Limits...
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

Peptide inhibition of the translocator:chaperone interaction. (a) Recombinant GST-CopD1–157 or GST-CopB1–200 was pre-incubated with 500 μM of the chaperone binding domain peptide mimetic (+) or vehicle alone (−). CopD1–157 and GST-CopB1–200 did not interact with its putative chaperone in the presence of the CBD peptide, but did so in the presence of a control peptide or vehicle alone. (b) Left image is C. pneumoniae incubated with vehicle alone (PBS), right image is C. pneumoniae incubated with 500 μM CBD Peptide, bottom image is C. pneumonia incubated with 50 uM anti-RSV peptide. Chlamydial inclusions are stained green, while HeLa cells are stained red by Evan’s blue counterstain
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Peptide inhibition of the translocator:chaperone interaction. (a) Recombinant GST-CopD1–157 or GST-CopB1–200 was pre-incubated with 500 μM of the chaperone binding domain peptide mimetic (+) or vehicle alone (−). CopD1–157 and GST-CopB1–200 did not interact with its putative chaperone in the presence of the CBD peptide, but did so in the presence of a control peptide or vehicle alone. (b) Left image is C. pneumoniae incubated with vehicle alone (PBS), right image is C. pneumoniae incubated with 500 μM CBD Peptide, bottom image is C. pneumonia incubated with 50 uM anti-RSV peptide. Chlamydial inclusions are stained green, while HeLa cells are stained red by Evan’s blue counterstain

Mentions: Given the necessity of the PxLxxP motif for the interaction between translocator proteins and their class II chaperones, a synthetic peptide containing the chaperone binding motif was synthesized and tested for its ability to block the interaction between LcrH_1 and both CopB and CopD. To determine whether a synthetic peptide consisting of a cell penetrating peptide sequence (YGRKKRRQRRR) and the 10 amino acids (ETPELPKPGV) encompassing the chaperone binding motif of CopB is capable of preventing the chaperone:translocator interaction, the peptide was incubated with GST-CopB1–200 or GST-CopD1–157 prior to the addition of His-LcrH_1. In the presence of the peptide, no interaction was observed between the putative translocators, CopB and CopD, and LcrH_1 under high salt conditions (Fig. 4a). To explore the hypothesis that the CopB:LcrH_1 and CopD:LcrH_1 interaction are essential for infection, we then tested the ability of the peptide to block C. pneumoniae infection. We pre-incubated C. pneumoniae with the peptide or vehicle alone and then infected host cells. The peptide inhibited infection by 90 % compared to the control infection with vehicle alone (Fig. 4b).Fig. 4


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)

Peptide inhibition of the translocator:chaperone interaction. (a) Recombinant GST-CopD1–157 or GST-CopB1–200 was pre-incubated with 500 μM of the chaperone binding domain peptide mimetic (+) or vehicle alone (−). CopD1–157 and GST-CopB1–200 did not interact with its putative chaperone in the presence of the CBD peptide, but did so in the presence of a control peptide or vehicle alone. (b) Left image is C. pneumoniae incubated with vehicle alone (PBS), right image is C. pneumoniae incubated with 500 μM CBD Peptide, bottom image is C. pneumonia incubated with 50 uM anti-RSV peptide. Chlamydial inclusions are stained green, while HeLa cells are stained red by Evan’s blue counterstain
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig4: Peptide inhibition of the translocator:chaperone interaction. (a) Recombinant GST-CopD1–157 or GST-CopB1–200 was pre-incubated with 500 μM of the chaperone binding domain peptide mimetic (+) or vehicle alone (−). CopD1–157 and GST-CopB1–200 did not interact with its putative chaperone in the presence of the CBD peptide, but did so in the presence of a control peptide or vehicle alone. (b) Left image is C. pneumoniae incubated with vehicle alone (PBS), right image is C. pneumoniae incubated with 500 μM CBD Peptide, bottom image is C. pneumonia incubated with 50 uM anti-RSV peptide. Chlamydial inclusions are stained green, while HeLa cells are stained red by Evan’s blue counterstain
Mentions: Given the necessity of the PxLxxP motif for the interaction between translocator proteins and their class II chaperones, a synthetic peptide containing the chaperone binding motif was synthesized and tested for its ability to block the interaction between LcrH_1 and both CopB and CopD. To determine whether a synthetic peptide consisting of a cell penetrating peptide sequence (YGRKKRRQRRR) and the 10 amino acids (ETPELPKPGV) encompassing the chaperone binding motif of CopB is capable of preventing the chaperone:translocator interaction, the peptide was incubated with GST-CopB1–200 or GST-CopD1–157 prior to the addition of His-LcrH_1. In the presence of the peptide, no interaction was observed between the putative translocators, CopB and CopD, and LcrH_1 under high salt conditions (Fig. 4a). To explore the hypothesis that the CopB:LcrH_1 and CopD:LcrH_1 interaction are essential for infection, we then tested the ability of the peptide to block C. pneumoniae infection. We pre-incubated C. pneumoniae with the peptide or vehicle alone and then infected host cells. The peptide inhibited infection by 90 % compared to the control infection with vehicle alone (Fig. 4b).Fig. 4

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