scholarly journals A 20-residue peptide of the inner membrane protein OutC mediates interaction with two distinct sites of the outer membrane secretin OutD and is essential for the functional type II secretion system in Erwinia chrysanthemi

2010 ◽  
Vol 76 (4) ◽  
pp. 944-955 ◽  
Author(s):  
Frédéric H. Login ◽  
Markus Fries ◽  
Xiaohui Wang ◽  
Richard W. Pickersgill ◽  
Vladimir E. Shevchik
Keyword(s):  
Membrane Protein ◽  
Outer Membrane ◽  
Secretion System ◽  
Erwinia Chrysanthemi ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Functional Type ◽  
Inner Membrane ◽  
Type Ii Secretion System ◽  
Inner Membrane Protein

scholarly journals Multiple approaches towards understanding the type II secretion system

2014 ◽  
Vol 70 (a1) ◽  
pp. C577-C577
Author(s):  
Connie Lu ◽  
Young-un Park ◽  
Konstantin Korotkov ◽  
Wei Mi ◽  
Stewart Turley ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Secretion System ◽  
Crystal Formation ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Inner Membrane ◽  
Membrane Pore ◽  
Membrane Complex ◽  
Type Ii Secretion System ◽  
Folded Proteins

Transport of folded proteins across membranes is a feat accomplished by few biomacromolecular machines. One of the machineries able to do so is the sophisticated type II secretion system (T2SS). It can translocate key virulence factors from the bacterial periplasm into the lumen of the gut of the human host. A prime example is the secretion of cholera toxin by Vibrio cholerae. The T2SS consists of ~12 different proteins, most of these present in multiple copies, organized into three subassemblies: (i) the Inner Membrane Platform; (ii) the Pseudopilus in the periplasm, which acts most likely as a piston pushing exoproteins through the outer membrane pore; (iii) the Outer Membrane Complex, allowing passage of ~100 kDa folded proteins. We have determined crystal structures from more than a dozen T2SS domains, yet, a full understanding of the architecture and mechanism of action of the T2SS remains a formidable challenge. Our approaches include the use of "assistant-multimers" to promote recalcitrant multimer formation and of nanobodies to overcome reluctant crystal formation. The Inner Membrane Platform is interacting with the secretion ATPase GspE which most likely needs to be hexameric for full activity. Full-length GspE co-crystallized with its major partner, the cytoplasmic domain of GspL, revealed a tremendous flexibility of this ATPase, and, most unexpectedly, also the organization of the same linear arrangement of cyto-GspL domains throughout three entirely different crystal forms. Two very different hexamers of GspE were elucidated by linking the GspE subunit to the subunit of Hcp1, which successfully acted as an "assistant hexamer", inducing hexamer formation by GspE. The dodecameric nature of the ~ 850 kDa GspD, the major component of the Outer Membrane Complex, evident in earlier electron microscopy studies, was observed in the dodecameric ring-like helix in crystals of its N-terminal domain. The contacts between GspD and the inner-membrane protein GspC will be discussed as well as the remarkably frequent occurrence of dimers of Inner Membrane Platform domains. How dimers are co-assembled with an ATPase hexamer with C6 symmetry and the Outer Membrane Complex dodecamer with C12 symmetry remains one of the many fascinating outstanding questions of the T2SS.

scholarly journals Type II Secretion System Secretin PulD Localizes in Clusters in the Escherichia coli Outer Membrane

2008 ◽  
Vol 191 (1) ◽  
pp. 161-168 ◽  
Author(s):  
Nienke Buddelmeijer ◽  
Martin Krehenbrink ◽  
Frédéric Pecorari ◽  
Anthony P. Pugsley
Keyword(s):  
Escherichia Coli ◽  
Fluorescence Microscopy ◽  
Outer Membrane ◽  
Cellular Localization ◽  
Fluorescent Protein ◽  
Sodium Dodecyl ◽  
Secretion System ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Type Ii Secretion System

