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A gatekeeper chaperone complex directs translocator secretion during type three secretion.

Archuleta TL, Spiller BW - PLoS Pathog. (2014)

Bottom Line: A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective.One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion.The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown.

View Article: PubMed Central - PubMed

Affiliation: Chemical and Physical Biology Program, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.

ABSTRACT
Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ∼20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.

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The gatekeeper-chaperone complex is needed for efficient translocator secretion.A. T3S was induced from wild type and mxiC mutant Shigella strains. M90T is a wild type strain. ΔmxiC is M90T derived, with mxiC deleted [18]. EV: empty vector. Strains were complemeted with mxiC, mxiC mutant (RDR), or empty vector (EV). Proteins were visualized by silver staining and identified by MS. IpaA, OspCs, and IpgB are effectors. IpaB, IpaC, and IpaD are translocators. Bottom: anti-MxiC blot of the same samples indicates that MxiC and MxiC-RDR are secreted normally. B. A secretion timecourse. The experiment shown in A. was repeated and samples taken a 10, 15, 30, and 60 minutes post induction. IpaA, an effector, is secreted at early time points in the absence of functional Mxic, but in the presence of MxiC it is not efficiently secreted until later time points.
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ppat-1004498-g004: The gatekeeper-chaperone complex is needed for efficient translocator secretion.A. T3S was induced from wild type and mxiC mutant Shigella strains. M90T is a wild type strain. ΔmxiC is M90T derived, with mxiC deleted [18]. EV: empty vector. Strains were complemeted with mxiC, mxiC mutant (RDR), or empty vector (EV). Proteins were visualized by silver staining and identified by MS. IpaA, OspCs, and IpgB are effectors. IpaB, IpaC, and IpaD are translocators. Bottom: anti-MxiC blot of the same samples indicates that MxiC and MxiC-RDR are secreted normally. B. A secretion timecourse. The experiment shown in A. was repeated and samples taken a 10, 15, 30, and 60 minutes post induction. IpaA, an effector, is secreted at early time points in the absence of functional Mxic, but in the presence of MxiC it is not efficiently secreted until later time points.

Mentions: To determine the importance of the gatekeeper-chaperone interaction during T3S, we disrupted the homologous gatekeeper-translocator chaperone interface in Shigella. Shigella, unlike Chlamydia, are genetically tractable allowing disruption of the endogenous mxiC gene (the copN homolog) and rescue with a plasmid expressing mutant or wild-type MxiC. This is a well-established strategy that has been used to study other MxiC mutants [18], [28]. The CopN and Scc3 homologs in Shigella, MxiC and IpgC, form a complex that includes the T3SS ATPase [28]. The Scc3-CopNΔ84 structure was disrupted by mutation of A362R, R365D, and G369R on CopN (Supporting Figure S4), supporting the idea that the homologous mutations would disrupt IpgC-MxiC interface. We expressed the E331R/R334D/I338R MxiC mutant (MxiC-RDR) from a plasmid in a previously described mxiC Shigella strain [18] and compared secretion profiles following Congo Red induction. MxiC-RDR is deficient in secretion of the translocators IpaB, IpaC, and IpaD, but efficiently secretes IpaA, an effector, and secretes elevated levels of the effectors OspC1-3 and IpgB (Figure 4A). IpaA is not secreted efficiently if wild type MxiC is present, but is secreted earlier and in greater quantities from ΔMxiC or MxiC-RDR strains (Figure 4B, compare IpaA secretion at 10 minutes and 60 minutes). The secretion profile of MxiC-RDR closely mimics that of the ΔMxiC strain (Figure 4A), highlighting the importance of gatekeeper-translocator chaperone complexes in translocator secretion. To further evaluate the importance of the conserved, central, arginine, we evaluated the secretion profile of a single point mutant, MxiC-R334D, revealing a phenotype similar to the triple mutant (Supporting Figure S6). Similar to CopN, MxiC is both the gatekeeper and a T3S substrate [18], [42], [43]. To verify that the mutations to MxiC did not prevent recognition and secretion of MxiC by the T3SS, we evaluated the secretion of MxiC-RDR, which is unaltered from wild-type MxiC (Figure 4A). To further confirm that the mutations didn't grossly alter the structure of MxiC, we compared circular dichroism specta of MxiCΔ74, MxiCΔ74-RDR, and MxiCΔ74-R334D, which indicated that all three proteins are similarly folded (Supporting Figure S3, Methods S1). In these constructs the first 74 residues of MxiC have been deleted to allow expression and purification from E. coli. MxiC-RDR is secreted in a similar manner to MxiC, indicating that the mutations do not disrupt its ability to interact with the T3SS, yet is unable to direct translocators for secretion, and is unable to prevent inappropriate secretion of the effectors, IpaA, OspC1-3, and IpgB.


A gatekeeper chaperone complex directs translocator secretion during type three secretion.

