The Experts below are selected from a list of 12468 Experts worldwide ranked by ideXlab platform
Gabriel Waksma - One of the best experts on this subject based on the ideXlab platform.
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a comprehensive guide to Pilus biogenesis in gram negative bacteria
Nature Reviews Microbiology, 2017Co-Authors: Manuela K Hospenthal, Tiago R D Costa, Gabriel WaksmaAbstract:Pili are crucial virulence factors for many Gram-negative pathogens. These surface structures provide bacteria with a link to their external environments by enabling them to interact with, and attach to, host cells, other surfaces or each other, or by providing a conduit for secretion. Recent high-resolution structures of Pilus filaments and the machineries that produce them, namely chaperone-usher pili, type IV pili, conjugative type IV secretion pili and type V pili, are beginning to explain some of the intriguing biological properties that pili exhibit, such as the ability of chaperone-usher pili and type IV pili to stretch in response to external forces. By contrast, conjugative pili provide a conduit for the exchange of genetic information, and recent high-resolution structures have revealed an integral association between the pilin subunit and a phospholipid molecule, which may facilitate DNA transport. In addition, progress in the area of cryo-electron tomography has provided a glimpse of the overall architecture of the type IV Pilus machinery. In this Review, we examine recent advances in our structural understanding of various Gram-negative Pilus systems and discuss their functional implications.
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structure of a chaperone usher Pilus reveals the molecular basis of rod uncoiling
Cell, 2016Co-Authors: Manuela K Hospenthal, Adam Redzej, Kare W Dodso, Marta Ukleja, Ando Frenz, Catarina Rodrigues, Sco J Hultgre, Frank Dimaio, Edward H Egelma, Gabriel WaksmaAbstract:Types 1 and P pili are prototypical bacterial cell-surface appendages playing essential roles in mediating adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of Pilus subunits assembling into two parts: a thin, short tip fibrillum at the top, mounted on a long Pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive Pilus extrusion by preventing backsliding of the nascent growing Pilus within the secretion pore; the rod also has striking spring-like properties, being able to uncoil and recoil depending on the intensity of shear forces generated by urine flow. Here, we present an atomic model of the P Pilus generated from a 3.8 A resolution cryo-electron microscopy reconstruction. This structure provides the molecular basis for the rod's remarkable mechanical properties and illuminates its role in Pilus secretion.
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chaperone usher pathways diversity and Pilus assembly mechanism
Philosophical Transactions of the Royal Society B, 2012Co-Authors: Andreas Usch, Gabriel WaksmaAbstract:Up to eight different types of secretion systems, and several more subtypes, have been described in Gram-negative bacteria. Here, we focus on the diversity and assembly mechanism of one of the best-studied secretion systems, the widespread chaperone–usher pathway known to assemble and secrete adhesive surface structures, called pili or fimbriae, which play essential roles in targeting bacterial pathogens to the host.
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structural biology of the chaperone usher pathway of Pilus biogenesis
Nature Reviews Microbiology, 2009Co-Authors: Gabriel Waksma, Sco J HultgreAbstract:The chaperone-usher (CU) pathway of Pilus biogenesis is the most widespread of the five pathways that assemble adhesive pili at the surface of Gram-negative bacteria. Recent progress in the study of the structural biology of the CU pathway has unravelled the molecular basis of chaperone function and elucidated the mechanisms of fibre assembly at the outer membrane, leading to a comprehensive description of each step in the biogenesis pathway. Other studies have provided the molecular basis of host recognition by CU pili. The knowledge that has been gathered about both the assembly of and host recognition by CU pili has been harnessed to design promising antibiotic compounds.
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rationally designed small compounds inhibit Pilus biogenesis in uropathogenic bacteria
Proceedings of the National Academy of Sciences of the United States of America, 2006Co-Authors: Jerome S Pinkne, Ha Remau, Floris Uelens, Eric Mille, Veronica Aberg, Nils Pemberto, Mattias Hedenstrom, Andreas Larsso, Patrick C Seed, Gabriel WaksmaAbstract:A chemical synthesis platform with broad applications and flexibility was rationally designed to inhibit biogenesis of adhesive pili assembled by the chaperone–usher pathway in Gram-negative pathogens. The activity of a family of bicyclic 2-pyridones, termed pilicides, was evaluated in two different Pilus biogenesis systems in uropathogenic Escherichia coli. Hemagglutination mediated by either type 1 or P pili, adherence to bladder cells, and biofilm formation mediated by type 1 pili were all reduced by ≈90% in laboratory and clinical E. coli strains. The structure of the pilicide bound to the P Pilus chaperone PapD revealed that the pilicide bound to the surface of the chaperone known to interact with the usher, the outer-membrane assembly platform where pili are assembled. Point mutations in the pilicide-binding site dramatically reduced Pilus formation but did not block the ability of PapD to bind subunits and mediate their folding. Surface plasmon resonance experiments confirmed that the pilicide interfered with the binding of chaperone–subunit complexes to the usher. These pilicides thus target key virulence factors in pathogenic bacteria and represent a promising proof of concept for developing drugs that function by targeting virulence factors.
