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Analysis of pmpD expression and PmpD post-translational processing during the life cycle of Chlamydia trachomatis serovars A, D, and L2.

Kiselev AO, Skinner MC, Lampe MF - PLoS ONE (2009)

Bottom Line: Each of these three serovars targets different human organs and tissues and encodes a different pmpD gene nucleotide sequence.Using mass spectrometry analysis, we identified the protein products of post-translational processing of PmpD of C. trachomatis serovar L2 and propose a double pathway model for PmpD processing, with one cleavage site between the passenger and autotransporter domains and the other site in the middle of the passenger domain.Notably, when Chlamydia infected culture cells were subjected to low (28 degrees C) temperature, PmpD post-translational processing and secretion was found to be uninhibited in the resulting persistent infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: The polymorphic membrane protein D (PmpD) in Chlamydia is structurally similar to autotransporter proteins described in other bacteria and may be involved in cellular and humoral protective immunity against Chlamydia. The mechanism of PmpD post-translational processing and the role of its protein products in the pathogenesis of chlamydial infection have not been very well elucidated to date.

Methodology/principal findings: Here we examined the expression and post-translational processing of the protein product of the pmpD gene during the life cycle of C. trachomatis serovars A, D, and L2. Each of these three serovars targets different human organs and tissues and encodes a different pmpD gene nucleotide sequence. Our quantitative real-time reverse transcription polymerase chain reaction results demonstrate that the pmpD gene is up-regulated at 12-24 hours after infection regardless of the Chlamydia serovar. This up-regulation is coincidental with the period of exponential growth and replication of reticulate bodies (RB) of Chlamydia and indicates a probable similarity in function of pmpD in serovars A, D, and L2 of Chlamydia. Using mass spectrometry analysis, we identified the protein products of post-translational processing of PmpD of C. trachomatis serovar L2 and propose a double pathway model for PmpD processing, with one cleavage site between the passenger and autotransporter domains and the other site in the middle of the passenger domain. Notably, when Chlamydia infected culture cells were subjected to low (28 degrees C) temperature, PmpD post-translational processing and secretion was found to be uninhibited in the resulting persistent infection. In addition, confocal microscopy of cells infected with Chlamydia confirms our earlier hypothesis that PmpD is secreted outside Chlamydia and its secretion increases with growth of the chlamydial inclusion.

Conclusion/significance: The results of this current study involving multiple Chlamydia serovars support the general consensus that the pmpD gene is maximally expressed at mid infection and provide new information about PmpD as an autotransporter protein which is post-translationally processed and secreted outside Chlamydia during normal and low temperature induced persistent chlamydial infection.

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The amino acid sequence of the PmpD fragments used to raise antibodies and antibody reaction with proteins present in the soluble fraction.A. The amino acid sequence of the PmpD fragments used to raise antibodies (Fragment 1, aa 17–517; fragment 2, aa 470–818; fragment 3, aa 819–1180; fragment 4, aa 1174–1531). B. Reaction of antibodies raised against PmpD fragments with proteins present in the soluble fraction. McCoy cells infected with C. trachomatis serovar L2 were harvested at 48 h p.i. and the soluble fraction was prepared as described in METHODS. Lane 1, pAb against fragment 1. Lane 2, pAb against fragment 2. Lane 3, pAb against fragment 3. Lane 4, pAb against fragment 4.
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pone-0005191-g008: The amino acid sequence of the PmpD fragments used to raise antibodies and antibody reaction with proteins present in the soluble fraction.A. The amino acid sequence of the PmpD fragments used to raise antibodies (Fragment 1, aa 17–517; fragment 2, aa 470–818; fragment 3, aa 819–1180; fragment 4, aa 1174–1531). B. Reaction of antibodies raised against PmpD fragments with proteins present in the soluble fraction. McCoy cells infected with C. trachomatis serovar L2 were harvested at 48 h p.i. and the soluble fraction was prepared as described in METHODS. Lane 1, pAb against fragment 1. Lane 2, pAb against fragment 2. Lane 3, pAb against fragment 3. Lane 4, pAb against fragment 4.

Mentions: In our earlier study using MS analysis, we identified one of the proteins which reacted with antibodies against fragment 2 of PmpD, the ≈157 kDa protein, as the nearly full length PmpD [19]. In this work, MS analysis of the ≈120 and 65 kDa proteins revealed that they lack the peptides located in the signal sequence and the transporter domain (TD) (the beta-barrel) (Fig 4 and 5). Moreover, the ≈65 kDa protein contained peptides identical to those located in the N-terminal half (aa 68–698) of the 120 kDa passenger domain of PmpD (aa 68–1016) (Fig 7). The peptides found in the ≈80 kDa protein began where the peptides of the ≈65 kDa protein ended and consisted of the C-terminal half of the PD and the full beta-barrel (aa 699–1482) (Fig 6 and 7). Analyzing the proteome of purified EBs of C. trachomatis serovars A and D, Shaw et al [35] found a protein with a very similar molecular weight (87.4 kDa) whose peptides “were all located in the C-terminal part” of PmpD. In addition to the results obtained from mass spectrometry, proteins present in the soluble fraction prepared after 48 h of infection of McCoy cells with C. trachomatis serovar L2 were probed with antibodies generated against other fragments of PmpD [19]. Each antibody reaction strongly correlated with the peptide fragment of PmpD against which the antibodies were generated (Fig. 8A). Thus, antibodies against fragment 1 did not react with the ≈80 kDa protein band, antibodies against fragment 3 did not react with the ≈65 kDa band, and antibodies against fragment 4 (the beta-barrel) reacted with neither the ≈120 nor 65 kDa proteins (Fig. 8B).


Analysis of pmpD expression and PmpD post-translational processing during the life cycle of Chlamydia trachomatis serovars A, D, and L2.

