Membrane Assembly Platform Research Articles

Refining the Dynamic Network of T2SS Endopilus Tip Heterocomplex Combining cw-EPR and Nitroxide-GdIII Distance Measurements.

The type 2 secretion system (T2SS) is a bacterial nanomachine composed of an inner membrane assembly platform, an outer membrane pore and a dynamic endopilus. T2SS endopili are organized into a homo-multimeric body formed by the major pilin capped by a heterocomplex of four minor pilins. The first model of the T2SS endopilus was recently released, even if structural dynamics insights are still required to decipher the role of each protein in the full tetrameric complex. Here, we applied continuous-wave and pulse EPR spectroscopy using nitroxide-gadolinium orthogonal labelling strategies to investigate the hetero-oligomeric assembly of the minor pilins. Overall, our data are in line with the endopilus model even if they evidenced conformational flexibility and alternative orientations at local scale of specific regions of minor pilins. The integration of different labelling strategies and EPR experiments demonstrates the pertinence of this approach to investigate protein-protein interactions in such multiprotein heterocomplexes.

The periplasmic coiled coil formed by the assembly platform proteins PulL and PulM is critical for function of the Klebsiella type II secretion system

Bacteria use type II secretion systems (T2SS) to secrete to their surface folded proteins that confer diverse functions, from nutrient acquisition to virulence. In the Klebsiella species, T2SS-mediated secretion of pullulanase (PulA) requires assembly of a dynamic filament called the endopilus. The inner membrane assembly platform (AP) subcomplex is essential for endopilus assembly and PulA secretion. AP components PulL and PulM interact with each other through their C-terminal globular domains and transmembrane segments. Here, we investigated the roles of their periplasmic helices, predicted to form a coiled coil, in assembly and function of the PulL–PulM complex. PulL and PulM variants lacking these periplasmic helices were defective for interaction in the bacterial two-hybrid (BACTH) assay. Their functions in PulA secretion and assembly of PulG subunits into endopilus filaments were strongly reduced. Interestingly, deleting the cytoplasmic peptide of PulM nearly abolished the function of variant PulMΔN and its interaction with PulG, but not with PulL, in the BACTH assay. Nevertheless, PulL was specifically proteolyzed in the presence of the PulMΔN variant, suggesting that PulM N-terminal peptide stabilizes PulL in the cytoplasm. We discuss the implications of these results for the T2S endopilus and type IV pilus assembly mechanisms.

The structure and mechanism of the bacterial type II secretion system.

The type II secretion system (T2SS) is a multi-protein complex used by many bacteria to move substrates across their cell membrane. Substrates released into the environment serve as local and long-range effectors that promote nutrient acquisition, biofilm formation, and pathogenicity. In both animals and plants, the T2SS is increasingly recognized as a key driver of virulence. The T2SS spans the bacterial cell envelope and extrudes substrates through an outer membrane secretin channel using a pseudopilus. An inner membrane assembly platform and a cytoplasmic motor controls pseudopilus assembly. This microreview focuses on the structure and mechanism of the T2SS. Advances in cryo-electron microscopy are enabling increasingly elaborate sub-complexes to be resolved. However, key questions remain regarding the mechanism of pseudopilus extension and retraction, and how this is coupled with the choreography of the substrate moving through the secretion system. The T2SS is part of an ancient type IV filament superfamily that may have been present within the last universal common ancestor (LUCA). Overall, mechanistic principles that underlie T2SS function have implication for other closely related systems such as the type IV and tight adherence pilus systems.

Core architecture of a bacterial type II secretion system

Bacterial type II secretion systems (T2SSs) translocate virulence factors, toxins and enzymes across the cell outer membrane. Here we use negative stain and cryo-electron microscopy to reveal the core architecture of an assembled T2SS from the pathogen Klebsiella pneumoniae. We show that 7 proteins form a ~2.4 MDa complex that spans the cell envelope. The outer membrane complex includes the secretin PulD, with all domains modelled, and the pilotin PulS. The inner membrane assembly platform components PulC, PulE, PulL, PulM and PulN have a relative stoichiometric ratio of 2:1:1:1:1. The PulE ATPase, PulL and PulM combine to form a flexible hexameric hub. Symmetry mismatch between the outer membrane complex and assembly platform is overcome by PulC linkers spanning the periplasm, with PulC HR domains binding independently at the secretin base. Our results show that the T2SS has a highly dynamic modular architecture, with implication for pseudo-pilus assembly and substrate loading.