Michael P Sheetz - One of the best experts on this subject based on the ideXlab platform.
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a force dependent switch reverses type iv Pilus retraction
Proceedings of the National Academy of Sciences of the United States of America, 2004Co-Authors: Erenike Maie, Michael Koomey, Michael P SheetzAbstract:Type IV Pilus dynamics is important for virulence, motility, and DNA transfer in a wide variety of prokaryotes. The type IV Pilus system constitutes a very robust and powerful molecular machine that transports Pilus polymers as well as DNA through the bacterial cell envelope. In Neisseria gonorrhoeae, Pilus retraction is a highly irreversible process that depends on PilT, an AAA ATPase family member. However, when levels of PilT are reduced, the application of high external forces (F = 110 ± 10 pN) induces processive Pilus elongation. At forces of >50 pN, single pili elongate at a rate of v = 350 ± 50 nm/s. For forces of <50 pN, elongation velocity depends strongly on force and relaxation causes immediate retraction. Both Pilus retraction and force-induced elongation can be modeled by chemical kinetics with same step length for the rate-limiting translocation step. The model implies that a force-dependent molecular switch can induce Pilus elongation by reversing the retraction mechanism.
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single Pilus motor forces exceed 100 pn
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Erenike Maie, Laura Potte, H S Seife, Michael P SheetzAbstract:Force production by type IV Pilus retraction is critical for infectivity of Neisseria gonorrhoeae and DNA transfer. We investigated the roles of Pilus number and the retraction motor, PilT, in force generation in vivo at the single-molecule level and found that individual retraction events are generated by a single Pilus fiber, and only one PilT complex powers retraction. Retraction velocity is constant at low forces but decreases at forces greater than 40 pN, giving a remarkably high average stall force of 110 ± 30 pN. Further insights into the molecular mechanism of force generation are gained from the effect of ATP-depletion, which reduces the rate of retraction but not the stall force. Energetic considerations suggest that more than one ATP is involved in the removal of a single pilin subunit from a Pilus. The results are most consistent with a model in which the ATPase PilT forms an oligomer that disassembles the Pilus by a cooperative conformational change.
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Pilus retraction powers bacterial twitching motility
Nature, 2000Co-Authors: Alexey J Merz, Michael P SheetzAbstract:Twitching and social gliding motility allow many Gram negative bacteria to crawl along surfaces, and are implicated in a wide range of biological functions1. Type IV pili (Tfp) are required for twitching and social gliding, but the mechanism by which these filaments promote motility has remained enigmatic1,2,3,4. Here we use laser tweezers5 to show that Tfp forcefully retract. Neisseria gonorrhoeae cells that produce Tfp actively crawl on a glass surface and form adherent microcolonies. When laser tweezers are used to place and hold cells near a microcolony, retractile forces pull the cells toward the microcolony. In quantitative experiments, the Tfp of immobilized bacteria bind to latex beads and retract, pulling beads from the tweezers at forces that can exceed 80 pN. Episodes of retraction terminate with release or breakage of the Tfp tether. Both motility and retraction mediated by Tfp occur at about 1 µm s-1 and require protein synthesis and function of the PilT protein. Our experiments establish that Tfp filaments retract, generate substantial force and directly mediate cell movement.
Sco J Hultgre - One of the best experts on this subject based on the ideXlab platform.
