Limits...
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.

Show MeSH

Related in: MedlinePlus

cHtrA is secreted via a sec-dependent pathway. (A) The SignalP 3.0 program with both the Neural Networks (NN) and Hidden Markov Model (HMM) algorithms http://www.expasy.ch was used to analyze the precursor cHtrA sequence from C. trachomatis serovar D http://stdgen.northwestern.edu/. The NN algorithm predicts a signal peptide from the first methionine residue (M1) to a serine residue at position 16 (S16) while the HMM-predicted signal peptide is M1-S23. (B) The M1-S23 peptide of cHtrA (cHtrAss) directed translocation of PhoA into bacterial periplasmic space (cHtrAss-'PhoA, slot 1, blue). Expression of the positive control full-length PhoA construct also led to the translocation of mature PhoA (with its intrinsic signal peptide, slot 3, blue) but the negative control mature PhoA construct failed to do so (without a signal peptide, 'PhoA, slot 2, white). (C) Bacterial transformants expressing the same three constructs were fractionated into periplasmic (per) and cytosolic (cyto) fractions and the fractions were detected with antibodies against a FLAG tag (anti-Flag, panel a) and GroEL (anti-GroEL, panel b) on a Western blot. Mature PhoA was secreted into the periplasm of bacteria expressing either the full-length PhoA construct or HtrAss-PhoA construct while mature PhoA stayed in the cytoplasm of the bacteria expressing the mature PhoA alone construct. (D) cHtrA secretion into the cytosol of chlamydia-infected cells is not inhibited by the type III secretion inhibitor C1 compound. HeLa monolayers infected with C. trachomatis L2 for 6 hr were treated with DMSO (panels a, c & e) or 50 μM C1 (b, d & f). Thirty-six hours after treatment, the cultures were processed for triply labeling with antibodies against IncA (green) and cHtrA, CT621 or CPAF (red) and DAPI for DNA (blue). C1 inhibited secretion of IncA and CT621 but not cHtrA or CPAF. Red arrows indicate chlamydia proteins that are secreted into host cell cytosol.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3107777&req=5

Figure 7: cHtrA is secreted via a sec-dependent pathway. (A) The SignalP 3.0 program with both the Neural Networks (NN) and Hidden Markov Model (HMM) algorithms http://www.expasy.ch was used to analyze the precursor cHtrA sequence from C. trachomatis serovar D http://stdgen.northwestern.edu/. The NN algorithm predicts a signal peptide from the first methionine residue (M1) to a serine residue at position 16 (S16) while the HMM-predicted signal peptide is M1-S23. (B) The M1-S23 peptide of cHtrA (cHtrAss) directed translocation of PhoA into bacterial periplasmic space (cHtrAss-'PhoA, slot 1, blue). Expression of the positive control full-length PhoA construct also led to the translocation of mature PhoA (with its intrinsic signal peptide, slot 3, blue) but the negative control mature PhoA construct failed to do so (without a signal peptide, 'PhoA, slot 2, white). (C) Bacterial transformants expressing the same three constructs were fractionated into periplasmic (per) and cytosolic (cyto) fractions and the fractions were detected with antibodies against a FLAG tag (anti-Flag, panel a) and GroEL (anti-GroEL, panel b) on a Western blot. Mature PhoA was secreted into the periplasm of bacteria expressing either the full-length PhoA construct or HtrAss-PhoA construct while mature PhoA stayed in the cytoplasm of the bacteria expressing the mature PhoA alone construct. (D) cHtrA secretion into the cytosol of chlamydia-infected cells is not inhibited by the type III secretion inhibitor C1 compound. HeLa monolayers infected with C. trachomatis L2 for 6 hr were treated with DMSO (panels a, c & e) or 50 μM C1 (b, d & f). Thirty-six hours after treatment, the cultures were processed for triply labeling with antibodies against IncA (green) and cHtrA, CT621 or CPAF (red) and DAPI for DNA (blue). C1 inhibited secretion of IncA and CT621 but not cHtrA or CPAF. Red arrows indicate chlamydia proteins that are secreted into host cell cytosol.

