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Updated: Jan 2, 2026
Live-Cell Forward Genetic Approach to Identify and Isolate Developmental Mutants in Chlamydia trachomatis
Published on: June 10, 2020
iTRAQ-Based Quantitative Proteomics Analysis of HeLa Cells Infected With Chlamydia muridarum TC0668 Mutant and
Yingzi Wang1,2, Emmanuel Wirekoh Arthur1,2, Na Liu1,2
1Institute of Pathogenic Biology, Hengyang Medical College, University of South China, Hengyang, China.
Abstract:
Chlamydia muridarum, an obligate intracellular pathogen, was used to establish a murine model of female upper genital tract infection by Chlamydia trachomatis. TC0668 in C. muridarum is a hypothetical chromosomal virulence protein that is involved in upper genital tract pathogenesis. The infection of mice with the C. muridarum TC0668-mutant (G216*) strain results in less pathological damage in the upper genital tract. In this study, an isobaric tags for relative and absolute quantitation (iTRAQ)-based quantitative proteomics analysis was performed to identify differentially expressed proteins between TC0668 wild-type (TC0668wt) and TC0668 mutant (TC0668mut) strains at 6, 12, 18, and 24 h post-infection (p.i.). Of the 550 proteins differentially expressed at 18 h p.i., 222 and 328 were up-regulated and down-regulated, respectively, inTC0668mut-infected cells. The expression of seven up-regulated proteins (encoded by SRPRB, JAK1, PMM1, HLA-DQB1, THBS1, ITPR1, and BCAP31) and three down-regulated proteins (encoded by MAPKAPK2, TRAFD1, and IFI16) from the iTRAQ analysis were validated using quantitative real-time (qRT)-PCR. The qRT-PCR results were consistent with those of iTRAQ. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses revealed that the differentially expressed proteins primarily participated in inflammatory responses, fibrosis, metabolic processes, and complement coagulation cascades, and were mainly enriched in the phosphatidylinositol 3'-kinase (PI3K)/Akt, nuclear factor kappa-B (NF-κB), and other signaling pathways. Using western-blotting and immunofluorescence detection, significant differences in activation of the PI3K/Akt and NF-κB signaling pathways were observed between the TC0668wt- and TC0668mut-infected cells. Differentially expressed proteins linked with inflammation and fibrosis were used in a protein-protein interaction network analysis. The results suggest that TC0668 may play a pivotal role in C. muridarum-induced genital pathology by inducing inflammatory responses and fibrosis, which may involve the activation of the PI3K/Akt and NF-κB signaling pathways.
Insights
Chlamydia muridarum virulence protein TC0668 drives genital tract pathology by promoting inflammation and fibrosis. Its absence reduces damage, implicating TC0668 in Chlamydia trachomatis pathogenesis.
Area of Science:
- Microbiology
- Immunology
- Proteomics
Background:
- Chlamydia muridarum establishes a murine model for Chlamydia trachomatis upper genital tract infection.
- TC0668 is a hypothetical virulence protein in C. muridarum implicated in pathogenesis.
- Mutant strains lacking TC0668 exhibit reduced pathological damage.
Purpose of the Study:
- To investigate the role of TC0668 in C. muridarum-induced genital tract pathogenesis.
- To identify differentially expressed proteins and affected signaling pathways using quantitative proteomics.
- To validate proteomic findings and analyze protein-protein interactions related to inflammation and fibrosis.
Main Methods:
- Isobaric tags for relative and absolute quantitation (iTRAQ) for quantitative proteomics.
- Quantitative real-time PCR (qRT-PCR) for gene expression validation.
- Western-blotting, immunofluorescence, Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses.
Main Results:
- Proteomics identified 550 differentially expressed proteins at 18 hours post-infection between wild-type and TC0668 mutant strains.
- qRT-PCR validated the differential expression of key inflammatory and fibrotic proteins.
- Pathway analysis revealed enrichment in inflammatory responses, fibrosis, and PI3K/Akt and NF-κB signaling pathways.
Conclusions:
- TC0668 plays a critical role in C. muridarum-induced genital pathology.
- TC0668 likely induces inflammation and fibrosis via PI3K/Akt and NF-κB signaling.
- Targeting TC0668 may offer therapeutic strategies for Chlamydia-related diseases.