ABSTRACT The cellular localization of a chimera formed by fusing a monomeric red fluorescent protein to the C terminus of the Klebsiella oxytoca type II secretion system outer membrane secretin PulD (PulD-mCherry) in Escherichia coli was determined in vivo by fluorescence microscopy. Like PulD, PulD-mCherry formed sodium dodecyl sulfate- and heat-resistant multimers and was functional in pullulanase secretion. Chromosome-encoded PulD-mCherry formed fluorescent foci on the periphery of the cell in the presence of high (plasmid-encoded) levels of its cognate chaperone, the pilotin PulS. Subcellular fractionation demonstrated that the chimera was located exclusively in the outer membrane under these circumstances. A similar localization pattern was observed by fluorescence microscopy of fixed cells treated with green fluorescent protein-tagged affitin, which binds with high affinity to an epitope in the N-terminal region of PulD. At lower levels of (chromosome-encoded) PulS, PulD-mCherry was less stable, was located mainly in the inner membrane, from which it could not be solubilized with urea, and did not induce the phage shock response, unlike PulD in the absence of PulS. The fluorescence pattern of PulD-mCherry under these conditions was similar to that observed when PulS levels were high. The complete absence of PulS caused the appearance of bright and almost exclusively polar fluorescent foci.

scholarly journals Inner-membrane GspF of the bacterial type II secretion system is a dimeric adaptor mediating pseudopilus biogenesis

2018 ◽  
Author(s):  
Wouter Van Putte ◽  
Tatjana De Vos ◽  
Wim Van Den Broeck ◽  
Henning Stahlberg ◽  
Misha Kudryashev ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Virulence Factors ◽  
Protein Complex ◽  
Secretion System ◽  
Structural Basis ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Secretion Systems ◽  
Inner Membrane ◽  
Type Ii Secretion System

AbstractThe type II secretion system (T2SS), a protein complex spanning the bacterial envelope, is pivotal to bacterial pathogenicity. Central to T2SS function is the extrusion of protein cargos from the periplasm into the extracellular environment mediated by a pseudopilus and motorized by a cytosolic ATPase. GspF, an inner-membrane component of T2SS has long been considered to be a key player in this process, yet the structural basis of its role had remained elusive. Here, we employed single-particle electron microscopy based on XcpS (GspF) from the T2SS of pathogenicP. aeruginosastabilized by a nanobody, to show that XcpS adopts a dimeric structure mediated by its transmembrane helices. This assembly matches in terms of overall organization and dimensions the basal inner-membrane cassette of a T2SS machinery. Thus, GspF is poised to serve as an adaptor involved in the mediation of propeller-like torque generated by the motor ATPase to the secretion pseudopilus.Non-technical author summaryAntibiotic resistance by bacteria imposes a worldwide threat that can only be overcome through a multi-front approach: preventive actions and the parallel development of novel molecular strategies to combat antibiotic resistance mechanisms. One such strategy might focus on antivirulence drugs that prevent host invasion and spreading by pathogenic bacteria, without shutting down essential functions related to bacterial survival. The rationale behind such an approach is that it might limit selective pressure leading to slower evolutionary rates of resistant bacterial strains. Bacterial secretion systems are an appropriate target for such therapeutic approaches as their impairment will inhibit the secretion of a multitude of virulence factors. This study focuses on the structural characterization of one of the proteins residing in the inner-membrane cassette of the type II secretion system (T2SS), a multi-protein complex in multiple opportunistic pathogens that secretes virulence factors. The targeted protein is essential for the assembly of the pseudopilus, a rod-like supramolecular structure that propels the secretion of virulence factors by pathogenic Gram-negative bacteria. Our study crucially complements growing evidence supporting a rotational assembly model of the pseudopilus and contributes to a better understanding of the functioning of the T2SS and the related secretion systems. We envisage that such knowledge will facilitate targeting of these systems for therapeutic purposes.

scholarly journals Signal Recognition Particle-Dependent Inner Membrane Targeting of the PulG Pseudopilin Component of a Type II Secretion System