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structure of a chaperone usher Pilus reveals the molecular basis of rod uncoiling
Cell, 2016Co-Authors: Manuela K Hospenthal, Adam Redzej, Kare W Dodso, Marta Ukleja, Ando Frenz, Catarina Rodrigues, Sco J Hultgre, Frank Dimaio, Edward H Egelma, Gabriel WaksmaAbstract:Types 1 and P pili are prototypical bacterial cell-surface appendages playing essential roles in mediating adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of Pilus subunits assembling into two parts: a thin, short tip fibrillum at the top, mounted on a long Pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive Pilus extrusion by preventing backsliding of the nascent growing Pilus within the secretion pore; the rod also has striking spring-like properties, being able to uncoil and recoil depending on the intensity of shear forces generated by urine flow. Here, we present an atomic model of the P Pilus generated from a 3.8 A resolution cryo-electron microscopy reconstruction. This structure provides the molecular basis for the rod's remarkable mechanical properties and illuminates its role in Pilus secretion.
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structural biology of the chaperone usher pathway of Pilus biogenesis
Nature Reviews Microbiology, 2009Co-Authors: Gabriel Waksma, Sco J HultgreAbstract:The chaperone-usher (CU) pathway of Pilus biogenesis is the most widespread of the five pathways that assemble adhesive pili at the surface of Gram-negative bacteria. Recent progress in the study of the structural biology of the CU pathway has unravelled the molecular basis of chaperone function and elucidated the mechanisms of fibre assembly at the outer membrane, leading to a comprehensive description of each step in the biogenesis pathway. Other studies have provided the molecular basis of host recognition by CU pili. The knowledge that has been gathered about both the assembly of and host recognition by CU pili has been harnessed to design promising antibiotic compounds.
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type 1 Pilus mediated bacterial invasion of bladder epithelial cells
The EMBO Journal, 2000Co-Authors: Jua J Martinez, Jerome S Pinkne, Matthew A Mulvey, Joel D Schilling, Sco J HultgreAbstract:Most strains of uropathogenic Escherichia coli (UPEC) encode filamentous adhesive organelles called type 1 pili. We have determined that the type 1 Pilus adhesin, FimH, mediates not only bacterial adherence, but also invasion of human bladder epithelial cells. In contrast, adherence mediated by another Pilus adhesin, PapG, did not initiate bacterial internalization. FimH-mediated invasion required localized host actin reorganization, phosphoinositide 3-kinase (PI 3-kinase) activation and host protein tyrosine phosphorylation, but not activation of Src-family tyrosine kinases. Phosphorylation of focal adhesin kinase (FAK) at Tyr397 and the formation of complexes between FAK and PI 3-kinase and between α-actinin and vinculin were found to correlate with type 1 Pilus-mediated bacterial invasion. Inhibitors that prevented bacterial invasion also blocked the formation of these complexes. Our results demonstrate that UPEC strains are not strictly extracellular pathogens and that the type 1 Pilus adhesin FimH can directly trigger host cell signaling cascades that lead to bacterial internalization.
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structural basis of chaperone function and Pilus biogenesis
Science, 1999Co-Authors: Frederic G Saue, Kare W Dodso, Sco J Hultgre, Klaus Futtere, Jerome S Pinkne, Gabriel WaksmaAbstract:Many Gram-negative pathogens assemble architecturally and functionally diverse adhesive pili on their surfaces by the chaperone-usher pathway. Immunoglobulin-like periplasmic chaperones escort Pilus subunits to the usher, a large protein complex that facilitates the translocation and assembly of subunits across the outer membrane. The crystal structure of the PapD-PapK chaperone-subunit complex, determined at 2.4 angstrom resolution, reveals that the chaperone functions by donating its G1 β strand to complete the immunoglobulin-like fold of the subunit via a mechanism termed donor strand complementation. The structure of the PapD-PapK complex also suggests that during Pilus biogenesis, every subunit completes the immunoglobulin-like fold of its neighboring subunit via a mechanism termed donor strand exchange.
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the papc usher forms an oligomeric channel implications for Pilus biogenesis across the outer membrane
Proceedings of the National Academy of Sciences of the United States of America, 1998Co-Authors: David G Thanassi, Evan T Saulino, Maryjane Lombardo, Roby Roth, Joh E Heuse, Sco J HultgreAbstract:Bacterial virulence factors are typically surface-associated or secreted molecules that in Gram-negative bacteria must cross the outer membrane (OM). Protein translocation across the bacterial OM is not well understood. To elucidate this process we studied P Pilus biogenesis in Escherichia coli. We present high-resolution electron micrographs of the OM usher PapC and show that it forms an oligomeric complex containing a channel approximately 2 nm in diameter. This is large enough to accommodate Pilus subunits or the linear tip fibrillum of the Pilus but not large enough to accommodate the final 6.8-nm-wide helical Pilus rod. We show that P Pilus rods can be unraveled into linear fibers by incubation in 50% glycerol. Thus, they are likely to pass through the usher in this unwound form. Packaging of these fibers into their final helical structure would only occur outside the cell, a process that may drive outward growth of the Pilus organelles. The usher complex appears to be similar to complexes formed by members of the PulD/pIV family of OM proteins, and thus these two protein families, previously thought to be unrelated, may share structural and functional homologies.