Mentions: To determine the secretion pathway that chlamydial organisms may use to secrete cHtrA, we analyzed the amino acid sequence of cHtrA for secretion signal sequences using the program SignalP version 3.0 with NN (neural network) and HMM (hidden markov model) algorithms http://www.expasy.ch. Both NN and HMM algorithms predict an N-terminal signal peptide in cHtrA but with different cleavage sites. NN predicts a cleavage between S16 and S17 while HMM predicts the cleavage site between S23 and A24 (Figure 7A). We then tested the functionality of the cHtrA N-terminal sequence M1-S23 using a bacterium-based phoA gene fusion system (Figure 7B &7C). This assay system takes advantage of two characteristics of PhoA: the enzyme is only active after translocation into the bacterial periplasm, and the phosphatase activity can be conveniently monitored with the chromogenic substrate BCIP. DNA coding for the cHtrA N-terminal signal sequence covering residues M1 to S23 (designated as cHtrAss) was fused to the DNA sequence coding for mature PhoA (designated as 'PhoA). The fusion construct was expressed in pFLAG-CTC vector which adds a Flag epitope to the C-terminus of 'PhoA. The mature 'PhoA alone construct was used as a negative control while the precursor full-length PhoA (with its native N-terminal signal peptide) served as a positive control. As shown in Figure 7B, in the presence of BCIP, bacteria expressing either the precursor PhoA or the cHtrAss-'PhoA fusion constructs turned blue whereas bacteria expressing the mature PhoA alone ('PhoA) remained white, indicating that both the native PhoA and cHtrA signal peptides directed the translocation of PhoA into periplasm. We further used a Western blot analysis to monitor the distribution of PhoA protein in periplasmic (per) and cytosolic (cyto) fractions (Figure 7C). Mature PhoA was detected in the periplasm of bacteria expressing either the precursor PhoA or HtrAss-'PhoA fusion constructs while mature PhoA was only detected in the cytoplasm of the bacteria expressing the leaderless PhoA. Thus, the cHtrA N-terminal signal peptide is sufficient for directing PhoA across the bacterial inner membrane. We further found that the secretion of cHtrA was not inhibited by the C1 compound, an inhibitor known to inhibit chlamydial type III secretion system [52]. As positive controls, C1 inhibited the secretion of both IncA and CT621, two known chlamydial type III secretion substrates [30,52]. Consistently, the secretion of CPAF was not affected by C1. This is because secretion of CPAF is dependent on type II secretion pathway [62].


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)

