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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 cHtrA but not CT067 is detected in the cytosolic fraction of the chlamydia-infected HeLa cells. HeLa cells infected with C. trachomatis organisms (Ct-HeLa) were fractionated into nuclear (Ct-HeLa pellet, containing chlamydial inclusions, lane 3) and cytosolic (Ct-HeLa S100, containing chlamydia-secreted proteins, lane 4) fractions. The cellular fractions along with total cell lysates (normal HeLa, lane 1 & Ct-HeLa, lane 2) and purified chlamydial RB (lane 5) and EB (lane 6) organisms as listed at the top were resolved in SDS-polyacrylamide gels. The resolved protein bands were blotted onto nitrocellulose membrane for reacting with antibodies (listed on the left) against cHtrA (panel a), CT067 (b, a periplasmic iron binding protein), CPAF (c, a chlamydia-secreted protein), MOMP (d, a chlamydial outer membrane protein) and human HSP70 (e, a host cell cytosolic protein). All antibodies detected their corresponding proteins in the HeLa-L2 whole-cell lysate sample (lane 2) and other corresponding samples (as indicated on the right). Note that both cHtrA and CPAF but not CT067 or MOMP were detected in the cytosolic fraction (lane 4). CPAFc represents the C-terminal fragment of CPAF processed during chlamydial infection. The cHtrA degradation fragments (likely produced during in vitro sample processing) can always be detected with varying levels as HtrA is a powerful serine protease known to cleave itself [61] under certain conditions.
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Figure 4: The cHtrA but not CT067 is detected in the cytosolic fraction of the chlamydia-infected HeLa cells. HeLa cells infected with C. trachomatis organisms (Ct-HeLa) were fractionated into nuclear (Ct-HeLa pellet, containing chlamydial inclusions, lane 3) and cytosolic (Ct-HeLa S100, containing chlamydia-secreted proteins, lane 4) fractions. The cellular fractions along with total cell lysates (normal HeLa, lane 1 & Ct-HeLa, lane 2) and purified chlamydial RB (lane 5) and EB (lane 6) organisms as listed at the top were resolved in SDS-polyacrylamide gels. The resolved protein bands were blotted onto nitrocellulose membrane for reacting with antibodies (listed on the left) against cHtrA (panel a), CT067 (b, a periplasmic iron binding protein), CPAF (c, a chlamydia-secreted protein), MOMP (d, a chlamydial outer membrane protein) and human HSP70 (e, a host cell cytosolic protein). All antibodies detected their corresponding proteins in the HeLa-L2 whole-cell lysate sample (lane 2) and other corresponding samples (as indicated on the right). Note that both cHtrA and CPAF but not CT067 or MOMP were detected in the cytosolic fraction (lane 4). CPAFc represents the C-terminal fragment of CPAF processed during chlamydial infection. The cHtrA degradation fragments (likely produced during in vitro sample processing) can always be detected with varying levels as HtrA is a powerful serine protease known to cleave itself [61] under certain conditions.

Mentions: To directly visualize the molecular basis of the anti-cHtrA antibody-labeled cytosolic signals in Chlamydia-infected cells, the infected cells were fractionated into cytosolic (S100) and nuclear/inclusion (pellet) fractions. The distribution of cHtrA and CT067 in different fractions was compared in a Western blot (Figure 4). As a control for chlamydial proteins that are secreted into the host cell cytosol, CPAF was only detected in either the Chlamydia-infected whole cell lysate (Ct-HeLa) or cytosolic fraction (Ct-HeLa S100) samples but not other samples, which is consistent with what has been described previously [26]. Interestingly, cHtrA and its cleavage fragments but not CT067 was also detected in the cytosolic fraction, suggesting that cHtrA but not CT067 is secreted into host cell cytosol although both are periplasmic proteins. The cHtrA degradation fragments are likely generated during in vitro sample processing as HtrA is a powerful serine protease that is known to cleave itself [61]. To monitor the quality of the fractionation, the anti-MOMP antibody was used to indicate the pellet fraction that contains the chlamydial inclusions while an anti-human HSP70 antibody was used to indicate the host cell cytosolic fraction that contains the Chlamydia-secreted proteins. Detection with these antibodies revealed no cross contamination between the pellet and cytosolic fractions. In addition, detection with the anti-MOMP antibody also showed that the amounts of chlamydial organisms in the infected HeLa whole cell lysate, the pellet fraction and purified EB and RB samples were equivalent. These results together have independently confirmed that cHtrA is secreted into cytoplasm of Chlamydia-infected cells although it is also associated with the chlamydial RB and EB 