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Biosynthesis and disulfide cross-linking of outer membrane components during the growth cycle of Chlamydia
Abstract:
The synthesis and accumulation of Chlamydia trachomatis outer membrane proteins within infected HeLa 229 host cells were monitored by assessing the uptake of [35S]cysteine into chlamydial proteins during the 48-h growth cycle of a lymphogranuloma venereum strain, L2/434/Bu. Synthesis of the major outer membrane protein, a protein that accounts for about 60% of the outer membrane protein mass of elementary bodies (EB), was first detected between 12 and 18 h after infection. The uptake of [35S]cysteine into the 60,000-molecular-weight doublet (60K doublet) and 12.5K cysteine-rich proteins was not observed until 30 h after infection, when the intracellularly dividing reticulate bodies were beginning to transform into infectious EBs. By using a more sensitive immunoblotting method in conjunction with monoclonal antibodies specific for the 60K doublet proteins, synthesis of these proteins was detected even earlier, by 18 h after infection. These data suggest that the time and extent of synthesis of these outer membrane proteins are regulated by processes that coincide in time with the transformation of reticulate bodies into EBs. Additional studies were performed to determine the extent of disulfide cross-linking of outer membrane proteins during the growth cycle. Both the major outer membrane protein and the 12.5K protein became progressively cross-linked to about 60% during the last 24 h of the growth cycle, whereas the 60K doublet proteins were extensively cross-linked during most of the cycle. These data may indicate an intracellular cross-linking mechanism, possibly enzymatic, that exists in addition to an auto-oxidation mechanism that occurs upon host cell lysis and exposure to the extracellular environment.
Insights
Chlamydia trachomatis outer membrane protein synthesis is timed with the host cell cycle. This study tracks major outer membrane protein and 60K doublet protein synthesis and cross-linking during infection.
Area of Science:
- Microbiology
- Cell Biology
- Bacterial Pathogenesis
Background:
- Chlamydia trachomatis is an obligate intracellular bacterium causing significant human disease.
- Outer membrane proteins (OMPs) are crucial for chlamydial structure and host cell interaction.
- Understanding OMP synthesis and modification is key to developing effective treatments.
Purpose of the Study:
- To investigate the temporal synthesis and accumulation of key Chlamydia trachomatis outer membrane proteins.
- To determine the timing of disulfide cross-linking for OMPs during the intracellular growth cycle.
- To elucidate potential mechanisms regulating OMP expression and modification.
Main Methods:
- Monitoring [35S]cysteine uptake into chlamydial proteins during a 48-h infection cycle in HeLa 229 cells.
- Employing immunoblotting with specific monoclonal antibodies to detect OMP synthesis.
- Assessing disulfide cross-linking of OMPs at various stages of the bacterial growth cycle.
Main Results:
- Major outer membrane protein synthesis initiated between 12-18 hours post-infection.
- 60K doublet and 12.5K proteins synthesis detected around 30 hours, coinciding with elementary body (EB) formation.
- Immunoblotting revealed earlier synthesis of 60K doublet proteins by 18 hours.
- Progressive disulfide cross-linking of major outer membrane protein and 12.5K protein occurred in the latter half of the growth cycle.
- 60K doublet proteins showed extensive cross-linking throughout most of the cycle.
Conclusions:
- OMP synthesis timing is regulated and linked to the developmental transition from reticulate bodies to elementary bodies.
- Evidence suggests an intracellular mechanism for OMP disulfide cross-linking, potentially enzymatic, in addition to extracellular auto-oxidation.
- These findings provide insights into Chlamydia trachomatis OMP regulation and structural maturation.