Architecture of the Vibrio cholerae toxin-coregulated pilus machine revealed by electron cryotomography

Type IV pili (T4P) are filamentous appendages found on many Bacteria and Archaea. They are helical fibres of pilin proteins assembled by a multi-component macromolecular machine we call the basal body. Based on pilin features, T4P are classified into type IVa pili (T4aP) and type IVb pili (T4bP)1,2. T4aP are more widespread and are involved in cell motility3, DNA transfer4, host predation5 and electron transfer6. T4bP are less prevalent and are mainly found in enteropathogenic bacteria, where they play key roles in host colonization7. Following similar work on T4aP machines8,9, here we use electron cryotomography10 to reveal the three-dimensional in situ structure of a T4bP machine in its piliated and non-piliated states. The specific machine we analyse is the Vibrio cholerae toxin-coregulated pilus machine (TCPM). Although only about half of the components of the TCPM show sequence homology to components of the previously analysed Myxococcus xanthus T4aP machine (T4aPM), we find that their structures are nevertheless remarkably similar. Based on homologies with components of the M. xanthus T4aPM and additional reconstructions of TCPM mutants in which the non-homologous proteins are individually deleted, we propose locations for all eight TCPM components within the complex. Non-homologous proteins in the T4aPM and TCPM are found to form similar structures, suggesting new hypotheses for their functions and evolutionary histories.

[1]  N. Bose,et al.  Secretion of a soluble colonization factor by the TCP type 4 pilus biogenesis pathway in Vibrio cholerae , 2003, Molecular microbiology.

[2]  Nicholas A. Frost,et al.  Outer Membrane Targeting, Ultrastructure, and Single Molecule Localization of the Enteropathogenic Escherichia coli Type IV Pilus Secretin BfpB , 2012, Journal of bacteriology.

[3]  J. Koo,et al.  PilN Binding Modulates the Structure and Binding Partners of the Pseudomonas aeruginosa Type IVa Pilus Protein PilM* , 2016, The Journal of Biological Chemistry.

[4]  C. van der Does,et al.  The Type IV Pilus Assembly ATPase PilB of Myxococcus xanthus Interacts with the Inner Membrane Platform Protein PilC and the Nucleotide-binding Protein PilM* , 2016, The Journal of Biological Chemistry.

[5]  Jun Zhu,et al.  Vibrio cholerae virulence regulator-coordinated evasion of host immunity , 2006, Proceedings of the National Academy of Sciences.

[6]  L. Søgaard-Andersen,et al.  Outside-In Assembly Pathway of the Type IV Pilus System in Myxococcus xanthus , 2013, Journal of bacteriology.

[7]  SödingJohannes.  Protein homology detection by HMM--HMM comparison , 2005 .

[8]  J. McIntosh,et al.  The Molecular Architecture of Axonemes Revealed by Cryoelectron Tomography , 2006, Science.

[9]  John A Tainer,et al.  Hexameric structures of the archaeal secretion ATPase GspE and implications for a universal secretion mechanism , 2007, The EMBO journal.

[10]  Ning Ma,et al.  BLAST+: architecture and applications , 2009, BMC Bioinformatics.

[11]  P. Manning,et al.  The tcp gene cluster of Vibrio cholerae. , 1997, Gene.

[12]  W. Hol,et al.  The three-dimensional structure of the cytoplasmic domains of EpsF from the type 2 secretion system of Vibrio cholerae. , 2009, Journal of structural biology.

[13]  W. Hol,et al.  The type II secretion system: biogenesis, molecular architecture and mechanism , 2012, Nature Reviews Microbiology.

[14]  W. Hol,et al.  Structural and Functional Studies on the Interaction of GspC and GspD in the Type II Secretion System , 2011, PLoS pathogens.

[15]  S. Lory,et al.  A single bifunctional enzyme, PilD, catalyzes cleavage and N-methylation of proteins belonging to the type IV pilin family. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[16]  M. Levine,et al.  Morphological studies on fimbriae expressed by Vibrio cholerae 01. , 1988, Microbial pathogenesis.

[17]  Ronald K. Taylor,et al.  Protection and Attachment of Vibrio cholerae Mediated by the Toxin-Coregulated Pilus in the Infant Mouse Model , 2011, Journal of bacteriology.