2006 ◽  
Vol 189 (5) ◽  
pp. 1783-1793 ◽  
Author(s):  
Olivera Francetic ◽  
Nienke Buddelmeijer ◽  
Shawn Lewenza ◽  
Carol A. Kumamoto ◽  
Anthony P. Pugsley
Keyword(s):  
Signal Sequence ◽  
Klebsiella Oxytoca ◽  
Signal Recognition Particle ◽  
Secretion System ◽  
Membrane Targeting ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Signal Recognition ◽  
Inner Membrane ◽  
Type Ii Secretion System

ABSTRACT The pseudopilin PulG is an essential component of the pullulanase-specific type II secretion system from Klebsiella oxytoca. PulG is the major subunit of a short, thin-filament pseudopilus, which presumably elongates and retracts in the periplasm, acting as a dynamic piston to promote pullulanase secretion. It has a signal sequence-like N-terminal segment that, according to studies with green and red fluorescent protein chimeras, anchors unassembled PulG in the inner membrane. We analyzed the early steps of PulG inner membrane targeting and insertion in Escherichia coli derivatives defective in different protein targeting and export factors. The β-galactosidase activity in strains producing a PulG-LacZ hybrid protein increased substantially when the dsbA, dsbB, or all sec genes tested except secB were compromised by mutations. To facilitate analysis of native PulG membrane insertion, a leader peptidase cleavage site was engineered downstream from the N-terminal transmembrane segment (PrePulG*). Unprocessed PrePulG* was detected in strains carrying mutations in secA, secY, secE, and secD genes, including some novel alleles of secY and secD. Furthermore, depletion of the Ffh component of the signal recognition particle (SRP) completely abolished PrePulG* processing, without affecting the Sec-dependent export of periplasmic MalE and RbsB proteins. Thus, PulG is cotranslationally targeted to the inner membrane Sec translocase by SRP.

scholarly journals A putative multicopper protein secreted by an atypical type II secretion system involved in the reduction of insoluble electron acceptors in Geobacter sulfurreducens

Microbiology ◽  
2006 ◽  
Vol 152 (8) ◽  
pp. 2257-2264 ◽  
Author(s):  
Teena Mehta ◽  
Susan E. Childers ◽  
Richard Glaven ◽  
Derek R. Lovley ◽  
Tünde Mester
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Secretion System ◽  
Geobacter Sulfurreducens ◽  
Extracellular Electron Transfer ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Oxide Reduction ◽  
Type Ii Secretion System

Extracellular electron transfer onto Fe(III) oxides in Geobacter sulfurreducens is considered to require proteins that must be exported to the outer surface of the cell. In order to investigate this, the putative gene for OxpG, the pseudopilin involved in a type II general secretion pathway of Gram-negative bacteria, was deleted. The mutant was unable to grow with insoluble Fe(III) oxide as the electron acceptor. Growth on soluble Fe(III) was not affected. An analysis of proteins that accumulated in the periplasm of the oxpG mutant, but not in the wild-type, led to the identification of a secreted protein, OmpB. OmpB is predicted to be a multicopper protein, with highest homology to the manganese oxidase, MofA, from Leptothrix discophora. OmpB contains a potential Fe(III)-binding site and a fibronectin type III domain, suggesting a possible role for this protein in accessing Fe(III) oxides. OmpB was localized to the membrane fraction of G. sulfurreducens and in the supernatant of growing cultures, consistent with the type II secretion system exporting OmpB. A mutant in which ompB was deleted had the same phenotype as the oxpG mutant, suggesting that the failure to export OmpB was responsible for the inability of the oxpG-deficient mutant to reduce Fe(III) oxide. This is the first report that proposes a role for a multicopper oxidase-like protein in an anaerobic organism. These results further emphasize the importance of outer-membrane proteins in Fe(III) oxide reduction and suggest that outer-membrane proteins other than c-type cytochromes are required for Fe(III) oxide reduction in Geobacter species.

scholarly journals The GspLM inner membrane complex from the bacterial type II secretion system is a dimer of dimers and interacts with the system ATPase with high affinity

