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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 passenger domain of PmpD is secreted outside Chlamydia and accumulated in the inclusion lumen during the life cycle of C. trachomatis.McCoy cells were infected with C. trachomatis serovar L2 and fixed with methanol at 16 (A and E), 24 (B and F), 36 (C and G), and 48 (D and H) h p.i. and reacted with mAb against chlamydial MOMP (green) in combination with pAb against fragment 2 of PmpD (red) (A–D) or pAb against chlamydial HSP60 protein (red) (E–H) (internal control). Arrow indicates localization of PmpD during the chlamydial life cycle on the surface of Chlamydia (A) or in the inclusion lumen outside Chlamydia (red spots) (B–D). When reacted with anti-HSP60 pAb, no material was visible outside Chlamydia. The photographs were made using a Leica SL confocal microscope.
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pone-0005191-g009: The passenger domain of PmpD is secreted outside Chlamydia and accumulated in the inclusion lumen during the life cycle of C. trachomatis.McCoy cells were infected with C. trachomatis serovar L2 and fixed with methanol at 16 (A and E), 24 (B and F), 36 (C and G), and 48 (D and H) h p.i. and reacted with mAb against chlamydial MOMP (green) in combination with pAb against fragment 2 of PmpD (red) (A–D) or pAb against chlamydial HSP60 protein (red) (E–H) (internal control). Arrow indicates localization of PmpD during the chlamydial life cycle on the surface of Chlamydia (A) or in the inclusion lumen outside Chlamydia (red spots) (B–D). When reacted with anti-HSP60 pAb, no material was visible outside Chlamydia. The photographs were made using a Leica SL confocal microscope.

Mentions: IMF microscopy of Chlamydia infected McCoy cells fixed at specified time points after infection and double stained with antibodies against MOMP and fragment 2 of PmpD or the HSP60 protein demonstrated that at 16 h p.i., PmpD was found strictly on the surface of chlamydial RBs (Fig. 9A). A small amount of reactive protein material was clearly visible outside chlamydial particles at 24 h p.i., but much more protein inside the inclusion lumen was visible at 48 h after infection (Fig. 9B–D), which is in agreement with the results of Swanson et al [18]. No such protein material was observed outside Chlamydia in infected culture cells stained with antibodies against MOMP (localized on the surface of Chlamydia), HSP60 (Fig 9E–H), PmpA (internal protein controls) (Fig 10C–D) or IncA, which is secreted outside Chlamydia and localized to the inclusion membrane (Fig 10E–F). Antibodies against other PmpD fragments, with the exception of fragment 4, stain McCoy cells infected with Chlamydia similarly to antibodies against fragment 2 (not shown). Antibodies against fragment 4 (the beta-barrel) showed that this portion of PmpD is not secreted and remains in the cell wall of Chlamydia (Fig 10A–B) as a part of the ≈80 kDa protein. Similarly to antibodies against fragments 1, 2, and 3, antibodies against fragment 4 stained RBs inside inclusions in host cells infected with Chlamydia and fixed with methanol in a doughnut-like staining pattern. However, contrary to antibodies against other PmpD fragments, antibodies against fragment 4 do not stain unfixed purified RBs (not shown), indicating that the beta-barrel is not localized on the surface of Chlamydia. In addition, no stained material was visible outside the inclusion.


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 passenger domain of PmpD is secreted outside Chlamydia and accumulated in the inclusion lumen during the life cycle of C. trachomatis.McCoy cells were infected with C. trachomatis serovar L2 and fixed with methanol at 16 (A and E), 24 (B and F), 36 (C and G), and 48 (D and H) h p.i. and reacted with mAb against chlamydial MOMP (green) in combination with pAb against fragment 2 of PmpD (red) (A–D) or pAb against chlamydial HSP60 protein (red) (E–H) (internal control). Arrow indicates localization of PmpD during the chlamydial life cycle on the surface of Chlamydia (A) or in the inclusion lumen outside Chlamydia (red spots) (B–D). When reacted with anti-HSP60 pAb, no material was visible outside Chlamydia. The photographs were made using a Leica SL confocal microscope.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2666266&req=5

pone-0005191-g009: The passenger domain of PmpD is secreted outside Chlamydia and accumulated in the inclusion lumen during the life cycle of C. trachomatis.McCoy cells were infected with C. trachomatis serovar L2 and fixed with methanol at 16 (A and E), 24 (B and F), 36 (C and G), and 48 (D and H) h p.i. and reacted with mAb against chlamydial MOMP (green) in combination with pAb against fragment 2 of PmpD (red) (A–D) or pAb against chlamydial HSP60 protein (red) (E–H) (internal control). Arrow indicates localization of PmpD during the chlamydial life cycle on the surface of Chlamydia (A) or in the inclusion lumen outside Chlamydia (red spots) (B–D). When reacted with anti-HSP60 pAb, no material was visible outside Chlamydia. The photographs were made using a Leica SL confocal microscope.
Mentions: IMF microscopy of Chlamydia infected McCoy cells fixed at specified time points after infection and double stained with antibodies against MOMP and fragment 2 of PmpD or the HSP60 protein demonstrated that at 16 h p.i., PmpD was found strictly on the surface of chlamydial RBs (Fig. 9A). A small amount of reactive protein material was clearly visible outside chlamydial particles at 24 h p.i., but much more protein inside the inclusion lumen was visible at 48 h after infection (Fig. 9B–D), which is in agreement with the results of Swanson et al [18]. No such protein material was observed outside Chlamydia in infected culture cells stained with antibodies against MOMP (localized on the surface of Chlamydia), HSP60 (Fig 9E–H), PmpA (internal protein controls) (Fig 10C–D) or IncA, which is secreted outside Chlamydia and localized to the inclusion membrane (Fig 10E–F). Antibodies against other PmpD fragments, with the exception of fragment 4, stain McCoy cells infected with Chlamydia similarly to antibodies against fragment 2 (not shown). Antibodies against fragment 4 (the beta-barrel) showed that this portion of PmpD is not secreted and remains in the cell wall of Chlamydia (Fig 10A–B) as a part of the ≈80 kDa protein. Similarly to antibodies against fragments 1, 2, and 3, antibodies against fragment 4 stained RBs inside inclusions in host cells infected with Chlamydia and fixed with methanol in a doughnut-like staining pattern. However, contrary to antibodies against other PmpD fragments, antibodies against fragment 4 do not stain unfixed purified RBs (not shown), indicating that the beta-barrel is not localized on the surface of Chlamydia. In addition, no stained material was visible outside the inclusion.

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