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The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol.

Wu X, Lei L, Gong S, Chen D, Flores R, Zhong G - BMC Microbiol. (2011)

Bottom Line: The periplasmic cHtrA protein appeared to be actively secreted into host cell cytosol since no other chlamydial periplasmic proteins were detected in the host cell cytoplasm.Most chlamydial species secreted cHtrA into host cell cytosol and the secretion was not inhibitable by a type III secretion inhibitor.Since it is hypothesized that chlamydial organisms possess a proteolysis strategy to manipulate host cell signaling pathways, secretion of the serine protease cHtrA into host cell cytosol suggests that the periplasmic cHtrA may also play an important role in chlamydial interactions with host cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA.

ABSTRACT

Background: The periplasmic High Temperature Requirement protein A (HtrA) plays important roles in bacterial protein folding and stress responses. However, the role of chlamydial HtrA (cHtrA) in chlamydial pathogenesis is not clear.

Results: The cHtrA was detected both inside and outside the chlamydial inclusions. The detection was specific since both polyclonal and monoclonal anti-cHtrA antibodies revealed similar intracellular labeling patterns that were only removed by absorption with cHtrA but not control fusion proteins. In a Western blot assay, the anti-cHtrA antibodies detected the endogenous cHtrA in Chlamydia-infected cells without cross-reacting with any other chlamydial or host cell antigens. Fractionation of the infected cells revealed cHtrA in the host cell cytosol fraction. The periplasmic cHtrA protein appeared to be actively secreted into host cell cytosol since no other chlamydial periplasmic proteins were detected in the host cell cytoplasm. Most chlamydial species secreted cHtrA into host cell cytosol and the secretion was not inhibitable by a type III secretion inhibitor.

Conclusion: Since it is hypothesized that chlamydial organisms possess a proteolysis strategy to manipulate host cell signaling pathways, secretion of the serine protease cHtrA into host cell cytosol suggests that the periplasmic cHtrA may also play an important role in chlamydial interactions with host cells.

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The anti-GST-cHtrA fusion protein antibodies specifically detected the endogenous cHtrA produced by chlamydial organisms. The anti-cHtrA antibodies with or without absorption with GST fusion proteins were used to detect the endogenous proteins in C. trachomatis-infected cells (A) and on nitrocellulose membranes (B). (A) C. trachomatis-infected cells were processed for immunostaining as described in Figure 1A legend. Note that the antibody labeling of endogenous antigens was blocked only by corresponding but not unrelated control fusion proteins. (B) In a Western blot assay, HeLa alone or HeLa infected with C. trachomatis (Ct-HeLa), GST-CPAF & GST-cHtrA fusion proteins were used as antigens as indicated on top of the figure. The antigens blotted onto nitrocellulose membrane were detected with mouse antibodies as displayed at the bottom of the figure. The anti-CPAF mAb 100a is specific to the C-terminal fragment of CPAF (CPAFc) and the full length CPAF is rapidly processed into the N- and C-terminal fragments to form intramolecular dimmers for activity during chlamydial infection. The control antibodies anti-MOMP and anti-human HSP70 were used to indicate that the Ct-HeLa samples contain chlamydial organisms and both HeLa and Ct-HeLa samples were loaded with similar amounts. Note that each antibody only detected a major protein band migrated at the molecular weight that matched the corresponding chlamydial or host proteins as indicated on the right side of the figure.
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Figure 2: The anti-GST-cHtrA fusion protein antibodies specifically detected the endogenous cHtrA produced by chlamydial organisms. The anti-cHtrA antibodies with or without absorption with GST fusion proteins were used to detect the endogenous proteins in C. trachomatis-infected cells (A) and on nitrocellulose membranes (B). (A) C. trachomatis-infected cells were processed for immunostaining as described in Figure 1A legend. Note that the antibody labeling of endogenous antigens was blocked only by corresponding but not unrelated control fusion proteins. (B) In a Western blot assay, HeLa alone or HeLa infected with C. trachomatis (Ct-HeLa), GST-CPAF & GST-cHtrA fusion proteins were used as antigens as indicated on top of the figure. The antigens blotted onto nitrocellulose membrane were detected with mouse antibodies as displayed at the bottom of the figure. The anti-CPAF mAb 100a is specific to the C-terminal fragment of CPAF (CPAFc) and the full length CPAF is rapidly processed into the N- and C-terminal fragments to form intramolecular dimmers for activity during chlamydial infection. The control antibodies anti-MOMP and anti-human HSP70 were used to indicate that the Ct-HeLa samples contain chlamydial organisms and both HeLa and Ct-HeLa samples were loaded with similar amounts. Note that each antibody only detected a major protein band migrated at the molecular weight that matched the corresponding chlamydial or host proteins as indicated on the right side of the figure.

