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The Chlamydia type III secretion system C-ring engages a chaperone-effector protein complex.

Spaeth KE, Chen YS, Valdivia RH - PLoS Pathog. (2009)

Bottom Line: In Gram-negative bacterial pathogens, specialized chaperones bind to secreted effector proteins and maintain them in a partially unfolded form competent for translocation by type III secretion systems/injectisomes.How diverse sets of effector-chaperone complexes are recognized by injectisomes is unclear.By yeast two-hybrid analysis we identified networks of Chlamydia-specific proteins that interacted with the basal structure of the injectisome, including two hubs of protein-protein interactions that linked known secreted effector proteins to CdsQ, the putative cytoplasmic C-ring component of the secretion apparatus.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology and Center for Microbial Pathogenesis, Duke University Medical Center, Durham, North Carolina, USA.

ABSTRACT
In Gram-negative bacterial pathogens, specialized chaperones bind to secreted effector proteins and maintain them in a partially unfolded form competent for translocation by type III secretion systems/injectisomes. How diverse sets of effector-chaperone complexes are recognized by injectisomes is unclear. Here we describe a new mechanism of effector-chaperone recognition by the Chlamydia injectisome, a unique and ancestral line of these evolutionarily conserved secretion systems. By yeast two-hybrid analysis we identified networks of Chlamydia-specific proteins that interacted with the basal structure of the injectisome, including two hubs of protein-protein interactions that linked known secreted effector proteins to CdsQ, the putative cytoplasmic C-ring component of the secretion apparatus. One of these protein-interaction hubs is defined by Ct260/Mcsc (Multiple cargo secretion chaperone). Mcsc binds to and stabilizes at least two secreted hydrophobic proteins, Cap1 and Ct618, that localize to the membrane of the pathogenic vacuole ("inclusion"). The resulting complexes bind to CdsQ, suggesting that in Chlamydia, the C-ring of the injectisome mediates the recognition of a subset of inclusion membrane proteins in complex with their chaperone. The selective recognition of inclusion membrane proteins by chaperones may provide a mechanism to co-ordinate the translocation of subsets of inclusion membrane proteins at different stages in infection.

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Ct260/Mcsc (Multiple cargo secretion chaperone) dimers bind to the inclusion membrane proteins Cap1 and Ct618.(A). Mcsc forms a dimer. Hexahistidine-tagged –Mcsc (Ct260) was treated with various concentrations of the reversible crosslinker DSP, resolved on a 4–20% gradient gel and probed with Mcsc specific antibodies. Arrow marks the dimeric form of Mcsc, which was reduced to monomeric form after boiling in the presence of DTT. (B–C). Mcsc-Cap1 and Mcsc-Ct618 co-purify as complexes. (B). Lysates of E. coli expressing 6xHis-tagged Mcsc or co-expressing Mcsc and 6xHis-tagged Ct618 (aa1–189) or Cap1 (aa1–298) were incubated with Ni2+-NTA agarose beads. Bound proteins were eluted with 150 mM imidazole and detected by SDS PAGE and Coomassie staining. Mcsc co-purified with Ct618-6xHis and Cap1-6xHis on Ni2+ beads. The numbers above the lanes represent fractions collected after incubation with wash buffer or elution buffers. Untagged Mcsc does not bind to the nickel columns non-specifically (Fig. 4E). (C). Proteins eluted from affinity columns were further fractionated by gel filtration chromatography. Mcsc eluted from the column at a size of ∼34 kDa. Mcsc-Cap1 and Mcsc/Ct618 eluted as complexes of ∼66 kDa and 54 kDa, respectively. The identity of all eluted proteins was confirmed by immunoblot analysis with anti Mcsc, Cap1 and hexahistidine tags. (D). Mcsc binds to endogenous Cap1 from infected cells. Lysate of infected HeLa cells were incubated with purified Mcsc dimers pre-bound to Ni2+-NTA agarose beads, and proteins were eluted with 150 mM imidazole. Cap1, but not IncA, preferentially co-eluted with Mcsc. Ponceau staining of nitrocellulose membrane indicate levels of Mcsc eluted from beads. (E). The central region of Ct618 and Cap1 mediate binding to Mcsc. The Mcsc binding domains of Ct618 and Cap1 were mapped by Y2H analysis. Positive interactions were assessed by activation of GAL4-dependent HIS3 and ADE2 reporter genes and growth in media lacking histidine (H) or adenine (A). Growth on media lacking tryptophan (T) and leucine (L) are shown as controls for maintenance of the Y2H vectors. Cartoon schematic shows Mcsc binding region maps adjacent to the large hydrophobic region at the COOH-terminus of these inclusion membrane proteins.
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ppat-1000579-g003: Ct260/Mcsc (Multiple cargo secretion chaperone) dimers bind to the inclusion membrane proteins Cap1 and Ct618.(A). Mcsc forms a dimer. Hexahistidine-tagged –Mcsc (Ct260) was treated with various concentrations of the reversible crosslinker DSP, resolved on a 4–20% gradient gel and probed with Mcsc specific antibodies. Arrow marks the dimeric form of Mcsc, which was reduced to monomeric form after boiling in the presence of DTT. (B–C). Mcsc-Cap1 and Mcsc-Ct618 co-purify as complexes. (B). Lysates of E. coli expressing 6xHis-tagged Mcsc or co-expressing Mcsc and 6xHis-tagged Ct618 (aa1–189) or Cap1 (aa1–298) were incubated with Ni2+-NTA agarose beads. Bound proteins were eluted with 150 mM imidazole and detected by SDS PAGE and Coomassie staining. Mcsc co-purified with Ct618-6xHis and Cap1-6xHis on Ni2+ beads. The numbers above the lanes represent fractions collected after incubation with wash buffer or elution buffers. Untagged Mcsc does not bind to the nickel columns non-specifically (Fig. 4E). (C). Proteins eluted from affinity columns were further fractionated by gel filtration chromatography. Mcsc eluted from the column at a size of ∼34 kDa. Mcsc-Cap1 and Mcsc/Ct618 eluted as complexes of ∼66 kDa and 54 kDa, respectively. The identity of all eluted proteins was confirmed by immunoblot analysis with anti Mcsc, Cap1 and hexahistidine tags. (D). Mcsc binds to endogenous Cap1 from infected cells. Lysate of infected HeLa cells were incubated with purified Mcsc dimers pre-bound to Ni2+-NTA agarose beads, and proteins were eluted with 150 mM imidazole. Cap1, but not IncA, preferentially co-eluted with Mcsc. Ponceau staining of nitrocellulose membrane indicate levels of Mcsc eluted from beads. (E). The central region of Ct618 and Cap1 mediate binding to Mcsc. The Mcsc binding domains of Ct618 and Cap1 were mapped by Y2H analysis. Positive interactions were assessed by activation of GAL4-dependent HIS3 and ADE2 reporter genes and growth in media lacking histidine (H) or adenine (A). Growth on media lacking tryptophan (T) and leucine (L) are shown as controls for maintenance of the Y2H vectors. Cartoon schematic shows Mcsc binding region maps adjacent to the large hydrophobic region at the COOH-terminus of these inclusion membrane proteins.