cHtrA is secreted via a sec-dependent pathway. (A) The SignalP 3.0 program with both the Neural Networks (NN) and Hidden Markov Model (HMM) algorithms http://www.expasy.ch was used to analyze the precursor cHtrA sequence from C. trachomatis serovar D http://stdgen.northwestern.edu/. The NN algorithm predicts a signal peptide from the first methionine residue (M1) to a serine residue at position 16 (S16) while the HMM-predicted signal peptide is M1-S23. (B) The M1-S23 peptide of cHtrA (cHtrAss) directed translocation of PhoA into bacterial periplasmic space (cHtrAss-'PhoA, slot 1, blue). Expression of the positive control full-length PhoA construct also led to the translocation of mature PhoA (with its intrinsic signal peptide, slot 3, blue) but the negative control mature PhoA construct failed to do so (without a signal peptide, 'PhoA, slot 2, white). (C) Bacterial transformants expressing the same three constructs were fractionated into periplasmic (per) and cytosolic (cyto) fractions and the fractions were detected with antibodies against a FLAG tag (anti-Flag, panel a) and GroEL (anti-GroEL, panel b) on a Western blot. Mature PhoA was secreted into the periplasm of bacteria expressing either the full-length PhoA construct or HtrAss-PhoA construct while mature PhoA stayed in the cytoplasm of the bacteria expressing the mature PhoA alone construct. (D) cHtrA secretion into the cytosol of chlamydia-infected cells is not inhibited by the type III secretion inhibitor C1 compound. HeLa monolayers infected with C. trachomatis L2 for 6 hr were treated with DMSO (panels a, c & e) or 50 μM C1 (b, d & f). Thirty-six hours after treatment, the cultures were processed for triply labeling with antibodies against IncA (green) and cHtrA, CT621 or CPAF (red) and DAPI for DNA (blue). C1 inhibited secretion of IncA and CT621 but not cHtrA or CPAF. Red arrows indicate chlamydia proteins that are secreted into host cell cytosol.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: cHtrA is secreted via a sec-dependent pathway. (A) The SignalP 3.0 program with both the Neural Networks (NN) and Hidden Markov Model (HMM) algorithms http://www.expasy.ch was used to analyze the precursor cHtrA sequence from C. trachomatis serovar D http://stdgen.northwestern.edu/. The NN algorithm predicts a signal peptide from the first methionine residue (M1) to a serine residue at position 16 (S16) while the HMM-predicted signal peptide is M1-S23. (B) The M1-S23 peptide of cHtrA (cHtrAss) directed translocation of PhoA into bacterial periplasmic space (cHtrAss-'PhoA, slot 1, blue). Expression of the positive control full-length PhoA construct also led to the translocation of mature PhoA (with its intrinsic signal peptide, slot 3, blue) but the negative control mature PhoA construct failed to do so (without a signal peptide, 'PhoA, slot 2, white). (C) Bacterial transformants expressing the same three constructs were fractionated into periplasmic (per) and cytosolic (cyto) fractions and the fractions were detected with antibodies against a FLAG tag (anti-Flag, panel a) and GroEL (anti-GroEL, panel b) on a Western blot. Mature PhoA was secreted into the periplasm of bacteria expressing either the full-length PhoA construct or HtrAss-PhoA construct while mature PhoA stayed in the cytoplasm of the bacteria expressing the mature PhoA alone construct. (D) cHtrA secretion into the cytosol of chlamydia-infected cells is not inhibited by the type III secretion inhibitor C1 compound. HeLa monolayers infected with C. trachomatis L2 for 6 hr were treated with DMSO (panels a, c & e) or 50 μM C1 (b, d & f). Thirty-six hours after treatment, the cultures were processed for triply labeling with antibodies against IncA (green) and cHtrA, CT621 or CPAF (red) and DAPI for DNA (blue). C1 inhibited secretion of IncA and CT621 but not cHtrA or CPAF. Red arrows indicate chlamydia proteins that are secreted into host cell cytosol.
Mentions: To determine the secretion pathway that chlamydial organisms may use to secrete cHtrA, we analyzed the amino acid sequence of cHtrA for secretion signal sequences using the program SignalP version 3.0 with NN (neural network) and HMM (hidden markov model) algorithms http://www.expasy.ch. Both NN and HMM algorithms predict an N-terminal signal peptide in cHtrA but with different cleavage sites. NN predicts a cleavage between S16 and S17 while HMM predicts the cleavage site between S23 and A24 (Figure 7A). We then tested the functionality of the cHtrA N-terminal sequence M1-S23 using a bacterium-based phoA gene fusion system (Figure 7B &7C). This assay system takes advantage of two characteristics of PhoA: the enzyme is only active after translocation into the bacterial periplasm, and the phosphatase activity can be conveniently monitored with the chromogenic substrate BCIP. DNA coding for the cHtrA N-terminal signal sequence covering residues M1 to S23 (designated as cHtrAss) was fused to the DNA sequence coding for mature PhoA (designated as 'PhoA). The fusion construct was expressed in pFLAG-CTC vector which adds a Flag epitope to the C-terminus of 'PhoA. The mature 'PhoA alone construct was used as a negative control while the precursor full-length PhoA (with its native N-terminal signal peptide) served as a positive control. As shown in Figure 7B, in the presence of BCIP, bacteria expressing either the precursor PhoA or the cHtrAss-'PhoA fusion constructs turned blue whereas bacteria expressing the mature PhoA alone ('PhoA) remained white, indicating that both the native PhoA and cHtrA signal peptides directed the translocation of PhoA into periplasm. We further used a Western blot analysis to monitor the distribution of PhoA protein in periplasmic (per) and cytosolic (cyto) fractions (Figure 7C). Mature PhoA was detected in the periplasm of bacteria expressing either the precursor PhoA or HtrAss-'PhoA fusion constructs while mature PhoA was only detected in the cytoplasm of the bacteria expressing the leaderless PhoA. Thus, the cHtrA N-terminal signal peptide is sufficient for directing PhoA across the bacterial inner membrane. We further found that the secretion of cHtrA was not inhibited by the C1 compound, an inhibitor known to inhibit chlamydial type III secretion system [52]. As positive controls, C1 inhibited the secretion of both IncA and CT621, two known chlamydial type III secretion substrates [30,52]. Consistently, the secretion of CPAF was not affected by C1. This is because secretion of CPAF is dependent on type II secretion pathway [62].

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