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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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Detection of cHtrA protease in the cytosol of C. trachomatis-infected cells. HeLa cells infected with C. trachomatis L2 organisms were processed for co-staining with mouse antibodies visualized with a goat anti-mouse IgG conjugated with Cy3 (red), rabbit antibodies visualized with a Cy2-conjugated goat anti-rabbit IgG (green) and the DNA dye Hoechst (blue). The mouse antibodies included an anti-cHtrA (CT823) antiserum (raised with GST-cHtrA fusion protein) at various dilutions (A), the anti-cHtrA antiserum at 1:1000 dilution (B, panels a & f), mAb 6A2 (b & g, also raised with the GST-cHtrA fusion protein), mAb (100a) against CPAF (c & h), mAb (BB2) against IncA (d & i) and mAb (1L11C3) against HSP60 (e & j). The mouse anti-cHtrA staining (red) was also co-labeled with a rabbit anti-IncA antibody (green; C). Note that the anti-cHtrA antibodies detected signals both inside the chlamydial inclusions with (yellow arrowheads) or without (red arrowheads) overlapping with the chlamydial organisms and in the host cell cytosol (red arrows) while the anti-CPAF antibody mainly detected signals in the host cell cytosol.
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Figure 1: Detection of cHtrA protease in the cytosol of C. trachomatis-infected cells. HeLa cells infected with C. trachomatis L2 organisms were processed for co-staining with mouse antibodies visualized with a goat anti-mouse IgG conjugated with Cy3 (red), rabbit antibodies visualized with a Cy2-conjugated goat anti-rabbit IgG (green) and the DNA dye Hoechst (blue). The mouse antibodies included an anti-cHtrA (CT823) antiserum (raised with GST-cHtrA fusion protein) at various dilutions (A), the anti-cHtrA antiserum at 1:1000 dilution (B, panels a & f), mAb 6A2 (b & g, also raised with the GST-cHtrA fusion protein), mAb (100a) against CPAF (c & h), mAb (BB2) against IncA (d & i) and mAb (1L11C3) against HSP60 (e & j). The mouse anti-cHtrA staining (red) was also co-labeled with a rabbit anti-IncA antibody (green; C). Note that the anti-cHtrA antibodies detected signals both inside the chlamydial inclusions with (yellow arrowheads) or without (red arrowheads) overlapping with the chlamydial organisms and in the host cell cytosol (red arrows) while the anti-CPAF antibody mainly detected signals in the host cell cytosol.

Mentions: A mouse antiserum raised with GST-cHtrA fusion protein detected the endogenous cHtrA protein both inside and outside of the chlamydial inclusions in C. trachomatis-infected HeLa cells (Figure 1A). The amount of intra-inclusion labeling appeared to be greater since the labeling in the host cell cytosol (outside inclusions) disappeared first as the dilution of the antiserum increased. Interestingly, some of the cHtrA-positive intra-inclusion granules appeared to be distinct from C. trachomatis organisms, suggesting that a portion of cHtrA may be secreted out of the organisms but still trapped inside the inclusions. Both the intra-inclusion and cytosolic distribution of cHtrA were confirmed with a mAb against cHtrA (Figure 1B). Similar intra-inclusion stainings that are free of organisms were reported previously [15,57,58]. In contrast, most CPAF molecules were secreted out of the inclusions without obvious intra-inclusion accumulation. As expected, most of the chlamydial HSP60 molecules co-localized with the chlamydial organisms. The secretion of cHtrA into host cell cytosol became more obvious when the chlamydial inclusion membrane was counter-labeled using an anti-inclusion membrane protein antibody (Figure 1C). The cHtrA molecules were detected both inside and outside the inclusion membrane. The above observations together suggested that cHtrA might be secreted into both intra-inclusion space and the host cell cytosol.


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)

Detection of cHtrA protease in the cytosol of C. trachomatis-infected cells. HeLa cells infected with C. trachomatis L2 organisms were processed for co-staining with mouse antibodies visualized with a goat anti-mouse IgG conjugated with Cy3 (red), rabbit antibodies visualized with a Cy2-conjugated goat anti-rabbit IgG (green) and the DNA dye Hoechst (blue). The mouse antibodies included an anti-cHtrA (CT823) antiserum (raised with GST-cHtrA fusion protein) at various dilutions (A), the anti-cHtrA antiserum at 1:1000 dilution (B, panels a & f), mAb 6A2 (b & g, also raised with the GST-cHtrA fusion protein), mAb (100a) against CPAF (c & h), mAb (BB2) against IncA (d & i) and mAb (1L11C3) against HSP60 (e & j). The mouse anti-cHtrA staining (red) was also co-labeled with a rabbit anti-IncA antibody (green; C). Note that the anti-cHtrA antibodies detected signals both inside the chlamydial inclusions with (yellow arrowheads) or without (red arrowheads) overlapping with the chlamydial organisms and in the host cell cytosol (red arrows) while the anti-CPAF antibody mainly detected signals in the host cell cytosol.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 1: Detection of cHtrA protease in the cytosol of C. trachomatis-infected cells. HeLa cells infected with C. trachomatis L2 organisms were processed for co-staining with mouse antibodies visualized with a goat anti-mouse IgG conjugated with Cy3 (red), rabbit antibodies visualized with a Cy2-conjugated goat anti-rabbit IgG (green) and the DNA dye Hoechst (blue). The mouse antibodies included an anti-cHtrA (CT823) antiserum (raised with GST-cHtrA fusion protein) at various dilutions (A), the anti-cHtrA antiserum at 1:1000 dilution (B, panels a & f), mAb 6A2 (b & g, also raised with the GST-cHtrA fusion protein), mAb (100a) against CPAF (c & h), mAb (BB2) against IncA (d & i) and mAb (1L11C3) against HSP60 (e & j). The mouse anti-cHtrA staining (red) was also co-labeled with a rabbit anti-IncA antibody (green; C). Note that the anti-cHtrA antibodies detected signals both inside the chlamydial inclusions with (yellow arrowheads) or without (red arrowheads) overlapping with the chlamydial organisms and in the host cell cytosol (red arrows) while the anti-CPAF antibody mainly detected signals in the host cell cytosol.
Mentions: A mouse antiserum raised with GST-cHtrA fusion protein detected the endogenous cHtrA protein both inside and outside of the chlamydial inclusions in C. trachomatis-infected HeLa cells (Figure 1A). The amount of intra-inclusion labeling appeared to be greater since the labeling in the host cell cytosol (outside inclusions) disappeared first as the dilution of the antiserum increased. Interestingly, some of the cHtrA-positive intra-inclusion granules appeared to be distinct from C. trachomatis organisms, suggesting that a portion of cHtrA may be secreted out of the organisms but still trapped inside the inclusions. Both the intra-inclusion and cytosolic distribution of cHtrA were confirmed with a mAb against cHtrA (Figure 1B). Similar intra-inclusion stainings that are free of organisms were reported previously [15,57,58]. In contrast, most CPAF molecules were secreted out of the inclusions without obvious intra-inclusion accumulation. As expected, most of the chlamydial HSP60 molecules co-localized with the chlamydial organisms. The secretion of cHtrA into host cell cytosol became more obvious when the chlamydial inclusion membrane was counter-labeled using an anti-inclusion membrane protein antibody (Figure 1C). The cHtrA molecules were detected both inside and outside the inclusion membrane. The above observations together suggested that cHtrA might be secreted into both intra-inclusion space and the host cell cytosol.

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