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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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PmpD processing and secretion at 28°C.McCoy cells infected with C. trachomatis serovar L2 were incubated at 28°C, harvested every 48 h, and soluble fractions prepared as described in METHODS. The proteins in each soluble fraction were reacted in an immunoblot with antibodies against fragment 2 of PmpD. Lane 1, 48 h p.i. Lane 2, 96 h p.i. Lane 3, 144 h p.i. Lane 4, 192 h p.i.
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pone-0005191-g013: PmpD processing and secretion at 28°C.McCoy cells infected with C. trachomatis serovar L2 were incubated at 28°C, harvested every 48 h, and soluble fractions prepared as described in METHODS. The proteins in each soluble fraction were reacted in an immunoblot with antibodies against fragment 2 of PmpD. Lane 1, 48 h p.i. Lane 2, 96 h p.i. Lane 3, 144 h p.i. Lane 4, 192 h p.i.

Mentions: Chlamydia infected McCoy cells were constantly monitored using inverted light microscopy during the time course of 192 h p.i., which revealed formation of multiple small non-fusogenic or single “empty” looking giant inclusions (Fig 11). IMF microscopy of infected McCoy cells fixed at 48 and 120 h p.i. and double stained with antibodies against MOMP and PmpD/HSP60 exposed inclusions filled with abnormally large aberrant RBs with a thin cell wall compared with uniformly sized and round shaped RBs produced by normal infection (Fig 12), suggesting persistent infection in culture cells. These RBs were stained in a pattern very similar to that observed in a normal infection, with HSP60 localized inside and PmpD and MOMP on the surface of RBs. In addition, a small amount of protein material stained with pAb against PmpD was clearly visible outside chlamydial RBs at 120 h p.i., indicating PmpD secretion, however at a much lower and slower rate compared with normal (37°C) chlamydial infection. An immunoblot with soluble fractions prepared from McCoy cells infected with Chlamydia, incubated at 28°C, and prepared at 48, 96, 144, and 192 h p.i., showed the ≈120 and 65 kDa protein bands in the 96–192 h p.i. materials reacted with anti-PmpD antibodies (Fig. 13), indicating that the PmpD protein was post-translationally processed and secreted outside Chlamydia and confirming the results of IMF microscopy. To ensure that the 192 h p.i. insoluble material contained no viable infectious particles (chlamydial EBs), it was used to infect McCoy cells in 96-well tissue culture plates (CORNING). Cells were stained with antibodies against MOMP and examined by IMF microscopy 48 h after infection. McCoy cells seeded with material obtained at 192 h p.i. after incubation at 28°C contained no inclusions. At the same time, chlamydial inclusions were seen in cells seeded with control material obtained from infected cells which were incubated for 48 h at 37°C.


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)

PmpD processing and secretion at 28°C.McCoy cells infected with C. trachomatis serovar L2 were incubated at 28°C, harvested every 48 h, and soluble fractions prepared as described in METHODS. The proteins in each soluble fraction were reacted in an immunoblot with antibodies against fragment 2 of PmpD. Lane 1, 48 h p.i. Lane 2, 96 h p.i. Lane 3, 144 h p.i. Lane 4, 192 h p.i.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0005191-g013: PmpD processing and secretion at 28°C.McCoy cells infected with C. trachomatis serovar L2 were incubated at 28°C, harvested every 48 h, and soluble fractions prepared as described in METHODS. The proteins in each soluble fraction were reacted in an immunoblot with antibodies against fragment 2 of PmpD. Lane 1, 48 h p.i. Lane 2, 96 h p.i. Lane 3, 144 h p.i. Lane 4, 192 h p.i.
Mentions: Chlamydia infected McCoy cells were constantly monitored using inverted light microscopy during the time course of 192 h p.i., which revealed formation of multiple small non-fusogenic or single “empty” looking giant inclusions (Fig 11). IMF microscopy of infected McCoy cells fixed at 48 and 120 h p.i. and double stained with antibodies against MOMP and PmpD/HSP60 exposed inclusions filled with abnormally large aberrant RBs with a thin cell wall compared with uniformly sized and round shaped RBs produced by normal infection (Fig 12), suggesting persistent infection in culture cells. These RBs were stained in a pattern very similar to that observed in a normal infection, with HSP60 localized inside and PmpD and MOMP on the surface of RBs. In addition, a small amount of protein material stained with pAb against PmpD was clearly visible outside chlamydial RBs at 120 h p.i., indicating PmpD secretion, however at a much lower and slower rate compared with normal (37°C) chlamydial infection. An immunoblot with soluble fractions prepared from McCoy cells infected with Chlamydia, incubated at 28°C, and prepared at 48, 96, 144, and 192 h p.i., showed the ≈120 and 65 kDa protein bands in the 96–192 h p.i. materials reacted with anti-PmpD antibodies (Fig. 13), indicating that the PmpD protein was post-translationally processed and secreted outside Chlamydia and confirming the results of IMF microscopy. To ensure that the 192 h p.i. insoluble material contained no viable infectious particles (chlamydial EBs), it was used to infect McCoy cells in 96-well tissue culture plates (CORNING). Cells were stained with antibodies against MOMP and examined by IMF microscopy 48 h after infection. McCoy cells seeded with material obtained at 192 h p.i. after incubation at 28°C contained no inclusions. At the same time, chlamydial inclusions were seen in cells seeded with control material obtained from infected cells which were incubated for 48 h at 37°C.

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