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Chlamydia Outer Protein (Cop) B from Chlamydia pneumoniae possesses characteristic features of a type III secretion (T3S) translocator protein.

Bulir DC, Waltho DA, Stone CB, Liang S, Chiang CK, Mwawasi KA, Nelson JC, Zhang SW, Mihalco SP, Scinocca ZC, Mahony JB - BMC Microbiol. (2015)

Bottom Line: Important early effector proteins of the type III secretion system (T3SS) are a class of proteins called the translocators.The translocator proteins insert into the host cell membrane to form a pore, allowing the injectisome to dock onto the host cell to facilitate translocation of effectors.The inhibition of the LcrH_1:CopB interaction with a cognate peptide and subsequent inhibition of host cell infection provides strong evidence that T3S is an essential virulence factor for chlamydial infection and pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: M. G. DeGroote Institute for Infectious Disease Research, Faculty of Health Sciences and Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada. bulirdc@mcmaster.ca.

ABSTRACT

Background: Chlamydia spp. are believed to use a conserved virulence factor called type III secretion (T3S) to facilitate the delivery of effector proteins from the bacterial pathogen to the host cell. Important early effector proteins of the type III secretion system (T3SS) are a class of proteins called the translocators. The translocator proteins insert into the host cell membrane to form a pore, allowing the injectisome to dock onto the host cell to facilitate translocation of effectors. CopB is a predicted hydrophobic translocator protein within the chlamydial T3SS.

Results: In this study, we identified a novel interaction between the hydrophobic translocator, CopB, and the putative filament protein, CdsF. Furthermore, we identified a conserved PxLxxP motif in CopB (amino acid residues 166-171), which is required for interaction with its cognate chaperone, LcrH_1. Using a synthetic peptide derived from the chaperone binding motif of CopB, we were able to block the LcrH_1 interaction with either CopB or CopD; this CopB peptide was capable of inhibiting C. pneumoniae infection of HeLa cells at micromolar concentrations. An antibody raised against the N-terminus of CopB was able to inhibit C. pneumoniae infection of HeLa cells.

Conclusion: The inhibition of the LcrH_1:CopB interaction with a cognate peptide and subsequent inhibition of host cell infection provides strong evidence that T3S is an essential virulence factor for chlamydial infection and pathogenesis. Together, these results support that CopB plays the role of a hydrophobic translocator.

No MeSH data available.


Related in: MedlinePlus

LcrH_1 (Cpn0811) interacts with CopB. (a) Recombinant LcrH_1 interacted with amino acids 1–200 of CopB. CopB mutants were created using Gblock synthesis: P166ACopB1–200, L168ACopB1–200, and P171ACopD1–200. (b & c) Mutations at the conserved amino acids within the predicted chaperone binding domain disrupted the interaction between CopB1–200 and the chaperone LcrH_1, but not other identified interactions
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Fig3: LcrH_1 (Cpn0811) interacts with CopB. (a) Recombinant LcrH_1 interacted with amino acids 1–200 of CopB. CopB mutants were created using Gblock synthesis: P166ACopB1–200, L168ACopB1–200, and P171ACopD1–200. (b & c) Mutations at the conserved amino acids within the predicted chaperone binding domain disrupted the interaction between CopB1–200 and the chaperone LcrH_1, but not other identified interactions

Mentions: Cpn0811 (LcrH_1) is a small, basic isoelectric protein located upstream in the same operon as CopB (Cpn0809) [20]. We explored the possible interaction between LcrH_1 and CopB and found that His-LcrH_1 interacts within the N-terminus of CopB (Fig. 3a). Both CopB1–200 and CopB1–255 interacted with His-LcrH_1, but CopB1–180 did not, suggesting the hydrophobic stretch of amino acids spanning residues 180–200 plays an important role in this interaction. Since CopB1–200 was the smallest truncation construct that maintained an interaction with His-LcrH_1, we examined the amino acid sequence for the presence of a conserved chaperone binding motif, PxLxxP, which begins at amino acid 166. To elucidate the importance of the conserved motif, we performed an alanine walkthrough of the conserved amino acids in the PxLxxP motif starting at amino acid 166(P166ACopB1–200, L168ACopB1–200, P171ACopB1–200). Mutation of the PxLxxP motif abrogated the interaction between His-LcrH_1 and CopB (Fig. 3b). To ensure that the absence of interaction was the result of the specific amino acid substitution, as opposed to gross misfolding of the mutant protein, L168ACopB1–200 was subjected to a GST pulldown against CdsF. As expected, L168ACopB1–200 maintained the interaction with HisMBP-CdsF (Fig. 3c), suggesting that the PxLxxP is a critical interaction domain between the chaperone and CopB.Fig. 3


Chlamydia Outer Protein (Cop) B from Chlamydia pneumoniae possesses characteristic features of a type III secretion (T3S) translocator protein.

