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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

Genetic organization and topographic overview of structural prediction of CopB. Solid black regions represent transmembrane domains. (a) Genetic organization of the ORF containing the putative translocator, CopB and CopD, and the TPR-domain containing chaperone, LcrH_1. (b) Diagonal stripes represent predicted coiled-coil domain in the C-terminus of the protein. Vertical stripes depict predicted Chaperone Binding Domain (CBD) spanning amino acid residues 168–171. The hydrophobic region is shown from amino acids 180–200
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Fig1: Genetic organization and topographic overview of structural prediction of CopB. Solid black regions represent transmembrane domains. (a) Genetic organization of the ORF containing the putative translocator, CopB and CopD, and the TPR-domain containing chaperone, LcrH_1. (b) Diagonal stripes represent predicted coiled-coil domain in the C-terminus of the protein. Vertical stripes depict predicted Chaperone Binding Domain (CBD) spanning amino acid residues 168–171. The hydrophobic region is shown from amino acids 180–200

Mentions: Translocator proteins have a conserved function across numerous bacterial species, facilitating the translocation of effector proteins from the bacterial cytosol to the host cell cytoplasm through formation of pores within the host cell membrane. However, there is limited sequence orthology between Chlamydia spp. translocators and other well-characterized bacterial translocator proteins. BLAST-P analysis identified potentially orthologous sequences in the recently sequenced genome of Bacteroides fragilis with an expect value of 6e−141 and percent identity of 54 %. CopB is a 493 amino acid protein with a predicted molecular weight of 50.5 kDa. Potential transmembrane domains were identified using online prediction software, TMpred, which suggests the presence of two transmembrane domains, spanning amino acids 256–274 and 383–406, respectively, and a hydrophobic stretch of amino acids from 180 to 200. COILS software identified three potential coiled-coil domains located at amino acids 117–140, 234–347, and 410–437. Sequence analysis of the N-terminal region of CopB identified a conserved chaperone binding motif of PxLxxP at amino acids 166–171 with the sequence of PELPKP (Fig. 1). Together, these results are consistent with features characteristically found in T3S translocator proteins [22, 23].Fig. 1


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)

Genetic organization and topographic overview of structural prediction of CopB. Solid black regions represent transmembrane domains. (a) Genetic organization of the ORF containing the putative translocator, CopB and CopD, and the TPR-domain containing chaperone, LcrH_1. (b) Diagonal stripes represent predicted coiled-coil domain in the C-terminus of the protein. Vertical stripes depict predicted Chaperone Binding Domain (CBD) spanning amino acid residues 168–171. The hydrophobic region is shown from amino acids 180–200
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4536800&req=5

Fig1: Genetic organization and topographic overview of structural prediction of CopB. Solid black regions represent transmembrane domains. (a) Genetic organization of the ORF containing the putative translocator, CopB and CopD, and the TPR-domain containing chaperone, LcrH_1. (b) Diagonal stripes represent predicted coiled-coil domain in the C-terminus of the protein. Vertical stripes depict predicted Chaperone Binding Domain (CBD) spanning amino acid residues 168–171. The hydrophobic region is shown from amino acids 180–200
Mentions: Translocator proteins have a conserved function across numerous bacterial species, facilitating the translocation of effector proteins from the bacterial cytosol to the host cell cytoplasm through formation of pores within the host cell membrane. However, there is limited sequence orthology between Chlamydia spp. translocators and other well-characterized bacterial translocator proteins. BLAST-P analysis identified potentially orthologous sequences in the recently sequenced genome of Bacteroides fragilis with an expect value of 6e−141 and percent identity of 54 %. CopB is a 493 amino acid protein with a predicted molecular weight of 50.5 kDa. Potential transmembrane domains were identified using online prediction software, TMpred, which suggests the presence of two transmembrane domains, spanning amino acids 256–274 and 383–406, respectively, and a hydrophobic stretch of amino acids from 180 to 200. COILS software identified three potential coiled-coil domains located at amino acids 117–140, 234–347, and 410–437. Sequence analysis of the N-terminal region of CopB identified a conserved chaperone binding motif of PxLxxP at amino acids 166–171 with the sequence of PELPKP (Fig. 1). Together, these results are consistent with features characteristically found in T3S translocator proteins [22, 23].Fig. 1

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