Unraveling the Self-Assembly of the
            <i>Pseudomonas aeruginosa</i>
            XcpQ Secretin Periplasmic Domain Provides New Molecular Insights into Type II Secretion System Secreton Architecture and Dynamics

Douzi, Badreddine; Trinh, Nhung Thi Trang; Michel-Souzy, Sandra; Desmyter, Aline; Ball, Geneviève; Barbier, Pascale; Kosta, Artémis; Durand, Éric; Forest, Katrina T.; Cambillau, Christian; Roussel, Alain; Voulhoux, Romé

doi:10.1128/mbio.01185-17

Cited by 21 publications

(30 citation statements)

References 47 publications

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“…These intimate interactions raise a further structural dilemma, in that substrates access the T2SS in the periplasm and must therefore interpose between the seals formed by the N domains of the secretin ( 5 , 7 ). Given the recent findings of Douzi et al ( 21 ), in combination with our data, we suggest a plausible scenario that would resolve this conundrum ( Fig. 2E ).…”

Section: Observationsupporting

confidence: 83%

Structural Basis of Type 2 Secretion System Engagement between the Inner and Outer Bacterial Membranes

Hay

Belousoff

Lithgow

2017

mBio

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Sophisticated nanomachines are used by bacteria for protein secretion. In Gram-negative bacteria, the type 2 secretion system (T2SS) is composed of a pseudopilus assembly platform in the inner membrane and a secretin complex in the outer membrane. The engagement of these two megadalton-sized complexes is required in order to secrete toxins, effectors, and hydrolytic enzymes. Pseudomonas aeruginosa has at least two T2SSs, with the ancestral nanomachine having a secretin complex composed of XcpQ. Until now, no high-resolution structural information was available to distinguish the features of this Pseudomonas-type secretin, which varies greatly in sequence from the well-characterized Klebsiella-type and Vibrio-type secretins. We have purified the ~1-MDa secretin complex and analyzed it by cryo-electron microscopy. Structural comparisons with the Klebsiella-type secretin complex revealed a striking structural homology despite the differences in their sequence characteristics. At 3.6-Å resolution, the secretin complex was found to have 15-fold symmetry throughout the membrane-embedded region and through most of the domains in the periplasm. However, the N1 domain and N0 domain were not well ordered into this 15-fold symmetry. We suggest a model wherein this disordering of the subunit symmetry for the periplasmic N domains provides a means to engage with the 6-fold symmetry in the inner membrane platform, with a metastable engagement that can be disrupted by substrate proteins binding to the region between XcpP, in the assembly platform, and the XcpQ secretin.

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Section: Observationsupporting

confidence: 83%

Structural Basis of Type 2 Secretion System Engagement between the Inner and Outer Bacterial Membranes

Hay

Belousoff

Lithgow

2017

mBio

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“…For CbpD, the extraction and purification protocols of the periplasmic material were described previously (5). Purity and quality of the purified proteins were checked by analyz- (dashed red arrows).…”

Section: Effector Recognition In Type 2 Secretionmentioning

confidence: 99%

“…In addition, it has been shown that the TMHR domain of GspC is indirectly involved in effector recognition specificity in Pseudomonas aeruginosa (17). Various GspD N-domains are also involved in effector binding, depending on the organism (5,24,33,34,36). Finally, a direct interaction has also been reported between the effector and the pseudopilus tip constituted by the periplasmic domains of GspHIJK (34).…”

mentioning

confidence: 99%

Direct interactions between the secreted effector and the T2SS components GspL and GspM reveal a new effector-sensing step during type 2 secretion