Bulir DC, Waltho DA, Stone CB, Liang S, Chiang CK, Mwawasi KA, Nelson JC, Zhang SW, Mihalco SP, Scinocca ZC, Mahony JB - BMC Microbiol. (2015)

LcrH_1 (Cpn0811) interacts with CopB. (a) Recombinant LcrH_1 interacted with amino acids 1–200 of CopB. CopB mutants were created using Gblock synthesis: P166ACopB1–200, L168ACopB1–200, and P171ACopD1–200. (b & c) Mutations at the conserved amino acids within the predicted chaperone binding domain disrupted the interaction between CopB1–200 and the chaperone LcrH_1, but not other identified interactions
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4536800&req=5

Fig3: LcrH_1 (Cpn0811) interacts with CopB. (a) Recombinant LcrH_1 interacted with amino acids 1–200 of CopB. CopB mutants were created using Gblock synthesis: P166ACopB1–200, L168ACopB1–200, and P171ACopD1–200. (b & c) Mutations at the conserved amino acids within the predicted chaperone binding domain disrupted the interaction between CopB1–200 and the chaperone LcrH_1, but not other identified interactions
Mentions: Cpn0811 (LcrH_1) is a small, basic isoelectric protein located upstream in the same operon as CopB (Cpn0809) [20]. We explored the possible interaction between LcrH_1 and CopB and found that His-LcrH_1 interacts within the N-terminus of CopB (Fig. 3a). Both CopB1–200 and CopB1–255 interacted with His-LcrH_1, but CopB1–180 did not, suggesting the hydrophobic stretch of amino acids spanning residues 180–200 plays an important role in this interaction. Since CopB1–200 was the smallest truncation construct that maintained an interaction with His-LcrH_1, we examined the amino acid sequence for the presence of a conserved chaperone binding motif, PxLxxP, which begins at amino acid 166. To elucidate the importance of the conserved motif, we performed an alanine walkthrough of the conserved amino acids in the PxLxxP motif starting at amino acid 166(P166ACopB1–200, L168ACopB1–200, P171ACopB1–200). Mutation of the PxLxxP motif abrogated the interaction between His-LcrH_1 and CopB (Fig. 3b). To ensure that the absence of interaction was the result of the specific amino acid substitution, as opposed to gross misfolding of the mutant protein, L168ACopB1–200 was subjected to a GST pulldown against CdsF. As expected, L168ACopB1–200 maintained the interaction with HisMBP-CdsF (Fig. 3c), suggesting that the PxLxxP is a critical interaction domain between the chaperone and CopB.Fig. 3

Bottom Line: Important early effector proteins of the type III secretion system (T3SS) are a class of proteins called the translocators.The translocator proteins insert into the host cell membrane to form a pore, allowing the injectisome to dock onto the host cell to facilitate translocation of effectors.The inhibition of the LcrH_1:CopB interaction with a cognate peptide and subsequent inhibition of host cell infection provides strong evidence that T3S is an essential virulence factor for chlamydial infection and pathogenesis.

View Article: PubMed Central - PubMed

Affiliation: M. G. DeGroote Institute for Infectious Disease Research, Faculty of Health Sciences and Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada. bulirdc@mcmaster.ca.

ABSTRACT

Background: Chlamydia spp. are believed to use a conserved virulence factor called type III secretion (T3S) to facilitate the delivery of effector proteins from the bacterial pathogen to the host cell. Important early effector proteins of the type III secretion system (T3SS) are a class of proteins called the translocators. The translocator proteins insert into the host cell membrane to form a pore, allowing the injectisome to dock onto the host cell to facilitate translocation of effectors. CopB is a predicted hydrophobic translocator protein within the chlamydial T3SS.

Results: In this study, we identified a novel interaction between the hydrophobic translocator, CopB, and the putative filament protein, CdsF. Furthermore, we identified a conserved PxLxxP motif in CopB (amino acid residues 166-171), which is required for interaction with its cognate chaperone, LcrH_1. Using a synthetic peptide derived from the chaperone binding motif of CopB, we were able to block the LcrH_1 interaction with either CopB or CopD; this CopB peptide was capable of inhibiting C. pneumoniae infection of HeLa cells at micromolar concentrations. An antibody raised against the N-terminus of CopB was able to inhibit C. pneumoniae infection of HeLa cells.

Conclusion: The inhibition of the LcrH_1:CopB interaction with a cognate peptide and subsequent inhibition of host cell infection provides strong evidence that T3S is an essential virulence factor for chlamydial infection and pathogenesis. Together, these results support that CopB plays the role of a hydrophobic translocator.

No MeSH data available.


Related in: MedlinePlus