The traffic ATPase PilF interacts with the inner membrane platform of the DNA translocator and type IV pili from Thermusthermophilus.

A major driving force for the adaptation of bacteria to changing environments is the uptake of naked DNA from the environment by natural transformation, which allows the acquisition of new capabilities. Uptake of the high molecular weight DNA is mediated by a complex transport machinery that spans the entire cell periphery. This DNA translocator catalyzes the binding and splitting of double‐stranded DNA and translocation of single‐stranded DNA into the cytoplasm, where it is recombined with the chromosome. The thermophilic bacterium Thermus thermophilus exhibits the highest transformation frequencies reported and is a model system to analyze the structure and function of this macromolecular transport machinery. Transport activity is powered by the traffic ATPase PilF, a soluble protein that forms hexameric complexes. Here, we demonstrate that PilF physically binds to an inner membrane assembly platform of the DNA translocator, comprising PilMNO, via the ATP‐binding protein PilM. Binding to PilMNO or PilMN stimulates the ATPase activity of PilF ~ 2‐fold, whereas there is no stimulation when binding to PilM or PilN alone. A PilMK26A variant defective in ATP binding still binds PilF and, together with PilN, stimulates PilF‐mediated ATPase activity. PilF is unique in having three conserved GSPII (general secretory pathway II) domains (A–C) at its N terminus. Deletion analyses revealed that none of the GSPII domains is essential for binding PilMN, but GSPIIC is essential for PilMN‐mediated stimulation of ATP hydrolysis by PilF. Our data suggest that PilM is a coupling protein that physically and functionally connects the soluble motor ATPase PilF to the DNA translocator via the PilMNO assembly platform.

Restoration of cellular integrity following “ballistic” pronuclear exchange during Tetrahymena conjugation

During sexual reproduction or conjugation, ciliates form a specialized cell adhesion zone for the purpose of exchanging gametic pronuclei. Hundreds of individual membrane fusion events transform the adhesion zone into a perforated membrane curtain, the mating junction. Pronuclei from each mating partner are propelled through this fenestrated membrane junction by a web of short, cris-crossing microtubules. Pronuclear passage results in the formation of two breaches in the membrane junction. Following pronuclear exchange and karyogamy (fertilization), cells seal these twin membrane breaches thereby re-establishing cellular independence. This would seem like a straightforward problem: simply grow membrane in from the edges of each breach in a fashion similar to how animal cells “grow” their cytokinetic furrows or how plant cells construct a cell wall during mitosis. Serial section electron microscopy and 3-D electron tomography reveal that the actual mechanism is less straightforward. Each of the two membrane breaches transforms into a bowed membrane assembly platform. The resulting membrane protrusions continue to grow into the cytoplasm of the mating partner, traverse the cytoplasm in anti-parallel directions and make contact with the plasma membrane that flanks the mating junction. This investigation reveals the details of a novel, developmentally-induced mechanism of membrane disruption and restoration associated with pronuclear exchange and fertilization in the ciliate, Tetrahymena thermophila.

The role of core and accessory type IV pilus genes in natural transformation and twitching motility in the bacterium Acinetobacter baylyi.

Here we present an examination of type IV pilus genes associated with competence and twitching in the bacterium Acinetobacter baylyi (strain ADP1, BD413). We used bioinformatics to identify potential competence and twitching genes and their operons. We measured the competence and twitching phenotypes of the bioinformatically-identified genes. These results demonstrate that competence and twitching in A. baylyi both rely upon a core of the same type IV pilus proteins. The core includes the inner membrane assembly platform (PilC), a periplasmic assemblage connecting the inner membrane assembly platform to the secretin (ComM), a secretin (ComQ) and its associated pilotin (PilF) that assists with secretin assembly and localization, both cytoplasmic pilus retraction ATPases (PilU, PilT), and pilins (ComP, ComB, PilX). Proteins not needed for both competence and twitching are instead found to specialize in either of the two traits. The pilins are varied in their specialization with some required for either competence (FimT) and others for twitching (ComE). The protein that transports DNA across the inner membrane (ComA) specializes in competence, while signal transduction proteins (PilG, PilS, and PilR) specialize in twitching. Taken together our results suggest that the function of accessory proteins should not be based on homology alone. In addition the results suggest that in A. baylyi the mechanisms of natural transformation and twitching are mediated by the same set of core Type IV pilus proteins with distinct specialized proteins required for each phenotype. Finally, since competence requires multiple pilins as well as both pilus retraction motors PilU and PilT, this suggests that A. baylyi employs a pilus in natural transformation.