Mentions: (A). Network of predicted Ct260 (Mcsc) protein-protein interactions. Interactions were defined by Y2H analysis as described in Fig. 1D. (B–C). Ct260 and CdsQ are expressed throughout infection. (B). HeLa cells were infected with C. trachomatis and total protein samples collected at various times post-infection. Ct260, the bacterial major outer membrane protein (MOMP), and the inclusion membrane proteins IncA and Cap1 were detected with specific antibodies. Tubulin levels were serve as protein loading controls. (C). Reticulate bodies (RBs) and EBs were purified from infected cells by density gradient and total proteins were analyzed by immunoblot analysis with antibodies against the indicated chlamydial proteins. (D–E). Ct260 and CdsQ partition with membranes and hydrophobic proteins. (D). EBs were extracted with Triton-X114 to separate membrane from non-membrane associated proteins. Ct260 partitioned with the detergent fraction along with integral membrane proteins CdsJ and MOMP, but not the cytoplasmic proteins Hsp60 and RpoD. CdsQ partitioned in both membrane and aqueous fractions. (E). Ct260 is sensitive to Sarcosyl extraction, a detergent that solubilizes all membrane proteins except COMC, suggesting a lack of association with chlamydia outer membrane complexes (COMC) (F–G). Ct260 is not a target of T3S. (F). Secretion of T3S was induced by treatment of EBs with 0.5% BSA and 10mM EGTA. Tarp, but not Ct260, was detected in the extracellular media. (G). CdsQ and Ct260 localize to bacteria while Cap1 and Ct618 localize to the inclusion membrane. Ct260 and CdsQ (red) were observed exclusively in association with MOMP or LPS-positive (green) bacteria at early (12 h) and late (24 h) post-infection. Ct618 and Cap1 (red) localize to the extra-bacterial structures, including inclusion membranes. Scale bar range: 1.5 µm (12 h) to 4 µm (24 h). Abbreviations: (h.p.i.) hours post infection, (T) total lysates from purified EBs, (Memb.) and (Aq.) represent the detergent and aqueous phase of Triton-X114 extraction, (S) and (P) denote the soluble and insoluble protein fractions extracted with Sarcosyl respectively. Mcsc: Functional nomenclature for Ct260 (See Fig. 3).


