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

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.

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The Structure of the Cytoplasmic Domain of EpsL, An Inner Membrane Component of the Type II Secretion System of Vibrio cholerae: An Unusual Member of the Actin-like ATPase Superfamily

Journal of Molecular Biology ◽

10.1016/j.jmb.2004.09.062 ◽

2004 ◽

Vol 344 (3) ◽

pp. 619-633 ◽

Cited By ~ 47

Author(s):

Jan Abendroth ◽

Michael Bagdasarian ◽

Maria Sandkvist ◽

Wim G.J. Hol

Keyword(s):

Vibrio Cholerae ◽

Cytoplasmic Domain ◽

Secretion System ◽

Type Ii Secretion ◽

Type Ii ◽

Membrane Component ◽

Inner Membrane ◽

Type Ii Secretion System

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Insights into minor pseudopilin complexes of the Type II secretion system

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314094145 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C585-C585

Author(s):

Yichen Zhang ◽

Frederick Faucher ◽

Zongchao Jia

Keyword(s):

Virulence Factors ◽

Secretion System ◽

Stable Complex ◽

Type Ii Secretion ◽

Type Ii ◽

Binding Model ◽

Binary And Ternary Complexes ◽

Type Ii Secretion System ◽

Wide Range ◽

Binding Mechanisms

The type II secretion system (T2SS) is sophisticated multiprotein machinery that enables Gram-negative pathogens to secrete a wide range of exoproteins, named virulence factors, into the extracellular environments. In Pseudomonas aeruginosa, the Xcp T2SS is responsible for secreting many virulence factors that induce severe infections. In T2SS, the recognition and binding of secreted exoproteins are conducted by a structure called the pseudopilius tip, which is formed by four minor pseudopilins, including XcpU, XcpV, XcpW and XcpX. These minor pseudopilins form a quaternary complex, which is also involved in the initiation and regulation of the pseudopilus assembly. Although individual structures of these four pseudopilins have been revealed in different organisms, the substrate recognition and binding mechanisms have not been clearly elucidated due to the lack of systematic studies on the whole structures of several complexes formed by these pseudopilins. As a result, the understanding of the structures of these protein complexes will provide useful information for unveiling the mystery of the recognition and binding mechanisms. The establishment of the substrate binding model requires the preparation of stable complex(es) of substrates and certain minor pseudopilin(s). In this work, we aim to gradually elucidate the secretion mechanisms by assembling each component to build up the whole architecture. The structure of XcpV in complex with XcpW has been determined, and other complexes, especially the XcpU-containing binary and ternary complexes, have been stably established and purified. The identification of these complex structures will significantly promote our understandings of the type II secretion mechanisms.

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

Journal of Bacteriology ◽

10.1128/jb.01230-06 ◽

2006 ◽

Vol 189 (5) ◽

pp. 1783-1793 ◽

Cited By ~ 51

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.

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Multiple approaches towards understanding the type II secretion system

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314094224 ◽

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.

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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

10.1101/2020.03.20.999888 ◽

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.

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