[18]  G. Schoehn,et al.  Structural similarity of secretins from type II and type III secretion systems. , 2014, Structure.

[19]  Jun Zhu,et al.  Post‐transcriptional cross‐talk between pro‐ and anti‐colonization pili biosynthesis systems in Vibrio cholerae , 2007, Molecular microbiology.

[20]  D. Motooka,et al.  Homo-trimeric Structure of the Type IVb Minor Pilin CofB Suggests Mechanism of CFA/III Pilus Assembly in Human Enterotoxigenic Escherichia coli. , 2016, Journal of molecular biology.

[21]  K. Satyshur,et al.  P. aeruginosa PilT structures with and without nucleotide reveal a dynamic type IV pilus retraction motor. , 2010, Journal of molecular biology.

[22]  V. L. Miller,et al.  Use of phoA gene fusions to identify a pilus colonization factor coordinately regulated with cholera toxin. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[23]  L. Craig,et al.  Crystal Structure of the Minor Pilin CofB, the Initiator of CFA/III Pilus Assembly in Enterotoxigenic Escherichia coli* , 2015, The Journal of Biological Chemistry.

[24]  G. Schoolnik,et al.  Chitin Induces Natural Competence in Vibrio cholerae , 2005, Science.

[25]  Ronald K. Taylor,et al.  Secretion of TcpF by the Vibrio cholerae Toxin-Coregulated Pilus Biogenesis Apparatus Requires an N-Terminal Determinant , 2013, Journal of bacteriology.

[26]  M. Donnenberg,et al.  Interaction and localization studies of enteropathogenic Escherichia coli type IV bundle-forming pilus outer membrane components. , 2006, Microbiology.

[27]  J. Forman-Kay,et al.  Characterization of the PilN, PilO and PilP type IVa pilus subcomplex , 2011, Molecular microbiology.

[28]  Ronald K. Taylor,et al.  Membrane Association and Multimerization of TcpT, the Cognate ATPase Ortholog of the Vibrio cholerae Toxin-Coregulated-Pilus Biogenesis Apparatus , 2007, Journal of bacteriology.

[29]  J. Derrick,et al.  Structure of the PilM-PilN Inner Membrane Type IV Pilus Biogenesis Complex from Thermus thermophilus , 2011, The Journal of Biological Chemistry.

[30]  R. Sockett,et al.  Predation by Bdellovibrio bacteriovorus HD100 Requires Type IV Pili , 2007, Journal of bacteriology.

[31]  Kelly G. Aukema,et al.  Crystal structures of the pilus retraction motor PilT suggest large domain movements and subunit cooperation drive motility. , 2007, Structure.

[32]  S. de Bentzmann,et al.  Neglected but amazingly diverse type IVb pili. , 2012, Research in microbiology.

[33]  G. Jensen,et al.  Architecture of the type IVa pilus machine , 2016, Science.

[34]  Igor B. Zhulin,et al.  CDvist: a webserver for identification and visualization of conserved domains in protein sequences , 2015, Bioinform..

[35]  W. Hol,et al.  Structure of the cholera toxin secretion channel in its closed state , 2010, Nature Structural &Molecular Biology.

[36]  T. Mehta,et al.  Extracellular electron transfer via microbial nanowires , 2005, Nature.

[37]  John W Sedat,et al.  UCSF tomography: an integrated software suite for real-time electron microscopic tomographic data collection, alignment, and reconstruction. , 2007, Journal of structural biology.

[38]  S. Roseman,et al.  The Vibrio cholerae chitin utilization program. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[39]  J. Derrick,et al.  Structure and oligomerization of the PilC type IV pilus biogenesis protein from Thermus thermophilus , 2010, Proteins.

[40]  W. Hol,et al.  The X-ray structure of the type II secretion system complex formed by the N-terminal domain of EpsE and the cytoplasmic domain of EpsL of Vibrio cholerae. , 2005, Journal of molecular biology.

[41]  Igor B. Zhulin,et al.  The MiST2 database: a comprehensive genomics resource on microbial signal transduction , 2009, Nucleic Acids Res..

[42]  D. Dubnau,et al.  DNA uptake during bacterial transformation , 2004, Nature Reviews Microbiology.