Sonjaverena Albers - One of the best experts on this subject based on the ideXlab platform.
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the archaellum a rotating type iv Pilus
Molecular Microbiology, 2014Co-Authors: Rajesh Shahapure, Rosalie P C Driesse, Florencia M Haura, Sonjaverena Albers, Remus T DameAbstract:Summary Microbes have evolved sophisticated mechanisms of motility allowing them to respond to changing environ- mental conditions. While this cellular process is well characterized in bacteria, the mode and mechanisms of motility are poorly understood in archaea. This study examines the motility of individual cells of the thermoacidophilic crenarchaeon Sulfolobus acido- caldarius. Specifically, we investigated motility of cells producing exclusively the archaeal swimming orga- nelle, the archaellum. Archaella are structurally and in sequence similar to bacterial type IV pili involved in surface motility via Pilus extension-retraction cycles and not to rotating bacterial flagella. Unexpectedly, our studies reveal a novel type of behaviour for type IV Pilus like structures: archaella rotate and their rota- tion drives swimming motility. Moreover, we demon- strate that temperature has a direct effect on rotation velocity explaining temperature-dependent swimming velocity.
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structure and function of the adhesive type iv Pilus of sulfolobus acidocaldarius
Environmental Microbiology, 2012Co-Authors: Annalena Henche, Abhrajyoti Ghosh, Edward H Egelma, Torste Jeske, Sonjaverena AlbersAbstract:Summary Archaea display a variety of type IV pili on their surface and employ them in different physiological functions. In the crenarchaeon Sulfolobus acidocaldarius the most abundant surface structure is the aap Pilus (archaeal adhesive Pilus). The construction of in frame deletions of the aap genes revealed that all the five genes (aapA, aapX, aapE, aapF, aapB) are indispensible for assembly of the Pilus and an impact on surface motility and biofilm formation was observed. Our analyses revealed that there exists a regulatory cross-talk between the expression of aap genes and archaella (formerly archaeal flagella) genes during different growth phases. The structure of the aap Pilus is entirely different from the known bacterial type IV pili as well as other archaeal type IV pili. An aap Pilus displayed 3 stranded helices where there is a rotation per subunit of ∼ 138° and a rise per subunit of ∼ 5.7 A. The filaments have a diameter of ∼ 110 A and the resolution was judged to be ∼ 9 A. We concluded that small changes in sequence might be amplified by large changes in higher-order packing. Our finding of an extraordinary stability of aap pili possibly represents an adaptation to harsh environments that S. acidocaldarius encounters.
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archaeal type iv Pilus like structures evolutionarily conserved prokaryotic surface organelles
Current Opinion in Microbiology, 2011Co-Authors: Mecky Pohlschrode, Abhrajyoti Ghosh, Manuela Tripepi, Sonjaverena AlbersAbstract:In both bacteria and Archaea, the biosynthesis of type IV Pilus-related structures involves a set of core components, including a prepilin peptidase that specifically processes precursors of pilin-like proteins. Although in silico analyses showed that most sequenced archaeal genomes encode predicted pilins and conserved Pilus biosynthesis components, recent in vivo analyses of archaeal pili in genetically tractable crenarchaea and euryarchaea revealed Archaea-specific type IV Pilus functions and biosynthesis components. Studies in a variety of archaeal species will reveal which type IV Pilus-like structures are common in Archaea and which are limited to certain species within this domain. The insights gleaned from these studies may also elucidate the roles played by these types of structures in adapting to specific environments.
Lisa Craig - One of the best experts on this subject based on the ideXlab platform.