The Chlamydia type III secretion system C-ring engages a chaperone-effector protein complex.

Spaeth KE, Chen YS, Valdivia RH - PLoS Pathog. (2009)

Ct260/Mcsc (Multiple cargo secretion chaperone) dimers bind to the inclusion membrane proteins Cap1 and Ct618.(A). Mcsc forms a dimer. Hexahistidine-tagged –Mcsc (Ct260) was treated with various concentrations of the reversible crosslinker DSP, resolved on a 4–20% gradient gel and probed with Mcsc specific antibodies. Arrow marks the dimeric form of Mcsc, which was reduced to monomeric form after boiling in the presence of DTT. (B–C). Mcsc-Cap1 and Mcsc-Ct618 co-purify as complexes. (B). Lysates of E. coli expressing 6xHis-tagged Mcsc or co-expressing Mcsc and 6xHis-tagged Ct618 (aa1–189) or Cap1 (aa1–298) were incubated with Ni2+-NTA agarose beads. Bound proteins were eluted with 150 mM imidazole and detected by SDS PAGE and Coomassie staining. Mcsc co-purified with Ct618-6xHis and Cap1-6xHis on Ni2+ beads. The numbers above the lanes represent fractions collected after incubation with wash buffer or elution buffers. Untagged Mcsc does not bind to the nickel columns non-specifically (Fig. 4E). (C). Proteins eluted from affinity columns were further fractionated by gel filtration chromatography. Mcsc eluted from the column at a size of ∼34 kDa. Mcsc-Cap1 and Mcsc/Ct618 eluted as complexes of ∼66 kDa and 54 kDa, respectively. The identity of all eluted proteins was confirmed by immunoblot analysis with anti Mcsc, Cap1 and hexahistidine tags. (D). Mcsc binds to endogenous Cap1 from infected cells. Lysate of infected HeLa cells were incubated with purified Mcsc dimers pre-bound to Ni2+-NTA agarose beads, and proteins were eluted with 150 mM imidazole. Cap1, but not IncA, preferentially co-eluted with Mcsc. Ponceau staining of nitrocellulose membrane indicate levels of Mcsc eluted from beads. (E). The central region of Ct618 and Cap1 mediate binding to Mcsc. The Mcsc binding domains of Ct618 and Cap1 were mapped by Y2H analysis. Positive interactions were assessed by activation of GAL4-dependent HIS3 and ADE2 reporter genes and growth in media lacking histidine (H) or adenine (A). Growth on media lacking tryptophan (T) and leucine (L) are shown as controls for maintenance of the Y2H vectors. Cartoon schematic shows Mcsc binding region maps adjacent to the large hydrophobic region at the COOH-terminus of these inclusion membrane proteins.
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ppat-1000579-g003: Ct260/Mcsc (Multiple cargo secretion chaperone) dimers bind to the inclusion membrane proteins Cap1 and Ct618.(A). Mcsc forms a dimer. Hexahistidine-tagged –Mcsc (Ct260) was treated with various concentrations of the reversible crosslinker DSP, resolved on a 4–20% gradient gel and probed with Mcsc specific antibodies. Arrow marks the dimeric form of Mcsc, which was reduced to monomeric form after boiling in the presence of DTT. (B–C). Mcsc-Cap1 and Mcsc-Ct618 co-purify as complexes. (B). Lysates of E. coli expressing 6xHis-tagged Mcsc or co-expressing Mcsc and 6xHis-tagged Ct618 (aa1–189) or Cap1 (aa1–298) were incubated with Ni2+-NTA agarose beads. Bound proteins were eluted with 150 mM imidazole and detected by SDS PAGE and Coomassie staining. Mcsc co-purified with Ct618-6xHis and Cap1-6xHis on Ni2+ beads. The numbers above the lanes represent fractions collected after incubation with wash buffer or elution buffers. Untagged Mcsc does not bind to the nickel columns non-specifically (Fig. 4E). (C). Proteins eluted from affinity columns were further fractionated by gel filtration chromatography. Mcsc eluted from the column at a size of ∼34 kDa. Mcsc-Cap1 and Mcsc/Ct618 eluted as complexes of ∼66 kDa and 54 kDa, respectively. The identity of all eluted proteins was confirmed by immunoblot analysis with anti Mcsc, Cap1 and hexahistidine tags. (D). Mcsc binds to endogenous Cap1 from infected cells. Lysate of infected HeLa cells were incubated with purified Mcsc dimers pre-bound to Ni2+-NTA agarose beads, and proteins were eluted with 150 mM imidazole. Cap1, but not IncA, preferentially co-eluted with Mcsc. Ponceau staining of nitrocellulose membrane indicate levels of Mcsc eluted from beads. (E). The central region of Ct618 and Cap1 mediate binding to Mcsc. The Mcsc binding domains of Ct618 and Cap1 were mapped by Y2H analysis. Positive interactions were assessed by activation of GAL4-dependent HIS3 and ADE2 reporter genes and growth in media lacking histidine (H) or adenine (A). Growth on media lacking tryptophan (T) and leucine (L) are shown as controls for maintenance of the Y2H vectors. Cartoon schematic shows Mcsc binding region maps adjacent to the large hydrophobic region at the COOH-terminus of these inclusion membrane proteins.
Mentions: (A). Network of predicted Ct260 (Mcsc) protein-protein interactions. Interactions were defined by Y2H analysis as described in Fig. 1D. (B–C). Ct260 and CdsQ are expressed throughout infection. (B). HeLa cells were infected with C. trachomatis and total protein samples collected at various times post-infection. Ct260, the bacterial major outer membrane protein (MOMP), and the inclusion membrane proteins IncA and Cap1 were detected with specific antibodies. Tubulin levels were serve as protein loading controls. (C). Reticulate bodies (RBs) and EBs were purified from infected cells by density gradient and total proteins were analyzed by immunoblot analysis with antibodies against the indicated chlamydial proteins. (D–E). Ct260 and CdsQ partition with membranes and hydrophobic proteins. (D). EBs were extracted with Triton-X114 to separate membrane from non-membrane associated proteins. Ct260 partitioned with the detergent fraction along with integral membrane proteins CdsJ and MOMP, but not the cytoplasmic proteins Hsp60 and RpoD. CdsQ partitioned in both membrane and aqueous fractions. (E). Ct260 is sensitive to Sarcosyl extraction, a detergent that solubilizes all membrane proteins except COMC, suggesting a lack of association with chlamydia outer membrane complexes (COMC) (F–G). Ct260 is not a target of T3S. (F). Secretion of T3S was induced by treatment of EBs with 0.5% BSA and 10mM EGTA. Tarp, but not Ct260, was detected in the extracellular media. (G). CdsQ and Ct260 localize to bacteria while Cap1 and Ct618 localize to the inclusion membrane. Ct260 and CdsQ (red) were observed exclusively in association with MOMP or LPS-positive (green) bacteria at early (12 h) and late (24 h) post-infection. Ct618 and Cap1 (red) localize to the extra-bacterial structures, including inclusion membranes. Scale bar range: 1.5 µm (12 h) to 4 µm (24 h). Abbreviations: (h.p.i.) hours post infection, (T) total lysates from purified EBs, (Memb.) and (Aq.) represent the detergent and aqueous phase of Triton-X114 extraction, (S) and (P) denote the soluble and insoluble protein fractions extracted with Sarcosyl respectively. Mcsc: Functional nomenclature for Ct260 (See Fig. 3).

