periplasmic domains (CTD1 and 2). Pore gating occurs by a mechanism whereby the PLUG resides stably within the TD pore when the usher is inactive and then upon activation is translocated into the periplasmic space, where it functions in pilus assembly. Using antibiotic sensitivity and electrophysiology experiments, a single salt bridge was shown to function in maintaining the PLUG in the TD channel of the P pilus usher PapC, and a loop between the 12th and 13th beta strands of the TD (β12-13 loop) was found to facilitate pore opening. Mutation of the β12-13 loop resulted in a closed PapC pore, which was unable to efficiently mediate pilus assembly. Deletion of the PapH terminator/anchor resulted in increased OM permeability, suggesting a role for the proper anchoring of pili in retaining OM integrity. Further, we introduced cysteine residues in the PLUG and N-terminal periplasmic domains that resulted in a FimD usher with a greater propensity to exist in an open conformation, resulting in increased OM permeability but no loss in type 1 pilus assembly. These studies provide insights into the molecular basis of usher pore gating and its roles in pilus biogenesis and OM permeability.
    Proceedings of the National Academy of Sciences 12/2013; 110(51). DOI:10.1073/pnas.1320528110 · 9.81 Impact Factor
  • Sebastian Geibel, Gabriel Waksman
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    ABSTRACT: Secretion systems are specialized in transport of proteins, DNA or nutrients across the cell envelope of bacteria and enable them to communicate with their environment. The chaperone-usher (CU) pathway is used for assembly and secretion of a large family of long adhesive protein polymers, termed pili, and is widespread among Gram-negative pathogens [1]. Moreover, recent evidence has indicated that CU secretion systems are also involved in sporulation [2,3]. In this review we focus on the structural biology of the paradigmatic type 1 and P pili CU systems encoded by uropathogenic Escherichia coli (UPEC), where recent progress has provided unprecedented insights into pilus assembly and secretion mechanism. This article is part of a Special Issue entitled:Protein trafficking & Secretion.
    Biochimica et Biophysica Acta 10/2013; 1843(8). DOI:10.1016/j.bbamcr.2013.09.023 · 4.66 Impact Factor

Publication Stats

8k Citations
1,351.31 Total Impact Points

Institutions

  • 2004–2015
    • The Institute of Structural and Molecular Biology
      Londinium, England, United Kingdom
  • 2004–2014
    • Birkbeck, University of London
      • Institute of Structural and Molecular Biology
      Londinium, England, United Kingdom
  • 2010
    • MRC National Institute for Medical Research
      • Division of Molecular Structure
      Londinium, England, United Kingdom
  • 2009
    • Harvard Medical School
      Boston, Massachusetts, United States
  • 1997–2009
    • Washington University in St. Louis
      • • Department of Pediatrics
      • • Department of Biochemistry and Molecular Biophysics
      San Luis, Missouri, United States
  • 2006–2008
    • University College London
      • Department of Structural and Molecular Biology
      Londinium, England, United Kingdom
  • 2005
    • Universität Basel
      Bâle, Basel-City, Switzerland
  • 1999
    • Howard Hughes Medical Institute
      Ashburn, Virginia, United States
  • 1992–1994
    • The Rockefeller University
      • Laboratory of Molecular Biophysics
      New York City, NY, United States