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structure of the neisseria meningitidis type iv Pilus
Nature Communications, 2016Co-Authors: Subramania Kolappa, Edward H Egelma, Mathieu Coureuil, Xavie Nassif, Lisa CraigAbstract:Neisseria meningitidis use Type IV pili (T4P) to adhere to endothelial cells and breach the blood brain barrier, causing cause fatal meningitis. T4P are multifunctional polymers of the major pilin protein, which share a conserved hydrophobic N terminus that is a curved extended α-helix, α1, in X-ray crystal structures. Here we report a 1.44 A crystal structure of the N. meningitidis major pilin PilE and a ∼6 A cryo-electron microscopy reconstruction of the intact Pilus, from which we built an atomic model for the filament. This structure reveals the molecular arrangement of the N-terminal α-helices in the filament core, including a melted central portion of α1 and a bridge of electron density consistent with a predicted salt bridge necessary for Pilus assembly. This structure has important implications for understanding Pilus biology. Type IV pili are present on a wide range of bacterial pathogens and mediate diverse functions. Here the authors report a high resolution crystal structure of the pilin subunit PilE, and a cryoEM reconstruction of the Type IV Pilus filament from N. meningitidisthat offer insight into Pilus assembly and functions.
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crystal structure of the minor pilin cofb the initiator of cfa iii Pilus assembly in enterotoxigenic escherichia coli
Journal of Biological Chemistry, 2015Co-Authors: Subramania Kolappa, Guixiang Yang, Lisa CraigAbstract:Type IV pili are extracellular polymers of the major pilin subunit. These subunits are held together in the Pilus filament by hydrophobic interactions among their N-terminal α-helices, which also anchor the pilin subunits in the inner membrane prior to Pilus assembly. Type IV Pilus assembly involves a conserved group of proteins that span the envelope of Gram-negative bacteria. Among these is a set of minor pilins, so named because they share their hydrophobic N-terminal polymerization/membrane anchor segment with the major pilins but are much less abundant. Minor pilins influence Pilus assembly and retraction, but their precise functions are not well defined. The Type IV Pilus systems of enterotoxigenic Escherichia coli and Vibrio cholerae are among the simplest of Type IV Pilus systems and possess only a single minor pilin. Here we show that the enterotoxigenic E. coli minor pilins CofB and LngB are required for assembly of their respective Type IV pili, CFA/III and Longus. Low levels of the minor pilins are optimal for Pilus assembly, and CofB can be detected in the Pilus fraction. We solved the 2.0 A crystal structure of N-terminally truncated CofB, revealing a pilin-like protein with an extended C-terminal region composed of two discrete domains connected by flexible linkers. The C-terminal region is required for CofB to initiate Pilus assembly. We propose a model for CofB-initiated Pilus assembly with implications for understanding filament growth in more complex Type IV Pilus systems as well as the related Type II secretion system.
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type iv Pilus structure by cryo electron microscopy and crystallography implications for Pilus assembly and functions
Molecular Cell, 2006Co-Authors: Lisa Craig, Niels Volkmann, A S Arvai, Michael E Pique, Mark Yeager, Edward H Egelman, John A TainerAbstract:Summary Type IV pili (T4P) are long, thin, flexible filaments on bacteria that undergo assembly-disassembly from inner membrane pilin subunits and exhibit astonishing multifunctionality. Neisseria gonorrhoeae (gonococcal or GC) T4P are prototypic virulence factors and immune targets for increasingly antibiotic-resistant human pathogens, yet detailed structures are unavailable for any T4P. Here, we determined a detailed experimental GC-T4P structure by quantitative fitting of a 2.3 A full-length pilin crystal structure into a 12.5 A resolution native GC-T4P reconstruction solved by cryo-electron microscopy (cryo-EM) and iterative helical real space reconstruction. Spiraling three-helix bundles form the filament core, anchor the globular heads, and provide strength and flexibility. Protruding hypervariable loops and posttranslational modifications in the globular head shield conserved functional residues in pronounced grooves, creating a surprisingly corrugated Pilus surface. These results clarify T4P multifunctionality and assembly-disassembly while suggesting unified assembly mechanisms for T4P, archaeal flagella, and type II secretion system filaments.
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type iv Pilus structure and bacterial pathogenicity
Nature Reviews Microbiology, 2004Co-Authors: Lisa Craig, Michael E Pique, Joh A TaineAbstract:Type IV pili are remarkably strong, flexible filaments with varied roles in bacterial pathogenicity. All Gram-negative bacterial surfaces have type IV pili, which are polymeric assemblies of the protein pilin that evoke the host immune response and are potential drug and vaccine targets. Pilin structures that have been solved using X-ray crystallography and nuclear magnetic resonance, together with models for Pilus architectures inferred from electron microscopy, fibre diffraction and computation, have established a molecular basis for assembly and multi-functionality, with implications for therapeutic interventions.
