The <i>Legionella pneumophila</i> IcmS–LvgA protein complex is important for Dot/Icm‐dependent intracellular growth

Vincent, Carr D.; Vogel, Joseph P.

doi:10.1111/j.1365-2958.2006.05243.x

Cited by 96 publications

(65 citation statements)

References 53 publications

(129 reference statements)

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“…Previous work indicated that the IcmS/W complex binds to several different IDTSs, and in the case of SidG, this appears to alter the conformational state of the protein (34). Furthermore, IcmS is necessary for the stability of the T4BSS coupling protein DotL and binds directly to DotL (32,64). Therefore, if folded DHFR precludes IcmS from binding the IDTS fusions, the folded moiety could restrict the substrate from forming a translocation-competent conformation with IcmS/W.…”

Section: Low-level Translocation Of Dhfr-mavu Requires Icms Thementioning

confidence: 99%

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Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System

2013

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Legionella pneumophila uses the Icm/Dot type 4B secretion system (T4BSS) to deliver translocated protein substrates to the host cell, promoting replication vacuole formation. The conformational state of the translocated substrates within the bacterial cell is unknown, so we sought to determine if folded substrates could be translocated via this system. Fusions of L. pneumophila Icm/ Dot-translocated substrates (IDTS) to dihydrofolate reductase (DHFR) or ubiquitin (Ub), small proteins known to fold rapidly, resulted in proteins with low translocation efficiencies. The folded moieties did not cause increased aggregation of the IDTS and did not impede interaction with the adaptor protein complex IcmS/IcmW, which is thought to form a soluble complex that promotes translocation. The translocation defect was alleviated with a Ub moiety harboring mutations known to destabilize its structure, indicating that unfolded proteins are preferred substrates. Real-time analysis of translocation, following movement during the first 30 min after bacterial contact with host cells, revealed that the folded moiety caused a kinetic defect in IDTS translocation. Expression of an IDTS fused to a folded moiety interfered with the translocation of other IDTS, consistent with it causing a blockage of the translocation channel. Furthermore, the folded protein fusions also interfered with intracellular growth, consistent with inefficient or impaired translocation of proteins critical for L. pneumophila intracellular growth. These studies indicate that substrates of the Icm/Dot T4SS are translocated to the host cytosol in an unfolded conformation and that folded proteins are stalled within the translocation channel, impairing the function of the secretion system.

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Section: Low-level Translocation Of Dhfr-mavu Requires Icms Thementioning

confidence: 99%

“…IcmS/W adaptor complex is necessary for translocation of a large subset of L. pneumophila effectors (63,64). Previous work indicated that the IcmS/W complex binds to several different IDTSs, and in the case of SidG, this appears to alter the conformational state of the protein (34).…”

Section: Low-level Translocation Of Dhfr-mavu Requires Icms Thementioning

confidence: 99%

Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System

2013

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“…However, a study by Vincent et al (15,37) indicated that other Dot/Icm component proteins, such as T4SS chaperones IcmS and IcmW, may be equally promising baits for this type of screen. Our bioinformatics approach achieved significant success in predicting C. burnetii type IV substrates, as 26 of 42 predicted candidates were confirmed as Dot/Icm substrates (Table S3).…”

Section: Discussionmentioning

confidence: 99%

Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii

Chen

Banga²,

Mertens³

et al. 2010

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

176

259

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Coxiella burnetii is an obligate intracellular bacterial pathogen responsible for acute and chronic Q fever. This bacterium harbors a type IV secretion system (T4SS) highly similar to the Dot/Icm of Legionella pneumophila that is believed to be essential for its infectivity. Protein substrates of the Coxiella T4SS are predicted to facilitate the biogenesis of a phagosome permissive for its intracellular growth. However, due to the lack of genetic systems, protein transfer by the C. burnetii Dot/Icm has not been demonstrated. In this study, we report the identification of 32 substrates of the C. burnetii Dot/Icm system using a fluorescence-based β-lactamase (TEM1) translocation assay as well as the calmodulin-dependent adenylate cyclase (CyaA) assay in the surrogate host L. pneumophila. Notably, 26 identified T4SS substrates are hypothetical proteins without predicted function. Candidate secretion substrates were obtained by using (i) a genetic screen to identify C. burnetii proteins interacting with DotF, a component of the T4SS, and (ii) bioinformatic approaches to retrieve candidate genes that harbor characteristics associated with previously reported substrates of the Dot/Icm system from both C. burnetii and L. pneumophila. Moreover, we have developed a shuttle plasmid that allows the expression of recombinant proteins in C. burnetii as TEM fusion products. Using this system, we demonstrated that a Dot/Icm substrate identified with L. pneumophila was also translocated by C. burnetii in a process that requires its C terminus, providing direct genetic evidence of a functional T4SS in C. burnetii.effectors | protein translocation | transporter

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“…At least three T4SS chaperones, IcmS, IcmW, and LvgA, are required for the translocation of effector proteins (44,203,271). Interestingly, IcmS forms heterodimeric interactions with IcmW or LvgA, and the resulting chaperone complexes interact with and mediate the translocation of many effectors through the Dot/Icm T4SS.…”

Section: Contributions Of Accessory Proteins To Substrate Recognitionmentioning

confidence: 99%

Biological Diversity of Prokaryotic Type IV Secretion Systems

Martı́nez

Christie

2009

Microbiol Mol Biol Rev

620

780

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SUMMARY Type IV secretion systems (T4SS) translocate DNA and protein substrates across prokaryotic cell envelopes generally by a mechanism requiring direct contact with a target cell. Three types of T4SS have been described: (i) conjugation systems, operationally defined as machines that translocate DNA substrates intercellularly by a contact-dependent process; (ii) effector translocator systems, functioning to deliver proteins or other macromolecules to eukaryotic target cells; and (iii) DNA release/uptake systems, which translocate DNA to or from the extracellular milieu. Studies of a few paradigmatic systems, notably the conjugation systems of plasmids F, R388, RP4, and pKM101 and the Agrobacterium tumefaciens VirB/VirD4 system, have supplied important insights into the structure, function, and mechanism of action of type IV secretion machines. Information on these systems is updated, with emphasis on recent exciting structural advances. An underappreciated feature of T4SS, most notably of the conjugation subfamily, is that they are widely distributed among many species of gram-negative and -positive bacteria, wall-less bacteria, and the Archaea. Conjugation-mediated lateral gene transfer has shaped the genomes of most if not all prokaryotes over evolutionary time and also contributed in the short term to the dissemination of antibiotic resistance and other virulence traits among medically important pathogens. How have these machines adapted to function across envelopes of distantly related microorganisms? A survey of T4SS functioning in phylogenetically diverse species highlights the biological complexity of these translocation systems and identifies common mechanistic themes as well as novel adaptations for specialized purposes relating to the modulation of the donor-target cell interaction.

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The Legionella pneumophila IcmS–LvgA protein complex is important for Dot/Icm‐dependent intracellular growth

Cited by 96 publications

References 53 publications

Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System

Poison Domains Block Transit of Translocated Substrates via the Legionella pneumophila Icm/Dot System

Large-scale identification and translocation of type IV secretion substrates by Coxiella burnetii

Biological Diversity of Prokaryotic Type IV Secretion Systems

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