Michel-Souzy

Douzi

Cadoret

et al. 2018

Journal of Biological Chemistry

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In many Gram-negative bacteria, the type 2 secretion system (T2SS) plays an important role in virulence because of its capacity to deliver a large amount of fully folded protein effectors to the extracellular milieu. Despite our knowledge of most T2SS components, the mechanisms underlying effector recruitment and secretion by the T2SS remain enigmatic. Using complementary biophysical and biochemical approaches, we identified here two direct interactions between the secreted effector CbpD and two components, XcpY L and XcpZ M , of the T2SS assembly platform (AP) in the opportunistic pathogen Pseudomonas aeruginosa. Competition experiments indicated that CbpD binding to XcpY L is XcpZ M -dependent, suggesting sequential recruitment of the effector by the periplasmic domains of these AP components. Using a bacterial two-hybrid system, we then tested the influence of the effector on the AP protein-protein interaction network. Our findings revealed that the presence of the effector modifies the AP interactome and, in particular, induces XcpZ M homodimerization and increases the affinity between XcpY L and XcpZ M . The observed direct relationship between effector binding and T2SS dynamics suggests an additional synchronizing step during the type 2 secretion process, where the activation of the AP of the T2SS nanomachine is triggered by effector binding.Type IV filament (Tff) 3 nanomachines are membrane-embedded macromolecular complexes organized around a char-acteristic helical pilus-like structure emerging from an assembly platform at the cytoplasmic membrane (1). Tffs are widespread in prokaryotes where they fulfill diverse cellular functions, in particular the type 2 secretion system (T2SS) that is found in many pathogenic Gram-negative proteobacteria (2). It is dedicated to the secretion of large folded periplasmic exoproteins via a piston-like apparatus called the secreton. Secretons are constituted by 12-15 different components organized into three subcomplexes: the outer-membrane pore belonging to the secretin family, the assembly platform (AP) or motor of the system, and the typical pseudopilus emerging from the AP up to the secretin pore (for the most recent reviews, see Refs. 3 and 4).T2SS secretins are homomultimers assembled into a giant gated ␤-barrel pore in the outer membrane connected to the AP by an N-terminal periplasmic extension (5-7). The Tff-specific structure of the T2SS is called the pseudopilus. It is constituted by the helical assembly of the five pseudopilins of the system. Chronologically, the four minor pseudopilins, GspHIJK, are first assembled by the AP and thus form the head of the structure. This step is followed by the addition, from the bottom of the filament, of the major pseudopilin GspG into a pseudopilus (8 -11). Pseudopilus assembly is energized and promoted by the AP, which is constituted by the oligomeric assembly of four membrane proteins, including the three bitopic proteins GspC, GspL, and GspM and the integral membrane protein GspF. The energy for pseudopilus assembly is p...

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“…Understanding the instrinsic oligomeric states of protein subassemblies of the bacterial T2SS is necessary for deciphering the architectures and assembly principles of the functional T2SS apparatus. With the exception of the outer membrane secretin [34][35][36], experimental data on the oligomeric state of the membrane-embedded components of the T2SS is scarce. Therefore, we addressed the question of the oligomeric state of the purified GspLM complex via SEC-MALLS.…”

Section: Gsplm Is a Dimer Of Dimers In Solutionmentioning

confidence: 99%

“…A nanobody consists of a single monomeric variable domain of a heavy-chain only antibody, of 12-15 kDa. Their high antigen specificity and low molecular weight together with ease of expression and purification make such antibody fragments very well suited for the structural characterization of membrane proteins and protein complexes [35,[41][42][43]. Following immunizations with the GspLM complex we obtained eight nanobody classes and opted to determine the binding epitopes of representatives from each class.…”

Section: Development and Characterization Of Vhh Single Domain Antibomentioning

confidence: 99%

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

Fulara

Ramou

Savvides

2020

Preprint

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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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Unraveling the Self-Assembly of the Pseudomonas aeruginosa XcpQ Secretin Periplasmic Domain Provides New Molecular Insights into Type II Secretion System Secreton Architecture and Dynamics

Cited by 21 publications

References 47 publications

Structural Basis of Type 2 Secretion System Engagement between the Inner and Outer Bacterial Membranes

Structural Basis of Type 2 Secretion System Engagement between the Inner and Outer Bacterial Membranes

Direct interactions between the secreted effector and the T2SS components GspL and GspM reveal a new effector-sensing step during type 2 secretion

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

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