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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

Inhibition of Chlamydia pneumoniae with CopB antibodies. Panels a-d show inhibition assay results performed with either no antibody (a), CopB antibody (b), pre-immune sera (c), or control antibody (α-GST) (d). Panel e shows the degree of inhibition by of CopB antibodies compared to control antibodies. Chlamydial inclusions are stained green, while HeLa cells are stained red by Evan’s blue counterstain. Panel f demonstrates reactivity of anti-CopB with (1) C. pneumoniae infected HeLa cell lysate, (2) uninfected HeLa cell lysate, (3) recombinant GST-CopB1–255 produced in E. coli, and (4) recombinant GST produced in E. coli. Experiments were performed in triplicate. Error bars represent 2 standard deviations. Images represent random fields of view. * = P < 0.0001
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Fig5: Inhibition of Chlamydia pneumoniae with CopB antibodies. Panels a-d show inhibition assay results performed with either no antibody (a), CopB antibody (b), pre-immune sera (c), or control antibody (α-GST) (d). Panel e shows the degree of inhibition by of CopB antibodies compared to control antibodies. Chlamydial inclusions are stained green, while HeLa cells are stained red by Evan’s blue counterstain. Panel f demonstrates reactivity of anti-CopB with (1) C. pneumoniae infected HeLa cell lysate, (2) uninfected HeLa cell lysate, (3) recombinant GST-CopB1–255 produced in E. coli, and (4) recombinant GST produced in E. coli. Experiments were performed in triplicate. Error bars represent 2 standard deviations. Images represent random fields of view. * = P < 0.0001

Mentions: Since T3S translocators are believed to be surface exposed proteins in other T3SS, we hypothesized that antibodies to CopB would inhibit infection [24–26]. We generated an antibody to a peptide (15-mer) in the N-terminal region of CopB and tested its ability to inhibit C. pneumoniae infection. To test whether this antibody could inhibit infection, we pre-incubated C. pneumoniae with the polyclonal antibody for 1 h at 37 °C prior to infection. C. pneumoniae infection was inhibited by the CopB antibody. (Fig. 5a-d), resulting in a 98 % reduction in inclusion forming units, as compared to control antibody (Fig. 5e). Using a Western blot, polyclonal antibodies were able to detect both recombinant and native CopB (Fig. 5f). The ability of the CopB antibody to block infection suggests that CopB is surfaced exposed, and plays a critical role in the infection process.Fig. 5


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)

Inhibition of Chlamydia pneumoniae with CopB antibodies. Panels a-d show inhibition assay results performed with either no antibody (a), CopB antibody (b), pre-immune sera (c), or control antibody (α-GST) (d). Panel e shows the degree of inhibition by of CopB antibodies compared to control antibodies. Chlamydial inclusions are stained green, while HeLa cells are stained red by Evan’s blue counterstain. Panel f demonstrates reactivity of anti-CopB with (1) C. pneumoniae infected HeLa cell lysate, (2) uninfected HeLa cell lysate, (3) recombinant GST-CopB1–255 produced in E. coli, and (4) recombinant GST produced in E. coli. Experiments were performed in triplicate. Error bars represent 2 standard deviations. Images represent random fields of view. * = P < 0.0001
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

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

Fig5: Inhibition of Chlamydia pneumoniae with CopB antibodies. Panels a-d show inhibition assay results performed with either no antibody (a), CopB antibody (b), pre-immune sera (c), or control antibody (α-GST) (d). Panel e shows the degree of inhibition by of CopB antibodies compared to control antibodies. Chlamydial inclusions are stained green, while HeLa cells are stained red by Evan’s blue counterstain. Panel f demonstrates reactivity of anti-CopB with (1) C. pneumoniae infected HeLa cell lysate, (2) uninfected HeLa cell lysate, (3) recombinant GST-CopB1–255 produced in E. coli, and (4) recombinant GST produced in E. coli. Experiments were performed in triplicate. Error bars represent 2 standard deviations. Images represent random fields of view. * = P < 0.0001
Mentions: Since T3S translocators are believed to be surface exposed proteins in other T3SS, we hypothesized that antibodies to CopB would inhibit infection [24–26]. We generated an antibody to a peptide (15-mer) in the N-terminal region of CopB and tested its ability to inhibit C. pneumoniae infection. To test whether this antibody could inhibit infection, we pre-incubated C. pneumoniae with the polyclonal antibody for 1 h at 37 °C prior to infection. C. pneumoniae infection was inhibited by the CopB antibody. (Fig. 5a-d), resulting in a 98 % reduction in inclusion forming units, as compared to control antibody (Fig. 5e). Using a Western blot, polyclonal antibodies were able to detect both recombinant and native CopB (Fig. 5f). The ability of the CopB antibody to block infection suggests that CopB is surfaced exposed, and plays a critical role in the infection process.Fig. 5

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