2020 ◽  
Author(s):  
Aleksandra Fulara ◽  
Ioanna Ramou ◽  
Savvas N. Savvides
Keyword(s):  
Multidrug Resistant ◽  
Secretion System ◽  
Klebsiella Pneumonia ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Acinetobacter Baumanii ◽  
Inner Membrane ◽  
Gram Negative ◽  
High Affinity ◽  
Type Ii Secretion System

ABSTACTThe type II secretion system (T2SS) is a multiprotein machinery spanning the diderm of gram-negative bacteria. T2SS contributes to the virulence of numerous gram-negative pathogens, including the multidrug resistant species Pseudomonas aeruginosa, Acinetobacter baumanii, Klebsiella pneumonia and Vibrio cholerae. Even though the T2SS has been studied extensively over the past three decades, our understanding of the molecular basis of its biogenesis and of its overall structure still remains unclear. Here we show that the core component of the inner membrane platform, the GspLM membrane protein complex, can be isolated as a dimer of dimers. Importantly, the complex is able to bind the T2SS ATPase, GspE, with high affinity. Finally, we have developed single domain VHH camelid antibodies (nanobodies) against the GspLM complex and have identified a nanobody that effectively prevents the cytoplasmic domain of GspL, GspLcyto, from binding to GspE. Our findings suggest that the T2SS ATPase is permanently associated with the inner membrane platform and that the GspELM complex should be considered as a key subassembly for the biogenesis of the T2SS apparatus.

scholarly journals Alterations in Peptidoglycan Cross-Linking Suppress the Secretin Assembly Defect Caused by Mutation of GspA in the Type II Secretion System

2017 ◽  
Vol 199 (8) ◽  
Author(s):  
Elizabeth M. Vanderlinde ◽  
Timothy G. Strozen ◽  
Sara B. Hernández ◽  
Felipe Cava ◽  
S. Peter Howard
Keyword(s):  
Outer Membrane ◽  
Secretion System ◽  
Cross Linking ◽  
Type Ii Secretion ◽  
Type Ii ◽  
Assembly Model ◽  
Gram Negative ◽  
Content Type ◽  
E Coli ◽  
Type Ii Secretion System

ABSTRACT In Gram-negative bacteria, the peptidoglycan (PG) cell wall is a significant structural barrier for outer membrane protein assembly. In Aeromonas hydrophila, outer membrane multimerization of the type II secretion system (T2SS) secretin ExeD requires the function of the inner membrane assembly factor complex ExeAB. The putative mechanism of the complex involves the reorganization of PG and localization of ExeD, whereby ExeA functions by interacting with PG to form a site for secretin assembly and ExeB forms an interaction with ExeD. This mechanism led us to hypothesize that increasing the pore size of PG would circumvent the requirement for ExeA in the assembly of the ExeD secretin. Growth of A. hydrophila in 270 mM Gly reduced PG cross-links by approximately 30% and led to the suppression of secretin assembly defects in exeA strains and in those expressing ExeA mutants by enabling localization of the secretin in the outer membrane. We also established a heterologous ExeD assembly system in Escherichia coli and showed that ExeAB and ExeC are the only A. hydrophila proteins required for the assembly of the ExeD secretin in E. coli and that ExeAB-independent assembly of ExeD can occur upon overexpression of the d,d-carboxypeptidase PBP 5. These results support an assembly model in which, upon binding to PG, ExeA induces multimerization and pore formation in the sacculus, which enables ExeD monomers to interact with ExeB and assemble into a secretin that both is inserted in the outer membrane and crosses the PG layer to interact with the inner membrane platform of the T2SS. IMPORTANCE The PG layer imposes a strict structural impediment for the assembly of macromolecular structures that span the cell envelope and serve as virulence factors in Gram-negative species. This work revealed that by decreasing PG cross-linking by growth in Gly, the absolute requirement for the PG-binding activity of ExeA in the assembly of the ExeD secretin was alleviated in A. hydrophila. In a heterologous assembly model in E. coli, expression of the carboxypeptidase PBP 5 could relieve the requirement for ExeAB in the assembly of the ExeD secretin. These results provide some mechanistic details of the ExeAB assembly complex function, in which the PG-binding and oligomerization functions of ExeAB are used to create a pore in the PG that is required for secretin assembly.

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