Mentions: We next confirmed the antibody binding specificity by using an absorption procedure (Figure 2A). Both the intra-inclusion and host cell cytosolic signals detected by the anti-cHtrA antiserum or anti-cHtrA mAb 6A2 were removed by absorption with GST-cHtrA but not GST-CPAF fusion proteins. Similarly, the cytosolic signal detected with the anti-CPAF antibody was removed by absorption with the GST-CPAF but not GST-cHtrA fusion proteins, demonstrating that the anti-cHtrA and anti-CPAF antibodies specifically labeled the corresponding endogenous proteins without cross-reacting with each other. In a Western blot assay (Figure 2B), the anti-cHtrA antibodies recognized both the GST-cHtrA fusion protein and the endogenous cHtrA from the C. trachomatis-infected HeLa cells (Ct-HeLa) while the various control antibodies recognized the corresponding antigens without any significant cross-reactivity with each other. The anti-CPAF antibody detected the GST-CPAF fusion protein and also the C-terminal fragment (CPAFc) of the endogenous CPAF from the Ct-HeLa sample. CPAF is rapidly processed into the N- and C-terminal fragments during chlamydial infection and the mAb 100a is specific to the 35 kDa C-terminal fragment [26]. The anti-MOMP antibody detected MOMP from Ct-HeLa, confirming the presence of whole chlamydial organisms in the sample while the anti-human HSP70 antibody detected similar amounts of HSP70 in the HeLa alone and Ct-HeLa samples, indicating that an equivalent amount of whole cell lysates was loaded in both samples. These observations together have demonstrated that the anti-cHtrA antibodies only recognized cHtrA without cross-reacting with any other chlamydial or host cell proteins, suggesting that the cellular signals detected with the anti-HtrA fusion protein antibodies in the immunofluorescence assay were specific to the endogenous cHtrA produced by chlamydial organisms.


The chlamydial periplasmic stress response serine protease cHtrA is secreted into host cell cytosol.

Wu X, Lei L, Gong S, Chen D, Flores R, Zhong G - BMC Microbiol. (2011)