Kiselev AO, Skinner MC, Lampe MF - PLoS ONE (2009)

The amino acid sequence of the PmpD fragments used to raise antibodies and antibody reaction with proteins present in the soluble fraction.A. The amino acid sequence of the PmpD fragments used to raise antibodies (Fragment 1, aa 17–517; fragment 2, aa 470–818; fragment 3, aa 819–1180; fragment 4, aa 1174–1531). B. Reaction of antibodies raised against PmpD fragments with proteins present in the soluble fraction. McCoy cells infected with C. trachomatis serovar L2 were harvested at 48 h p.i. and the soluble fraction was prepared as described in METHODS. Lane 1, pAb against fragment 1. Lane 2, pAb against fragment 2. Lane 3, pAb against fragment 3. Lane 4, pAb against fragment 4.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2666266&req=5

pone-0005191-g008: The amino acid sequence of the PmpD fragments used to raise antibodies and antibody reaction with proteins present in the soluble fraction.A. The amino acid sequence of the PmpD fragments used to raise antibodies (Fragment 1, aa 17–517; fragment 2, aa 470–818; fragment 3, aa 819–1180; fragment 4, aa 1174–1531). B. Reaction of antibodies raised against PmpD fragments with proteins present in the soluble fraction. McCoy cells infected with C. trachomatis serovar L2 were harvested at 48 h p.i. and the soluble fraction was prepared as described in METHODS. Lane 1, pAb against fragment 1. Lane 2, pAb against fragment 2. Lane 3, pAb against fragment 3. Lane 4, pAb against fragment 4.
Mentions: In our earlier study using MS analysis, we identified one of the proteins which reacted with antibodies against fragment 2 of PmpD, the ≈157 kDa protein, as the nearly full length PmpD [19]. In this work, MS analysis of the ≈120 and 65 kDa proteins revealed that they lack the peptides located in the signal sequence and the transporter domain (TD) (the beta-barrel) (Fig 4 and 5). Moreover, the ≈65 kDa protein contained peptides identical to those located in the N-terminal half (aa 68–698) of the 120 kDa passenger domain of PmpD (aa 68–1016) (Fig 7). The peptides found in the ≈80 kDa protein began where the peptides of the ≈65 kDa protein ended and consisted of the C-terminal half of the PD and the full beta-barrel (aa 699–1482) (Fig 6 and 7). Analyzing the proteome of purified EBs of C. trachomatis serovars A and D, Shaw et al [35] found a protein with a very similar molecular weight (87.4 kDa) whose peptides “were all located in the C-terminal part” of PmpD. In addition to the results obtained from mass spectrometry, proteins present in the soluble fraction prepared after 48 h of infection of McCoy cells with C. trachomatis serovar L2 were probed with antibodies generated against other fragments of PmpD [19]. Each antibody reaction strongly correlated with the peptide fragment of PmpD against which the antibodies were generated (Fig. 8A). Thus, antibodies against fragment 1 did not react with the ≈80 kDa protein band, antibodies against fragment 3 did not react with the ≈65 kDa band, and antibodies against fragment 4 (the beta-barrel) reacted with neither the ≈120 nor 65 kDa proteins (Fig. 8B).

Bottom Line: Each of these three serovars targets different human organs and tissues and encodes a different pmpD gene nucleotide sequence.Using mass spectrometry analysis, we identified the protein products of post-translational processing of PmpD of C. trachomatis serovar L2 and propose a double pathway model for PmpD processing, with one cleavage site between the passenger and autotransporter domains and the other site in the middle of the passenger domain.Notably, when Chlamydia infected culture cells were subjected to low (28 degrees C) temperature, PmpD post-translational processing and secretion was found to be uninhibited in the resulting persistent infection.

View Article: PubMed Central - PubMed

Affiliation: Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America.

ABSTRACT

Background: The polymorphic membrane protein D (PmpD) in Chlamydia is structurally similar to autotransporter proteins described in other bacteria and may be involved in cellular and humoral protective immunity against Chlamydia. The mechanism of PmpD post-translational processing and the role of its protein products in the pathogenesis of chlamydial infection have not been very well elucidated to date.

Methodology/principal findings: Here we examined the expression and post-translational processing of the protein product of the pmpD gene during the life cycle of C. trachomatis serovars A, D, and L2. Each of these three serovars targets different human organs and tissues and encodes a different pmpD gene nucleotide sequence. Our quantitative real-time reverse transcription polymerase chain reaction results demonstrate that the pmpD gene is up-regulated at 12-24 hours after infection regardless of the Chlamydia serovar. This up-regulation is coincidental with the period of exponential growth and replication of reticulate bodies (RB) of Chlamydia and indicates a probable similarity in function of pmpD in serovars A, D, and L2 of Chlamydia. Using mass spectrometry analysis, we identified the protein products of post-translational processing of PmpD of C. trachomatis serovar L2 and propose a double pathway model for PmpD processing, with one cleavage site between the passenger and autotransporter domains and the other site in the middle of the passenger domain. Notably, when Chlamydia infected culture cells were subjected to low (28 degrees C) temperature, PmpD post-translational processing and secretion was found to be uninhibited in the resulting persistent infection. In addition, confocal microscopy of cells infected with Chlamydia confirms our earlier hypothesis that PmpD is secreted outside Chlamydia and its secretion increases with growth of the chlamydial inclusion.

Conclusion/significance: The results of this current study involving multiple Chlamydia serovars support the general consensus that the pmpD gene is maximally expressed at mid infection and provide new information about PmpD as an autotransporter protein which is post-translationally processed and secreted outside Chlamydia during normal and low temperature induced persistent chlamydial infection.

Show MeSH
Related in: MedlinePlus