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Published on: June 10, 2020
Analysis of pmpD expression and PmpD post-translational processing during the life cycle of Chlamydia trachomatis
Andrey O Kiselev1, Megan C Skinner, Mary F Lampe
1Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America.
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.
Insights
Polymorphic membrane protein D (PmpD) in Chlamydia is upregulated during infection and secreted outside the bacteria. This study elucidates PmpD processing and secretion, crucial for understanding chlamydial pathogenesis.
Area of Science:
- Microbiology
- Immunology
- Molecular Biology
Background:
- Chlamydia polymorphic membrane protein D (PmpD) shares structural similarities with bacterial autotransporter proteins.
- PmpD's role in protective immunity and its post-translational processing in Chlamydia pathogenesis remain unclear.
Purpose of the Study:
- To investigate the expression and post-translational processing of PmpD during the Chlamydia trachomatis life cycle.
- To elucidate the mechanism of PmpD processing and its secretion pathway.
- To determine PmpD's role in both normal and persistent chlamydial infections.
Main Methods:
- Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) for gene expression analysis.
- Mass spectrometry for identifying protein products of PmpD.
- Confocal microscopy to visualize PmpD secretion.
- Infection of cell cultures at normal and low temperatures (28°C) to study persistent infection.
Main Results:
- The pmpD gene is upregulated 12-24 hours post-infection across C. trachomatis serovars A, D, and L2, coinciding with reticulate body replication.
- Mass spectrometry identified PmpD processing products, suggesting a double pathway model with specific cleavage sites.
- PmpD processing and secretion are uninhibited during low-temperature induced persistent chlamydial infections.
- Confocal microscopy confirmed PmpD secretion outside Chlamydia, increasing with inclusion growth.
Conclusions:
- The pmpD gene exhibits maximal expression during mid-infection, consistent across multiple Chlamydia serovars.
- PmpD functions as an autotransporter protein that undergoes post-translational modification and is secreted extracellularly.
- PmpD secretion occurs during both normal and persistent chlamydial infections, highlighting its potential role in pathogenesis and host interaction.
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