Archuleta TL, Spiller BW - PLoS Pathog. (2014)

The gatekeeper-chaperone complex is needed for efficient translocator secretion.A. T3S was induced from wild type and mxiC mutant Shigella strains. M90T is a wild type strain. ΔmxiC is M90T derived, with mxiC deleted [18]. EV: empty vector. Strains were complemeted with mxiC, mxiC mutant (RDR), or empty vector (EV). Proteins were visualized by silver staining and identified by MS. IpaA, OspCs, and IpgB are effectors. IpaB, IpaC, and IpaD are translocators. Bottom: anti-MxiC blot of the same samples indicates that MxiC and MxiC-RDR are secreted normally. B. A secretion timecourse. The experiment shown in A. was repeated and samples taken a 10, 15, 30, and 60 minutes post induction. IpaA, an effector, is secreted at early time points in the absence of functional Mxic, but in the presence of MxiC it is not efficiently secreted until later time points.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4222845&req=5

ppat-1004498-g004: The gatekeeper-chaperone complex is needed for efficient translocator secretion.A. T3S was induced from wild type and mxiC mutant Shigella strains. M90T is a wild type strain. ΔmxiC is M90T derived, with mxiC deleted [18]. EV: empty vector. Strains were complemeted with mxiC, mxiC mutant (RDR), or empty vector (EV). Proteins were visualized by silver staining and identified by MS. IpaA, OspCs, and IpgB are effectors. IpaB, IpaC, and IpaD are translocators. Bottom: anti-MxiC blot of the same samples indicates that MxiC and MxiC-RDR are secreted normally. B. A secretion timecourse. The experiment shown in A. was repeated and samples taken a 10, 15, 30, and 60 minutes post induction. IpaA, an effector, is secreted at early time points in the absence of functional Mxic, but in the presence of MxiC it is not efficiently secreted until later time points.
Mentions: To determine the importance of the gatekeeper-chaperone interaction during T3S, we disrupted the homologous gatekeeper-translocator chaperone interface in Shigella. Shigella, unlike Chlamydia, are genetically tractable allowing disruption of the endogenous mxiC gene (the copN homolog) and rescue with a plasmid expressing mutant or wild-type MxiC. This is a well-established strategy that has been used to study other MxiC mutants [18], [28]. The CopN and Scc3 homologs in Shigella, MxiC and IpgC, form a complex that includes the T3SS ATPase [28]. The Scc3-CopNΔ84 structure was disrupted by mutation of A362R, R365D, and G369R on CopN (Supporting Figure S4), supporting the idea that the homologous mutations would disrupt IpgC-MxiC interface. We expressed the E331R/R334D/I338R MxiC mutant (MxiC-RDR) from a plasmid in a previously described mxiC Shigella strain [18] and compared secretion profiles following Congo Red induction. MxiC-RDR is deficient in secretion of the translocators IpaB, IpaC, and IpaD, but efficiently secretes IpaA, an effector, and secretes elevated levels of the effectors OspC1-3 and IpgB (Figure 4A). IpaA is not secreted efficiently if wild type MxiC is present, but is secreted earlier and in greater quantities from ΔMxiC or MxiC-RDR strains (Figure 4B, compare IpaA secretion at 10 minutes and 60 minutes). The secretion profile of MxiC-RDR closely mimics that of the ΔMxiC strain (Figure 4A), highlighting the importance of gatekeeper-translocator chaperone complexes in translocator secretion. To further evaluate the importance of the conserved, central, arginine, we evaluated the secretion profile of a single point mutant, MxiC-R334D, revealing a phenotype similar to the triple mutant (Supporting Figure S6). Similar to CopN, MxiC is both the gatekeeper and a T3S substrate [18], [42], [43]. To verify that the mutations to MxiC did not prevent recognition and secretion of MxiC by the T3SS, we evaluated the secretion of MxiC-RDR, which is unaltered from wild-type MxiC (Figure 4A). To further confirm that the mutations didn't grossly alter the structure of MxiC, we compared circular dichroism specta of MxiCΔ74, MxiCΔ74-RDR, and MxiCΔ74-R334D, which indicated that all three proteins are similarly folded (Supporting Figure S3, Methods S1). In these constructs the first 74 residues of MxiC have been deleted to allow expression and purification from E. coli. MxiC-RDR is secreted in a similar manner to MxiC, indicating that the mutations do not disrupt its ability to interact with the T3SS, yet is unable to direct translocators for secretion, and is unable to prevent inappropriate secretion of the effectors, IpaA, OspC1-3, and IpgB.

Bottom Line: A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective.One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion.The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown.

View Article: PubMed Central - PubMed

Affiliation: Chemical and Physical Biology Program, Vanderbilt University School of Medicine, Nashville, Tennessee, United States of America.

ABSTRACT
Many Gram-negative bacteria use Type Three Secretion Systems (T3SS) to deliver effector proteins into host cells. These protein delivery machines are composed of cytosolic components that recognize substrates and generate the force needed for translocation, the secretion conduit, formed by a needle complex and associated membrane spanning basal body, and translocators that form the pore in the target cell. A defined order of secretion in which needle component proteins are secreted first, followed by translocators, and finally effectors, is necessary for this system to be effective. While the secreted effectors vary significantly between organisms, the ∼20 individual protein components that form the T3SS are conserved in many pathogenic bacteria. One such conserved protein, referred to as either a plug or gatekeeper, is necessary to prevent unregulated effector release and to allow efficient translocator secretion. The mechanism by which translocator secretion is promoted while effector release is inhibited by gatekeepers is unknown. We present the structure of the Chlamydial gatekeeper, CopN, bound to a translocator-specific chaperone. The structure identifies a previously unknown interface between gatekeepers and translocator chaperones and reveals that in the gatekeeper-chaperone complex the canonical translocator-binding groove is free to bind translocators. Structure-based mutagenesis of the homologous complex in Shigella reveals that the gatekeeper-chaperone-translocator complex is essential for translocator secretion and for the ordered secretion of translocators prior to effectors.

Show MeSH
Related in: MedlinePlus