Structure and Function of the Dot/Icm T4SS

Dutka, Przemysław; Liu, Yuxi; Maggi, Stefano; Ghosal, Debnath; Wang, Jue; Carter, Stephen D.; Wei, Zhao; Vijayrajratnam, Sukhithasri; Vogel, Joseph P.; Jensen, Grant J.

doi:10.1101/2023.03.22.533729

Cited by 3 publications

(3 citation statements)

References 92 publications

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“…Assembled CagC pentamers may serve as a scaffold for biogenesis of heteromeric complexes that incorporate the proposed VirB5-like minor pilin CagL 70,73,74 to form the cone-shaped stalk density that bridges the IMC and PRC 17,37 . Stalk-like structures with similar sizes and geometries have been identified in cryo-ET atomic models of other T4SS machineries including the R388 conjugative system 9 and the L. pneumophila dot/icm T4SS [75][76][77] . Alternatively, CagC may form the rod structure observed spanning the periplasm and originating from inner membrane platforms within the cag T4SS architecture in situ 37 .…”

Section: Discussionmentioning

confidence: 78%

Architectural asymmetry enables DNA transport through theHelicobacter pylori cagtype IV secretion system

Ryan

Damke

Bryant

et al. 2023

Preprint

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Structural asymmetry within secretion system architecture is fundamentally important for apparatus diversification and biological function. However, the mechanism by which symmetry mismatch contributes to nanomachine assembly and interkingdom effector translocation are undefined. Here, we show that architectural asymmetry orchestrates dynamic substrate selection and enables trans-kingdom DNA conjugation through theHelicobacter pylori cagtype IV secretion system (cagT4SS). Structural analyses of asymmetric units within thecagT4SS periplasmic ring complex (PRC) revealed intermolecular π-π stacking interactions that coordinate DNA binding and license trans-kingdom conjugation without disrupting the translocation of protein and peptidoglycan effector molecules. Additionally, we identified a novel proximal translocation channel gating mechanism that regulates cargo loading and governs substrate transport across the outer membrane. We thus propose a model whereby the organization and geometry of architectural symmetry mismatch exposes π−π interfaces within the PRC to facilitate DNA transit through thecagT4SS translocation channel.

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Section: Discussionmentioning

confidence: 78%

Architectural asymmetry enables DNA transport through theHelicobacter pylori cagtype IV secretion system

Ryan

Damke

Bryant

et al. 2023

Preprint

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“…This could be achieved by lateral access in the plane of the membrane or after membrane extraction of the TMD-effectors towards the periplasmic or cytoplasmic side. As the T4bSS core secretion channel is fenced by TMDs, a lateral access is unlikely [9]. Extraction of transmembrane proteins from the lipid bilayer is highly energy-consuming.…”

Section: Tmd-effectors Exhibit Short Periplasmic Loopsmentioning

confidence: 99%

“…like L. pneumophila to infect alveolar macrophages, leading to Legionnaires' disease. Evolutionary and structurally related to bacterial conjugation machines, the T4bSS of Legionella comprises at least 32 different structural components of which most are critical for secretion function of this molecular machine [6][7][8][9]. The T4bSS consists of a core complex spanning the entire bacterial cell envelope and of a type IV coupling complex (T4CC) in the inner membrane.…”

Section: Introductionmentioning

confidence: 99%

The T4bSS ofLegionellafeatures a two-step secretion pathway with an inner membrane intermediate for secretion of transmembrane effectors

Malmsheimer,

Grin,

Bohn

et al. 2024

Preprint

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To promote intracellular survival and infection, Legionella spp. translocate hundreds of effector proteins into eukaryotic host cells using a type IV b protein secretion system (T4bSS). T4bSS are well known to translocate soluble as well as transmembrane domain-containing effector proteins (TMD-effectors) but the mechanisms of secretion are still poorly understood. Herein we investigated the secretion of hydrophobic TMD-effectors, of which about 80 were previously reported to be encoded by L. pneumophila. A proteomic analysis of fractionated membranes revealed that TMD-effectors are targeted to and inserted into the bacterial inner membranes of L. pneumophila independent of the presence of a functional T4bSS. While the T4bSS chaperones IcmS and IcmW were critical for secretion of all tested TMD-effectors, they did not influence inner membrane targeting of these proteins. As for soluble effector proteins, translocation of TMD-effectors into host cells depended on a C-terminal secretion signal and this signal needed to be presented towards the cytoplasmic side of the inner membrane. A different secretion behavior of TMD- and soluble effectors and the need for small periplasmic loops within TMD-effectors provided strong evidence that TMD-effectors are secreted in a two-step secretion process: Initially, an inner membrane intermediate is formed, that is extracted towards the cytoplasmic side, possibly by the help of the type IV coupling protein complex and subsequently secreted into eukaryotic host cells by the T4bSS core complex. Overall, our study highlights the amazing versatility of T4bSS to secrete soluble and TMD-effectors from different subcellular locations of the bacterial cell.

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Membrane association and polar localization of the Legionella pneumophila T4SS DotO ATPase mediated by two nonredundant receptors

Vijayrajratnam,

Milek,

Maggi

et al. 2024

Proc. Natl. Acad. Sci. U.S.A.

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The Legionella pneumophila Dot/Icm type IVB secretion system (T4BSS) is a large, multisubunit complex that exports a vast array of substrates into eukaryotic host cells. DotO, a distant homolog of the T4ASS ATPase VirB4, associates with the bacterial inner membrane despite lacking hydrophobic transmembrane domains. Employing a genetic approach, we found DotO’s membrane association is mediated by three inner-membrane Dot/Icm components, IcmT, and a combined DotJ–DotI complex (referred to as DotJI). Although deletion of icmT or dotJI individually does not affect DotO’s membrane association, the simultaneous inactivation of all three genes results in increased amounts of soluble DotO. Nevertheless, deleting each receptor separately profoundly affects positioning of DotO, disrupting its link with the Dot/Icm complex at the bacterial poles, rendering the receptors nonredundant. Furthermore, a collection of dotO point mutants that we isolated established that DotO’s N-terminal domain interacts with the membrane receptors and is involved in dimerization, whereas DotO’s C-terminal ATPase domain primarily contributes to the protein’s formation of oligomers. Modeling data revealed the complex interaction between DotO and its receptors is responsible for formation of DotO’s unique “hexamer of dimers” configuration, which is a defining characteristic of VirB4 family members.

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Structure and Function of the Dot/Icm T4SS

Cited by 3 publications

References 92 publications

Architectural asymmetry enables DNA transport through theHelicobacter pylori cagtype IV secretion system

Architectural asymmetry enables DNA transport through theHelicobacter pylori cagtype IV secretion system

The T4bSS ofLegionellafeatures a two-step secretion pathway with an inner membrane intermediate for secretion of transmembrane effectors

Membrane association and polar localization of the Legionella pneumophila T4SS DotO ATPase mediated by two nonredundant receptors

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