[43]  J R Kremer,et al.  Computer visualization of three-dimensional image data using IMOD. , 1996, Journal of structural biology.

[44]  Koichiro Yamamoto,et al.  Gene Cluster for Assembly of Pilus Colonization Factor Antigen III of Enterotoxigenic Escherichia coli , 2001, Infection and Immunity.

[45]  José-Jesús Fernández,et al.  Fast tomographic reconstruction on multicore computers , 2011, Bioinform..

[46]  A. Krogh,et al.  Prediction of lipoprotein signal peptides in Gram‐negative bacteria , 2003, Protein science : a publication of the Protein Society.

[47]  R. Kulkarni,et al.  Direct Regulation by the Vibrio cholerae Regulator ToxT To Modulate Colonization and Anticolonization Pilus Expression , 2009, Infection and Immunity.

[48]  J. Derrick,et al.  Structure and assembly of an inner membrane platform for initiation of type IV pilus biogenesis , 2013, Proceedings of the National Academy of Sciences.

[49]  S. Lory,et al.  Structure-function and biogenesis of the type IV pili. , 1993, Annual review of microbiology.

[50]  E. Sokurenko,et al.  Genetic Diversity of the Gene Cluster Encoding Longus, a Type IV Pilus of Enterotoxigenic Escherichia coli , 2007, Journal of bacteriology.

[51]  G. Jensen,et al.  An Improved Cryogen for Plunge Freezing , 2008, Microscopy and Microanalysis.

[52]  Catherine M. Oikonomou,et al.  A new view into prokaryotic cell biology from electron cryotomography , 2016, Nature Reviews Microbiology.

[53]  Ronald K. Taylor,et al.  Effects of tcpB Mutations on Biogenesis and Function of the Toxin-Coregulated Pilus, the Type IVb Pilus of Vibrio cholerae , 2016, Journal of bacteriology.

[54]  E. Egelman,et al.  Structure of the Neisseria meningitidis Type IV pilus , 2016, Nature Communications.

[55]  John A. Tainer,et al.  Type IV pilus structure and bacterial pathogenicity , 2004, Nature Reviews Microbiology.

[56]  P. Manning,et al.  Translocation failure in a type-4 pilin operon: rfb and tcpT mutants in Vibrio cholerae. , 1997, Gene.

[57]  Maxuel O. Andrade,et al.  A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins , 2011, PLoS pathogens.

[58]  N. Bose,et al.  Identification of a TcpC-TcpQ Outer Membrane Complex Involved in the Biogenesis of the Toxin-Coregulated Pilus of Vibrio cholerae , 2005, Journal of bacteriology.

[59]  E. Egelman,et al.  Structure of the Vibrio cholerae Type IVb Pilus and stability comparison with the Neisseria gonorrhoeae type IVa pilus. , 2012, Journal of molecular biology.

[60]  S. Almo,et al.  Periplasmic domains of Pseudomonas aeruginosa PilN and PilO form a stable heterodimeric complex. , 2009, Journal of molecular biology.

[61]  John A Tainer,et al.  Type IV pilus structure by cryo-electron microscopy and crystallography: implications for pilus assembly and functions. , 2006, Molecular cell.

[62]  R. Owens,et al.  Structure and Assembly of a Trans-Periplasmic Channel for Type IV Pili in Neisseria meningitidis , 2012, PLoS pathogens.

[63]  W. Kühlbrandt,et al.  Figures and figure supplements Structure of a type IV pilus machinery in the open and closed state , 2015 .

[64]  W. Hol,et al.  Hexamers of the type II secretion ATPase GspE from Vibrio cholerae with increased ATPase activity. , 2013, Structure.

[65]  L. Craig,et al.  Structure of the cytoplasmic domain of TcpE, the inner membrane core protein required for assembly of the Vibrio cholerae toxin-coregulated pilus. , 2013, Acta crystallographica. Section D, Biological crystallography.

[66]  J. Mekalanos,et al.  Identification of a pilus colonization factor that is coordinately regulated with cholera toxin. , 1986, Annali Sclavo. Collana monografica.

[67]  J. Mattick.  Type IV pili and twitching motility. , 2002, Annual review of microbiology.