Related Experiment Video
Updated: Dec 4, 2025
Using Fluorescent Proteins to Visualize and Quantitate Chlamydia Vacuole Growth Dynamics in Living Cells
Published on: October 13, 2015
Super-Resolution Fluorescence Microscopy Reveals Clustering Behaviour of Chlamydia pneumoniae's Major Outer Membrane
Amy E Danson1,2,3, Alex McStea4, Lin Wang4
1School of Biological Sciences, University of Reading, Berkshire RG6 6AS, UK.
Abstract:
Chlamydia pneumoniae is a Gram-negative bacterium responsible for a number of human respiratory diseases and linked to some chronic inflammatory diseases. The major outer membrane protein (MOMP) of Chlamydia is a conserved immunologically dominant protein located in the outer membrane, which, together with its surface exposure and abundance, has led to MOMP being the main focus for vaccine and antimicrobial studies in recent decades. MOMP has a major role in the chlamydial outer membrane complex through the formation of intermolecular disulphide bonds, although the exact interactions formed are currently unknown. Here, it is proposed that due to the large number of cysteines available for disulphide bonding, interactions occur between cysteine-rich pockets as opposed to individual residues. Such pockets were identified using a MOMP homology model with a supporting low-resolution (~4 Å) crystal structure. The localisation of MOMP in the E. coli membrane was assessed using direct stochastic optical reconstruction microscopy (dSTORM), which showed a decrease in membrane clustering with cysteine-rich regions containing two mutations. These results indicate that disulphide bond formation was not disrupted by single mutants located in the cysteine-dense regions and was instead compensated by neighbouring cysteines within the pocket in support of this cysteine-rich pocket hypothesis.
Insights
Chlamydia pneumoniae outer membrane protein (MOMP) interactions were studied. Cysteine-rich pockets, not individual residues, form disulphide bonds, impacting bacterial membrane structure and potential antimicrobial targets.
Area of Science:
- Microbiology
- Structural Biology
- Biochemistry
Background:
- Chlamydia pneumoniae causes respiratory and chronic inflammatory diseases.
- Its major outer membrane protein (MOMP) is crucial for vaccine and antimicrobial research.
- MOMP's role in disulphide bond formation within the chlamydial outer membrane is not fully understood.
Purpose of the Study:
- To investigate the hypothesis that cysteine-rich pockets, rather than individual residues, mediate disulphide bonding in Chlamydia pneumoniae MOMP.
- To elucidate the structural basis of MOMP interactions in the bacterial outer membrane.
Main Methods:
- Homology modeling and analysis of a low-resolution crystal structure to identify cysteine-rich pockets in MOMP.
- Direct stochastic optical reconstruction microscopy (dSTORM) to assess MOMP localization and clustering in E. coli membranes.
- Site-directed mutagenesis of cysteine-rich regions in MOMP.
Main Results:
- Identification of cysteine-rich pockets within the MOMP structure.
- dSTORM revealed decreased membrane clustering in MOMP mutants within these regions.
- Single mutations in cysteine-dense regions did not disrupt disulphide bond formation, suggesting compensation by neighboring cysteines.
Conclusions:
- Disulphide bonds in Chlamydia pneumoniae MOMP are likely formed within cysteine-rich pockets.
- This pocket-based interaction model provides new insights into MOMP structure and function.
- Findings support the cysteine-rich pocket hypothesis and offer potential targets for novel antimicrobials.
