Single Molecule Fluorescence Study of the Bacillus thuringiensis Toxin Cry1Aa Reveals Tetramerization

Groulx, Nicolas; McGuire, Hugo; Laprade, Raynald; Schwartz, Jean-Louis; Blunck, Rikard

doi:10.1074/jbc.m111.296103

Cited by 40 publications

(40 citation statements)

References 59 publications

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“…We therefore employed TIRF microscopy and single molecule photobleaching (30,40,41) to determine the stoichiometry of homo-and hetero-oligomers formed by PopB and PopD inserted in membranes. These experiments clearly showed that a unique hetero-oligomer is formed when PopB and PopD are added together.…”

Section: Discussionmentioning

confidence: 99%

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Type 3 Secretion Translocators Spontaneously Assemble a Hexadecameric Transmembrane Complex

Romano¹,

Tang²,

Rossi³

et al. 2016

Journal of Biological Chemistry

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A type 3 secretion system is used by many bacterial pathogens to inject proteins into eukaryotic cells. Pathogens insert a translocon complex into the target eukaryotic membrane by secreting two proteins known as translocators. How these translocators form a translocon in the lipid bilayer and why both proteins are required remains elusive. Pseudomonas aeruginosa translocators PopB and PopD insert pores into membranes forming homo-or hetero-complexes of undetermined stoichiometry. Single-molecule fluorescence photobleaching experiments revealed that PopD formed mostly hexameric structures in membranes, whereas PopB displayed a bi-modal distribution with 6 and 12 subunits peaks. However, individually the proteins are not functional for effector translocation. We have found that when added together, the translocators formed distinct heterocomplexes containing 8 PopB and 8 PopD molecules. Thus, the interaction between PopB and PopD guide the assembly of a unique hetero-oligomer in membranes.The transport of proteins across membranes is essential at many stages of pathogen infection and colonization of human cells. This process typically involves the discharge of proteins from the pathogen (secretion) and the introduction of these secreted toxins/effectors into the cytosol of the target cell (translocation). Many pathogens, including the Shigella, Salmonella, Yersinia, and Pseudomonas species, exploit a sophisticated and efficient mechanism of protein secretion and translocation known as type III secretion (T3S) 3 system (1, 2). The T3S system is a syringe-like macromolecular machine formed by more than 20 different proteins organized in three major structures to span: (i) the inner bacterial membrane, the periplasmic space, and the outer bacterial membrane (the secreton); (ii) the extracellular space (the needle); and (iii) the host cellular membrane (the translocon) (3-6).A phylogenetic analysis of bacterial T3S systems based on conservation of their basal body ATPase indicates the presence of at least 7 families of T3S machines. The Pseudomonas aeruginosa genome encodes a single T3S system grouped within the Ysc family, named after the Yersinia spp. T3S system (the archetypical T3S system in this family) (7). The Ysc family includes pathogens like Yersinia pestis, Y. pseudotuberculosis, Y. enterocolitica, Bordetella pertussis, Vibrio parahemeolyticus, and P. aeruginosa, among others. The Ysc family shares structural similarity with the Inv-Mxi-Spa family of T3S systems, which includes the secretion systems used by Salmonella enterica and Shigella spp. (8).Great progress has been made in the structural characterization of the secreton and the needle for different T3S system families (9). However, little is known about how T3S-secreted proteins are translocated across the plasma membrane of the target cell to alter the normal function of the host (4). Two T3S-secreted proteins, known as the T3S translocators, insert into the target membrane to facilitate effector translocation. P. aeruginosa translocators PopB/PopD...

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Section: Discussionmentioning

confidence: 99%

“…We adapted the technique to quantify proteins in their native lipid bilayer environment (i.e. in the absence of detergents) by using SLB (30). PopB BpyFL or PopD BpyFL were reconstituted into liposomes using previously optimized conditions for membrane association and pore formation (14).…”

Section: Popb Assists the Association Of Popd With Membranes-bothmentioning

confidence: 99%

Type 3 Secretion Translocators Spontaneously Assemble a Hexadecameric Transmembrane Complex

Romano¹,

Tang²,

Rossi³

et al. 2016

Journal of Biological Chemistry

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“…Alternatively, the activated toxins could individually insert into the target membrane, where they subsequently assemble into an oligomeric pore complex, causing cell lysis (13,20). However, the precise molecular description of either model still remains to be critically verified.…”

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confidence: 99%

Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin

Sriwimol

Aroonkesorn

Sakdee

et al. 2015

Journal of Biological Chemistry

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Background: The molecular description of oligomeric pore formation by B. thuringiensis insecticidal toxins remains unclear.Results: Cry4Ba mosquito-active toxins assemble into a stable prepore trimer upon interaction with non-ionic micelles or lipid membranes. Conclusion: A membrane-bound state of monomers is required for facilitating a potential trimer assembly. Significance: This study reveals a requirement of membrane-bound monomers for forming a prepore trimer capable of perturbing target membranes.

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“…However, we cannot rule out the possibility that buried disulfide bridges may not be exposed to the reducing environment of the midgut lumen. In this case, a plausible explanation is that the entire domain I inserts into membrane as previously proposed, either as a whole protein [7,[12][13][14][15][16][17] or as a member of an oligomeric complex [18,23,35,36]. This would also result in no difference in toxicity between the wild-type and disulfide proteins.…”

Section: Discussionmentioning

confidence: 70%

“…Several disulfide-bridge mutations generated by protein engineering which are related to the membrane partitioning mechanisms of Cry toxins are reviewed in [21]. An intermediate step has been proposed in which the oligomerization occurs in tetramers after the hairpins from α-helices 3 and 4 are inserted into the membrane [22,23]. At the present time no single model can account for all the experimental observations.…”

Section: Introductionmentioning

confidence: 99%

Two disulfide mutants in domain I of <i>Bacillus thuringiensis</i> Cry3Aa δ-endotoxin increase stability with no effect on toxicity

Wu¹,

Florez²,

Homoelle³

et al. 2012

ABC

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To increase protein stability and test protein function, three double-cysteine mutations were individually introduced by protein engineering into the cysteinefree Cry3Aa δ-endotoxin from Bacillus thuringiensis. Despite the increase in stability and structural changes introduced by the disulfide bonds, no effect on toxicity was observed. A possible mechanism involving the insertion of all of domain I of Cry3Aa toxin into the target membrane accounts for these observations.

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Single Molecule Fluorescence Study of the Bacillus thuringiensis Toxin Cry1Aa Reveals Tetramerization

Cited by 40 publications

References 59 publications

Type 3 Secretion Translocators Spontaneously Assemble a Hexadecameric Transmembrane Complex

Type 3 Secretion Translocators Spontaneously Assemble a Hexadecameric Transmembrane Complex

Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin

Two disulfide mutants in domain I of <i>Bacillus thuringiensis</i> Cry3Aa δ-endotoxin increase stability with no effect on toxicity

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Single Molecule Fluorescence Study of the Bacillus thuringiensis Toxin Cry1Aa Reveals Tetramerization

Cited by 40 publications

References 59 publications

Type 3 Secretion Translocators Spontaneously Assemble a Hexadecameric Transmembrane Complex

Type 3 Secretion Translocators Spontaneously Assemble a Hexadecameric Transmembrane Complex

Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin

Two disulfide mutants in domain I of &lt;i&gt;Bacillus thuringiensis&lt;/i&gt; Cry3Aa δ-endotoxin increase stability with no effect on toxicity

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Two disulfide mutants in domain I of <i>Bacillus thuringiensis</i> Cry3Aa δ-endotoxin increase stability with no effect on toxicity