The anti-GST-cHtrA fusion protein antibodies specifically detected the endogenous cHtrA produced by chlamydial organisms. The anti-cHtrA antibodies with or without absorption with GST fusion proteins were used to detect the endogenous proteins in C. trachomatis-infected cells (A) and on nitrocellulose membranes (B). (A) C. trachomatis-infected cells were processed for immunostaining as described in Figure 1A legend. Note that the antibody labeling of endogenous antigens was blocked only by corresponding but not unrelated control fusion proteins. (B) In a Western blot assay, HeLa alone or HeLa infected with C. trachomatis (Ct-HeLa), GST-CPAF & GST-cHtrA fusion proteins were used as antigens as indicated on top of the figure. The antigens blotted onto nitrocellulose membrane were detected with mouse antibodies as displayed at the bottom of the figure. The anti-CPAF mAb 100a is specific to the C-terminal fragment of CPAF (CPAFc) and the full length CPAF is rapidly processed into the N- and C-terminal fragments to form intramolecular dimmers for activity during chlamydial infection. The control antibodies anti-MOMP and anti-human HSP70 were used to indicate that the Ct-HeLa samples contain chlamydial organisms and both HeLa and Ct-HeLa samples were loaded with similar amounts. Note that each antibody only detected a major protein band migrated at the molecular weight that matched the corresponding chlamydial or host proteins as indicated on the right side of the figure.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 2: The anti-GST-cHtrA fusion protein antibodies specifically detected the endogenous cHtrA produced by chlamydial organisms. The anti-cHtrA antibodies with or without absorption with GST fusion proteins were used to detect the endogenous proteins in C. trachomatis-infected cells (A) and on nitrocellulose membranes (B). (A) C. trachomatis-infected cells were processed for immunostaining as described in Figure 1A legend. Note that the antibody labeling of endogenous antigens was blocked only by corresponding but not unrelated control fusion proteins. (B) In a Western blot assay, HeLa alone or HeLa infected with C. trachomatis (Ct-HeLa), GST-CPAF & GST-cHtrA fusion proteins were used as antigens as indicated on top of the figure. The antigens blotted onto nitrocellulose membrane were detected with mouse antibodies as displayed at the bottom of the figure. The anti-CPAF mAb 100a is specific to the C-terminal fragment of CPAF (CPAFc) and the full length CPAF is rapidly processed into the N- and C-terminal fragments to form intramolecular dimmers for activity during chlamydial infection. The control antibodies anti-MOMP and anti-human HSP70 were used to indicate that the Ct-HeLa samples contain chlamydial organisms and both HeLa and Ct-HeLa samples were loaded with similar amounts. Note that each antibody only detected a major protein band migrated at the molecular weight that matched the corresponding chlamydial or host proteins as indicated on the right side of the figure.
Mentions: We next confirmed the antibody binding specificity by using an absorption procedure (Figure 2A). Both the intra-inclusion and host cell cytosolic signals detected by the anti-cHtrA antiserum or anti-cHtrA mAb 6A2 were removed by absorption with GST-cHtrA but not GST-CPAF fusion proteins. Similarly, the cytosolic signal detected with the anti-CPAF antibody was removed by absorption with the GST-CPAF but not GST-cHtrA fusion proteins, demonstrating that the anti-cHtrA and anti-CPAF antibodies specifically labeled the corresponding endogenous proteins without cross-reacting with each other. In a Western blot assay (Figure 2B), the anti-cHtrA antibodies recognized both the GST-cHtrA fusion protein and the endogenous cHtrA from the C. trachomatis-infected HeLa cells (Ct-HeLa) while the various control antibodies recognized the corresponding antigens without any significant cross-reactivity with each other. The anti-CPAF antibody detected the GST-CPAF fusion protein and also the C-terminal fragment (CPAFc) of the endogenous CPAF from the Ct-HeLa sample. CPAF is rapidly processed into the N- and C-terminal fragments during chlamydial infection and the mAb 100a is specific to the 35 kDa C-terminal fragment [26]. The anti-MOMP antibody detected MOMP from Ct-HeLa, confirming the presence of whole chlamydial organisms in the sample while the anti-human HSP70 antibody detected similar amounts of HSP70 in the HeLa alone and Ct-HeLa samples, indicating that an equivalent amount of whole cell lysates was loaded in both samples. These observations together have demonstrated that the anti-cHtrA antibodies only recognized cHtrA without cross-reacting with any other chlamydial or host cell proteins, suggesting that the cellular signals detected with the anti-HtrA fusion protein antibodies in the immunofluorescence assay were specific to the endogenous cHtrA produced by chlamydial organisms.

Bottom Line: The periplasmic cHtrA protein appeared to be actively secreted into host cell cytosol since no other chlamydial periplasmic proteins were detected in the host cell cytoplasm.Most chlamydial species secreted cHtrA into host cell cytosol and the secretion was not inhibitable by a type III secretion inhibitor.Since it is hypothesized that chlamydial organisms possess a proteolysis strategy to manipulate host cell signaling pathways, secretion of the serine protease cHtrA into host cell cytosol suggests that the periplasmic cHtrA may also play an important role in chlamydial interactions with host cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78229, USA.

ABSTRACT

Background: The periplasmic High Temperature Requirement protein A (HtrA) plays important roles in bacterial protein folding and stress responses. However, the role of chlamydial HtrA (cHtrA) in chlamydial pathogenesis is not clear.

Results: The cHtrA was detected both inside and outside the chlamydial inclusions. The detection was specific since both polyclonal and monoclonal anti-cHtrA antibodies revealed similar intracellular labeling patterns that were only removed by absorption with cHtrA but not control fusion proteins. In a Western blot assay, the anti-cHtrA antibodies detected the endogenous cHtrA in Chlamydia-infected cells without cross-reacting with any other chlamydial or host cell antigens. Fractionation of the infected cells revealed cHtrA in the host cell cytosol fraction. The periplasmic cHtrA protein appeared to be actively secreted into host cell cytosol since no other chlamydial periplasmic proteins were detected in the host cell cytoplasm. Most chlamydial species secreted cHtrA into host cell cytosol and the secretion was not inhibitable by a type III secretion inhibitor.

Conclusion: Since it is hypothesized that chlamydial organisms possess a proteolysis strategy to manipulate host cell signaling pathways, secretion of the serine protease cHtrA into host cell cytosol suggests that the periplasmic cHtrA may also play an important role in chlamydial interactions with host cells.

Show MeSH
Related in: MedlinePlus