Synergistic Pore Formation by Type III Toxin Translocators of<i>Pseudomonas aeruginosa</i>

Faudry, Eric; Vernier, Grégory; Neumann, Emmanuelle; Forge, Vincent; Attrée, Ina

doi:10.1021/bi060452+

Cited by 59 publications

(94 citation statements)

References 42 publications

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“…In addition, this observation also supports in vitro studies performed on other translocon molecules of Salmonella (40), Yersinia (41,43), and Shigella (42). These results suggest that, in Pseudomonas, PopB could be the major translocon component that binds most stably to target membranes upon infection, whereas PopD remains superficially associated to the bilayer, a suggestion that is in agreement with model membrane studies which showed that PopB plays the central role in pore formation (44,45).…”

Section: Resultssupporting

confidence: 87%

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

Discola

Förster

Boulay

et al. 2014

Journal of Biological Chemistry

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Background:The type III secretion system allows bacteria to inject effectors directly into the host cytoplasm through a translocation pore. Results: Translocator proteins were characterized in lipid-bound and chaperone-associated forms. Conclusion: Pseudomonas translocators use different regions to recognize their common chaperone and the eukaryotic membrane. Significance: Mapping of key regions in translocators provides a starting point for the potential development of novel antibacterials.

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Section: Resultssupporting

confidence: 87%

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

Discola

Förster

Boulay

et al. 2014

Journal of Biological Chemistry

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“…LUV Leakage Assay-LUV were prepared with 5 mM phosphate-citrate, pH 7, containing 50 mM sulforhodamine B (SRB) (Molecular Probes) as previously described (27). After filtration, unincorporated dye was removed by size exclusion chromatography on a PD10 column (Amersham Biosciences) equilibrated with 5 mM phosphate-citrate, 50 mM or 200 mM NaCl, pH 7.…”

Section: Methodsmentioning

confidence: 99%

Membrane Interaction of Botulinum Neurotoxin A Translocation (T) Domain

Galloux

Vitrac

Montagner

et al. 2008

Journal of Biological Chemistry

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The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH. We found that the interaction with membranes does not involve major secondary or tertiary structural changes, as reported for other toxins like diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage, the T domain becomes able to permeabilize lipid vesicles. This occurs for pH values lower than 5.5, in agreement with the pH encountered by the toxin within endosomes. Electrostatic interactions are also important for the process. The role of the so-called belt region was investigated with four variant proteins presenting different lengths of the N-extremity of the T domain. We observed that this part of the T domain, which contains numerous negatively charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. Overall, the data suggest that acidification eliminates repulsive electrostatic interactions between the T domain and the membrane, allowing its penetration into the membrane without triggering detectable structural changes.

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“…Buffer only, buffer containing the appropriate detergents, IpgC, or IpaB/IpgC were used as negative controls for liposome disruption, as appropriate. Release of 3, 10, 40, and 70 kDa fluorescein-labeled dextrans from lipid vesicles was monitored using a modified protocol described previously 37 following the addition of 15 nM oligomeric IpaB. It is important to note that all of these release assays are strongly dependent on protein and liposome concentration 25 as well as the final concentration of detergent.…”

Section: Preparation Of Phospholipid Vesiclesmentioning

confidence: 99%

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

et al. 2013

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The Shigella flexneri Type III secretion system (T3SS) senses contact with human intestinal cells and injects effector proteins that promote pathogen entry as the first step in causing life threatening bacillary dysentery (shigellosis). The Shigella Type III secretion apparatus (T3SA) consists of an anchoring basal body, an exposed needle, and a temporally assembled tip complex. Exposure to environmental small molecules recruits IpaB, the first hydrophobic translocator protein, to the maturing tip complex. IpaB then senses contact with a host cell membrane, forming the translocon pore through which effectors are delivered to the host cytoplasm. Within the bacterium, IpaB exists as a heterodimer with its chaperone IpgC; however, IpaB's structural state following secretion is unknown due to difficulties isolating stable protein.We have overcome this by coexpressing the IpaB/IpgC heterodimer and isolating IpaB by incubating the complex in mild detergents. Interestingly, preparation of IpaB with n-octyl-oligooxyethylene (OPOE) results in the assembly of discrete oligomers while purification in N,Ndimethyldodecylamine N-oxide (LDAO) maintains IpaB as a monomer. In this study, we demonstrate that IpaB tetramers penetrate phospholipid membranes to allow a size-dependent release of small molecules, suggesting the formation of discrete pores. Monomeric IpaB also interacts with liposomes but fails to disrupt them. From these and additional findings, we propose Abbreviations: AUC, analytical ultracentrifugation; CD, circular dichroism; CMC, critical micelle concentration; DGS-NTA, 1,2-dioleoylsn-glycero-3-[(N-(5-amino-1-carboxypentyl) iminodiacetic acid)succinyl]; DLS, dynamic light scattering; DMSO, dimethyl sulfoxide; DOPC, dioleoylphosphatidylcholine; DOPG, dioleoylphosphatidylglycerol; DSP, dithiobis[succinimidyl proprionate; FM, fluorescein maleimide; FRET, F-rster resonance energy transfer; Ipa, invasion plasmid antigen; Ipg, invasion plasmid gene; LDAO, N,N-dimethyldodecylamine N-oxide; Mxi, major exporter of Ipas; OPOE, n-Octyl-oligo-oxyethylene; SATA, N-succinimidyl-S-acetylthioacetate; SEC, size exclusion chromatography; SRB, sulforhodamine B; T m , thermal unfolding midpoint; TCEP, (tris (2-carboxyethyl)phosphine; TRITC DHPE, (N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine); T3SS, type-III secretion system; T3SA, type III secretion apparatus.Additional Supporting Information may be found in the online version of this article. that IpaB can exist as a tetramer having inherent flexibility, which allows it to cooperatively interact with and insert into host cell membranes. This event may then lay the foundation for formation of the Shigella T3SS translocon pore.

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Synergistic Pore Formation by Type III Toxin Translocators ofPseudomonas aeruginosa

Cited by 59 publications

References 42 publications

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

Membrane and Chaperone Recognition by the Major Translocator Protein PopB of the Type III Secretion System of Pseudomonas aeruginosa

Membrane Interaction of Botulinum Neurotoxin A Translocation (T) Domain

Oligomeric states of the Shigella translocator protein IpaB provide structural insights into formation of the type III secretion translocon

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