EssE Promotes Staphylococcus aureus ESS-Dependent Protein Secretion To Modify Host Immune Responses during Infection.

Staphylococcus aureus and other firmicutes evolved a specialized ESS (EsxA/ESAT-6-like secretion system) pathway for the secretion of small subsets of proteins lacking canonical signal peptides. The molecular mechanisms for ESS-dependent secretion and their functional purpose are still unknown. We demonstrate here that S. aureus EssE functions as a membrane assembly platform for elements of the secretion machinery and their substrates. Furthermore, S. aureus EssE-mediated secretion contributes to the production or the suppression of specific cytokines during host infection, thereby modifying immune responses toward this pathogen.

Structural and energetic basis of folded-protein transport by the FimD usher

Type 1 pili, produced by uropathogenic Escherichia coli (UPEC), are multisubunit fibers that play crucial roles in recognition of and adhesion to host tissues1. During pilus biogenesis, subunits are recruited to an outer membrane assembly platform, the FimD usher, which catalyzes their polymerization and mediates pilus secretion2. The recent crystal structure of an initiation complex provided insights into the initiation step of pilus biogenesis resulting in pore activation, but very little is known about the elongation steps that follow3. To address this question, we determined the structure of an elongation complex whereby FimD is traversed by the tip complex assembly composed of FimC:FimF:FimG:FimH. This structure reveals the conformational changes required to prevent backsliding of the nascent pilus through the FimD pore and also reveals unexpected properties of the usher pore. We show that the usher pore is energetically designed to facilitate substrate passage within the pore and that the circular binding interface between the pore lumen and the folded substrate plays a role in transport by defining a low energy pathway along which the nascent pilus polymer is guided during secretion.

Structural Homology between the C-Terminal Domain of the PapC Usher and Its Plug

P pili are extracellular appendages responsible for the targeting of uropathogenic Escherichia coli to the kidney. They are assembled by the chaperone-usher (CU) pathway of pilus biogenesis involving two proteins, the periplasmic chaperone PapD and the outer membrane assembly platform, PapC. Many aspects of the structural biology of the Pap CU pathway have been elucidated, except for the C-terminal domain of the PapC usher, the structure of which is unknown. In this report, we identify a stable and folded fragment of the C-terminal region of the PapC usher and determine its structure using both X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy. These structures reveal a beta-sandwich fold very similar to that of the plug domain, a domain of PapC obstructing its translocation domain. This structural similarity suggests similar functions in usher-mediated pilus biogenesis, playing out at different stages of the process. This structure paves the way for further functional analysis targeting surfaces common to both the plug and the C-terminal domain of PapC.

Fiber Formation across the Bacterial Outer Membrane by the Chaperone/Usher Pathway

Gram-negative pathogens commonly exhibit adhesive pili on their surfaces that mediate specific attachment to the host. A major class of pili is assembled via the chaperone/usher pathway. Here, the structural basis for pilus fiber assembly and secretion performed by the outer membrane assembly platform--the usher--is revealed by the crystal structure of the translocation domain of the P pilus usher PapC and single particle cryo-electron microscopy imaging of the FimD usher bound to a translocating type 1 pilus assembly intermediate. These structures provide molecular snapshots of a twinned-pore translocation machinery in action. Unexpectedly, only one pore is used for secretion, while both usher protomers are used for chaperone-subunit complex recruitment. The translocating pore itself comprises 24 beta strands and is occluded by a folded plug domain, likely gated by a conformationally constrained beta-hairpin. These structures capture the secretion of a virulence factor across the outer membrane of gram-negative bacteria.