The Legionella IcmSW Complex Directly Interacts with DotL to Mediate Translocation of Adaptor-Dependent Substrates

Sutherland, Molly C.; Nguyễn, Thùy Linh; Tseng, Victor; Vogel, Joseph P.

doi:10.1371/journal.ppat.1002910

Cited by 62 publications

(89 citation statements)

References 50 publications

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“…These results support a model in which VirD4, in the absence of other Lvh T4ASS proteins, functionally substitutes for DotA. While the mechanism of this effect is unclear, the observation suggests that the VirD4 protein of Legionella T4ASS can interact with components of the T4BSS, perhaps by promoting protein rearrangements of the Dot/Icm complex (60,61).…”

Section: Discussionsupporting

confidence: 73%

“…The significantly increased translocation in ⌬lvh and ⌬virD4 mutants suggests that absence of a functional Lvh T4ASS or its coupling protein, VirD4, changes the rate, temporal sequence, or access of SidD and RalF effectors to the two T4SSs so that translocation via the Dot/Icm T4BSS is enhanced. Alternatively, the absence of a functional Lvh T4ASS may change the assembly or subunit interaction of the Dot/Icm T4BSS (60,61) to increase translocation of those effectors. Regardless of the mechanism, our data support a role for the Lvh T4ASS in translocation of Dot/Icm effectors.…”

Section: Discussionmentioning

confidence: 99%

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Implication of the VirD4 Coupling Protein of the Lvh Type 4 Secretion System in Virulence Phenotypes of Legionella pneumophila

et al. 2013

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The genome of the Philadelphia-1 strain of Legionella pneumophila, the causative organism of Legionnaires' disease, encodes two virulence-associated type 4 secretion systems (T4SSs), the Dot/Icm type 4B (T4BSS) and the Lvh type 4A (T4ASS). Broth stationary-phase cultures of most dot/icm mutants are defective in entry and evasion of phagosome acidification. However, those virulence defects can be reversed by incubating broth cultures of dot/icm mutants in water, termed water stress (WS). WS reversal requires the lvh T4ASS locus, suggesting an interaction between the two T4SSs in producing Legionella virulence phenotypes. In the current work, the loss of WS reversal in a dotA ⌬lvh mutant of strain JR32 was shown to be attributable to loss of the lvh virD4 gene, encoding the putative coupling protein of the T4ASS. Transformation of a dotA ⌬lvh mutant with virD4 also reversed entry and phagosome acidification defects in broth cultures. In addition, broth cultures of ⌬lvh and ⌬virD4 mutants, which were dot/icm ؉ , showed 5-fold and >6-fold increases in translocation of the Dot/Icm translocation substrates, proteins RalF and SidD, respectively. These data demonstrate that the Lvh T4ASS functions in both broth stationary-phase cultures conventionally used for infection and cultures exposed to WS treatment. Our studies in a dotA ⌬lvh mutant and in a dot/icm ؉ background establish that VirD4 and the Lvh T4ASS contribute to virulence phenotypes and are consistent with independent functioning of Dot/Icm and Lvh T4SSs or functional substitution of the Lvh VirD4 protein for a component(s) of the Dot/Icm T4BSS.

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Section: Discussionsupporting

confidence: 73%

Section: Discussionmentioning

confidence: 99%

Implication of the VirD4 Coupling Protein of the Lvh Type 4 Secretion System in Virulence Phenotypes of Legionella pneumophila

et al. 2013

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“…Normally, chaperones carry their cognate substrates to the T3SS in a partially unfolded conformation, thus "priming" them for complete unfolding by the ATPase before translocation (45,46). In the Icm/Dot system it has been proposed that DotL, as part of a subcomplex with the ATPases DotM and DotN, acts as a regulator of IDTS entry (67). DotL has sequence similarity to type IV secretion system coupling proteins, which are proposed to link substrates to the rest of the secretion complex (68).…”