Bottom Line: In Gram-negative bacterial pathogens, specialized chaperones bind to secreted effector proteins and maintain them in a partially unfolded form competent for translocation by type III secretion systems/injectisomes.How diverse sets of effector-chaperone complexes are recognized by injectisomes is unclear.By yeast two-hybrid analysis we identified networks of Chlamydia-specific proteins that interacted with the basal structure of the injectisome, including two hubs of protein-protein interactions that linked known secreted effector proteins to CdsQ, the putative cytoplasmic C-ring component of the secretion apparatus.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Genetics and Microbiology and Center for Microbial Pathogenesis, Duke University Medical Center, Durham, North Carolina, USA.

ABSTRACT
In Gram-negative bacterial pathogens, specialized chaperones bind to secreted effector proteins and maintain them in a partially unfolded form competent for translocation by type III secretion systems/injectisomes. How diverse sets of effector-chaperone complexes are recognized by injectisomes is unclear. Here we describe a new mechanism of effector-chaperone recognition by the Chlamydia injectisome, a unique and ancestral line of these evolutionarily conserved secretion systems. By yeast two-hybrid analysis we identified networks of Chlamydia-specific proteins that interacted with the basal structure of the injectisome, including two hubs of protein-protein interactions that linked known secreted effector proteins to CdsQ, the putative cytoplasmic C-ring component of the secretion apparatus. One of these protein-interaction hubs is defined by Ct260/Mcsc (Multiple cargo secretion chaperone). Mcsc binds to and stabilizes at least two secreted hydrophobic proteins, Cap1 and Ct618, that localize to the membrane of the pathogenic vacuole ("inclusion"). The resulting complexes bind to CdsQ, suggesting that in Chlamydia, the C-ring of the injectisome mediates the recognition of a subset of inclusion membrane proteins in complex with their chaperone. The selective recognition of inclusion membrane proteins by chaperones may provide a mechanism to co-ordinate the translocation of subsets of inclusion membrane proteins at different stages in infection.

Show MeSH
Related in: MedlinePlus