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 cHtrA but not CT067 is detected in the cytosolic fraction of the chlamydia-infected HeLa cells. HeLa cells infected with C. trachomatis organisms (Ct-HeLa) were fractionated into nuclear (Ct-HeLa pellet, containing chlamydial inclusions, lane 3) and cytosolic (Ct-HeLa S100, containing chlamydia-secreted proteins, lane 4) fractions. The cellular fractions along with total cell lysates (normal HeLa, lane 1 & Ct-HeLa, lane 2) and purified chlamydial RB (lane 5) and EB (lane 6) organisms as listed at the top were resolved in SDS-polyacrylamide gels. The resolved protein bands were blotted onto nitrocellulose membrane for reacting with antibodies (listed on the left) against cHtrA (panel a), CT067 (b, a periplasmic iron binding protein), CPAF (c, a chlamydia-secreted protein), MOMP (d, a chlamydial outer membrane protein) and human HSP70 (e, a host cell cytosolic protein). All antibodies detected their corresponding proteins in the HeLa-L2 whole-cell lysate sample (lane 2) and other corresponding samples (as indicated on the right). Note that both cHtrA and CPAF but not CT067 or MOMP were detected in the cytosolic fraction (lane 4). CPAFc represents the C-terminal fragment of CPAF processed during chlamydial infection. The cHtrA degradation fragments (likely produced during in vitro sample processing) can always be detected with varying levels as HtrA is a powerful serine protease known to cleave itself [61] under certain conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3107777&req=5

Figure 4: The cHtrA but not CT067 is detected in the cytosolic fraction of the chlamydia-infected HeLa cells. HeLa cells infected with C. trachomatis organisms (Ct-HeLa) were fractionated into nuclear (Ct-HeLa pellet, containing chlamydial inclusions, lane 3) and cytosolic (Ct-HeLa S100, containing chlamydia-secreted proteins, lane 4) fractions. The cellular fractions along with total cell lysates (normal HeLa, lane 1 & Ct-HeLa, lane 2) and purified chlamydial RB (lane 5) and EB (lane 6) organisms as listed at the top were resolved in SDS-polyacrylamide gels. The resolved protein bands were blotted onto nitrocellulose membrane for reacting with antibodies (listed on the left) against cHtrA (panel a), CT067 (b, a periplasmic iron binding protein), CPAF (c, a chlamydia-secreted protein), MOMP (d, a chlamydial outer membrane protein) and human HSP70 (e, a host cell cytosolic protein). All antibodies detected their corresponding proteins in the HeLa-L2 whole-cell lysate sample (lane 2) and other corresponding samples (as indicated on the right). Note that both cHtrA and CPAF but not CT067 or MOMP were detected in the cytosolic fraction (lane 4). CPAFc represents the C-terminal fragment of CPAF processed during chlamydial infection. The cHtrA degradation fragments (likely produced during in vitro sample processing) can always be detected with varying levels as HtrA is a powerful serine protease known to cleave itself [61] under certain conditions.
Mentions: To directly visualize the molecular basis of the anti-cHtrA antibody-labeled cytosolic signals in Chlamydia-infected cells, the infected cells were fractionated into cytosolic (S100) and nuclear/inclusion (pellet) fractions. The distribution of cHtrA and CT067 in different fractions was compared in a Western blot (Figure 4). As a control for chlamydial proteins that are secreted into the host cell cytosol, CPAF was only detected in either the Chlamydia-infected whole cell lysate (Ct-HeLa) or cytosolic fraction (Ct-HeLa S100) samples but not other samples, which is consistent with what has been described previously [26]. Interestingly, cHtrA and its cleavage fragments but not CT067 was also detected in the cytosolic fraction, suggesting that cHtrA but not CT067 is secreted into host cell cytosol although both are periplasmic proteins. The cHtrA degradation fragments are likely generated during in vitro sample processing as HtrA is a powerful serine protease that is known to cleave itself [61]. To monitor the quality of the fractionation, the anti-MOMP antibody was used to indicate the pellet fraction that contains the chlamydial inclusions while an anti-human HSP70 antibody was used to indicate the host cell cytosolic fraction that contains the Chlamydia-secreted proteins. Detection with these antibodies revealed no cross contamination between the pellet and cytosolic fractions. In addition, detection with the anti-MOMP antibody also showed that the amounts of chlamydial organisms in the infected HeLa whole cell lysate, the pellet fraction and purified EB and RB samples were equivalent. These results together have independently confirmed that cHtrA is secreted into cytoplasm of Chlamydia-infected cells although it is also associated with the chlamydial RB and EB 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