Section: Discussionmentioning

confidence: 99%

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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“…The results of our studies suggest that E. coli conjugation systems configured with chimeric T4CPs offer distinct advantages over other surrogate systems in ease of use, genetic manipulation, and, most importantly, fidelity with respect to recruitment of cognate substrates. One possible limitation, common to all surrogate systems (97,98) and evidenced here with A. tumefaciens VirE2 ( Fig. 2A), is that effectors requiring a chaperone or adaptor for translocation are not efficiently delivered via chimeric T4CPs through E. coli conjugation channels.…”

Section: Discussionmentioning

confidence: 99%

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

Whitaker¹,

Berry²,

Rosenthal³

et al. 2016

J Bacteriol

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Bacterial type IV secretion systems (T4SSs) are composed of two major subfamilies, conjugation machines dedicated to DNA transfer and effector translocators for protein transfer. We show here that the Escherichia coli pKM101-encoded conjugation system, coupled with chimeric substrate receptors, can be repurposed for transfer of heterologous effector proteins. The chimeric receptors were composed of the N-terminal transmembrane domain of pKM101-encoded TraJ fused to soluble domains of VirD4 homologs functioning in Agrobacterium tumefaciens, Anaplasma phagocytophilum, or Wolbachia pipientis. A chimeric receptor assembled from A. tumefaciens VirD4 (VirD4 At ) mediated transfer of a MOBQ plasmid (pML122) and A. tumefaciens effector proteins (VirE2, VirE3, and VirF) through the pKM101 transfer channel. Equivalent chimeric receptors assembled from the rickettsial VirD4 homologs similarly supported the transfer of known or candidate effectors from rickettsial species. These findings establish a proof of principle for use of the dedicated pKM101 conjugation channel, coupled with chimeric substrate receptors, to screen for translocation competency of protein effectors from recalcitrant species. Many T4SS receptors carry sequence-variable C-terminal domains (CTDs) with unknown function. While VirD4 At and the TraJ/VirD4 At chimera with their CTDs deleted supported pML122 transfer at wild-type levels, ⌬CTD variants supported transfer of protein substrates at strongly diminished or elevated levels. We were unable to detect binding of VirD4 At 's CTD to the VirE2 effector, although other VirD4 At domains bound this substrate in vitro. We propose that CTDs evolved to govern the dynamics of substrate presentation to the T4SS either through transient substrate contacts or by controlling substrate access to other receptor domains. IMPORTANCEBacterial type IV secretion systems (T4SSs) display striking versatility in their capacity to translocate DNA and protein substrates to prokaryotic and eukaryotic target cells. A hexameric ATPase, the type IV coupling protein (T4CP), functions as a substrate receptor for nearly all T4SSs. Here, we report that chimeric T4CPs mediate transfer of effector proteins through the Escherichia coli pKM101-encoded conjugation system. Studies with these repurposed conjugation systems established a role for acidic C-terminal domains of T4CPs in regulating substrate translocation. Our findings advance a mechanistic understanding of T4CP receptor activity and, further, support a model in which T4SS channels function as passive conduits for any DNA or protein substrates that successfully engage with and pass through the T4CP specificity checkpoint.T he type IV secretion systems (T4SSs) display striking versatility among the known bacterial translocation systems in their capacity to translocate DNA and protein substrates to bacterial or eukaryotic target cells (1, 2). Members of one major T4SS subfamily, the conjugation machines, mediate transfer of mobile DNA elements and are responsible for widespr...

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The Legionella IcmSW Complex Directly Interacts with DotL to Mediate Translocation of Adaptor-Dependent Substrates

Cited by 62 publications

References 50 publications

Implication of the VirD4 Coupling Protein of the Lvh Type 4 Secretion System in Virulence Phenotypes of Legionella pneumophila

Implication of the VirD4 Coupling Protein of the Lvh Type 4 Secretion System in Virulence Phenotypes of Legionella pneumophila

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

Chimeric Coupling Proteins Mediate Transfer of Heterologous Type IV Effectors through the Escherichia coli pKM101-Encoded Conjugation Machine

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