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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

Chlamydia Outer Protein (Cop) B Interacts with T3S proteins. (a) GST-CopB1–255 or GST-CopB275–385 bound to glutathione-agarose beads (bait) pulled HisMBP-CdsF (prey) out of an E. coli lysate in the presence of a high salt wash buffer (500 mM NaCl). (b & c) Furthermore, GST-CopB fragments did not pull His-CopN or Cpn0803 out of an E. coli lysate in the presence of a high salt wash buffer
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Fig2: Chlamydia Outer Protein (Cop) B Interacts with T3S proteins. (a) GST-CopB1–255 or GST-CopB275–385 bound to glutathione-agarose beads (bait) pulled HisMBP-CdsF (prey) out of an E. coli lysate in the presence of a high salt wash buffer (500 mM NaCl). (b & c) Furthermore, GST-CopB fragments did not pull His-CopN or Cpn0803 out of an E. coli lysate in the presence of a high salt wash buffer

Mentions: CopB is believed to be a T3S protein, and thus it should interact with other proteins within the T3SS [10]. Cloning fragments of CopB lacking the transmembrane domains allowed us to identify specific domains of CopB that are responsible for interactions with other type III secretion components. GST pulldowns were performed between CopB and Cpn0803, CdsF, and CopN. No interactions were observed between any fragments of CopB and Cpn0803 or CopN (Fig. 2a and b). There was a positive interaction between the N-terminal (amino acids 1–255) and middle fragment of CopB and CdsF, but not the C-terminus of CopB (Fig. 2c). These observations are consistent with a role in the T3S apparatus of Chlamydia pneumonia, since translocator proteins from orthologous systems have been shown to interact with the needle filament protein.Fig. 2


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)

Chlamydia Outer Protein (Cop) B Interacts with T3S proteins. (a) GST-CopB1–255 or GST-CopB275–385 bound to glutathione-agarose beads (bait) pulled HisMBP-CdsF (prey) out of an E. coli lysate in the presence of a high salt wash buffer (500 mM NaCl). (b & c) Furthermore, GST-CopB fragments did not pull His-CopN or Cpn0803 out of an E. coli lysate in the presence of a high salt wash buffer
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig2: Chlamydia Outer Protein (Cop) B Interacts with T3S proteins. (a) GST-CopB1–255 or GST-CopB275–385 bound to glutathione-agarose beads (bait) pulled HisMBP-CdsF (prey) out of an E. coli lysate in the presence of a high salt wash buffer (500 mM NaCl). (b & c) Furthermore, GST-CopB fragments did not pull His-CopN or Cpn0803 out of an E. coli lysate in the presence of a high salt wash buffer
Mentions: CopB is believed to be a T3S protein, and thus it should interact with other proteins within the T3SS [10]. Cloning fragments of CopB lacking the transmembrane domains allowed us to identify specific domains of CopB that are responsible for interactions with other type III secretion components. GST pulldowns were performed between CopB and Cpn0803, CdsF, and CopN. No interactions were observed between any fragments of CopB and Cpn0803 or CopN (Fig. 2a and b). There was a positive interaction between the N-terminal (amino acids 1–255) and middle fragment of CopB and CdsF, but not the C-terminus of CopB (Fig. 2c). These observations are consistent with a role in the T3S apparatus of Chlamydia pneumonia, since translocator proteins from orthologous systems have been shown to interact with the needle filament protein.Fig. 2

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