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Sequence analysis and lipid modification of the cysteine-rich envelope proteins of Chlamydia psittaci 6BC
1Department of Microbiology and Immunology, University of Tennesse, Memphis 38163.
Abstract:
The envelopes of elementary bodies of Chlamydia spp. consist largely of disulfide-cross-linked major outer membrane protein (MOMP) and two cysteine-rich proteins (CRPs). The MOMP gene of Chlamydia psittaci 6BC has been sequenced previously, and the genes encoding the small and large CRPs from this strain were cloned and sequenced in this study. The CRP genes were found to be tandemly arranged on the chlamydial chromosome but could be independently expressed in Escherichia coli. The deduced 87-amino-acid sequence of the small-CRP gene (envA) contains 15 cysteine residues, a potential signal peptide, and a potential signal peptidase II-lipid modification site. Hydropathy plot and conformation analysis of the small-CRP amino acid sequence indicated that the protein was unlikely to be associated with a membrane. However, the small CRP was specifically labeled in host cells incubated with [3H]palmitic acid and may therefore be associated with a membrane through a covalently attached lipid portion of the molecule. The deduced 557-amino-acid sequence of the large-CRP gene (envB) contains 37 cysteine residues and a single putative signal peptidase I cleavage site. In one recombinant clone the large CRP appeared to be posttranslationally cleaved at two sites, forming a doublet in a manner similar to the large-CRP doublet made in native C. psittaci 6BC. Comparison of the deduced amino acid sequences of the CRPs from chlamydial strains indicated that the small CRP is moderately conserved, with 54% identity between C. psittaci 6BC and Chlamydia trachomatis, and the large CRP is highly conserved, with 71% identity between C. psittaci and C. trachomatis and 85% identity between C. psittaci 6BC and Chlamydia pneumoniae. The positions of the cysteine residues in both CRPs are highly conserved in Chlamydia spp. From the number of cysteine residues in the MOMP and the CRPs and the relative incorporation of [35S]cysteine into these proteins, it was calculated that the molar ratio of C. psittaci 6BC elementary body envelope proteins is about one large-CRP molecule to two small-CRP molecules to five MOMP molecules.
Insights
The study sequenced cysteine-rich protein (CRP) genes from Chlamydia psittaci 6BC, revealing their arrangement and expression. CRPs, along with major outer membrane protein (MOMP), form the chlamydial elementary body envelope.
Area of Science:
- Microbiology
- Molecular Biology
- Structural Biology
Background:
- The envelopes of Chlamydia elementary bodies are primarily composed of disulfide-cross-linked major outer membrane protein (MOMP) and two cysteine-rich proteins (CRPs).
- Understanding the genetic and structural basis of these envelope proteins is crucial for elucidating Chlamydia pathogenesis and developing targeted interventions.
Purpose of the Study:
- To clone and sequence the genes encoding the small and large CRPs from Chlamydia psittaci 6BC.
- To analyze the deduced amino acid sequences, potential post-translational modifications, and conservation of these CRPs across different Chlamydia species.
- To determine the molar ratios of MOMP and CRPs in the C. psittaci 6BC elementary body envelope.
Main Methods:
- Gene cloning and sequencing of small and large CRP genes (envA and envB) from Chlamydia psittaci 6BC.
- Expression of CRP genes in Escherichia coli.
- Sequence analysis, including hydropathy plots, conformation analysis, and comparison with CRP sequences from other Chlamydia strains.
- Metabolic labeling with [3H]palmitic acid to assess membrane association of small CRP.
- Quantification of cysteine incorporation to estimate protein molar ratios.
Main Results:
- The CRP genes (envA and envB) were found to be tandemly arranged but independently expressible in E. coli.
- The small CRP (87 amino acids) contains numerous cysteine residues and a potential lipid modification site, suggesting membrane association despite initial predictions.
- The large CRP (557 amino acids) contains many cysteine residues and undergoes post-translational cleavage, forming a doublet similar to native C. psittaci 6BC.
- Small CRP showed moderate conservation (54% identity with C. trachomatis), while large CRP was highly conserved (71-85% identity with C. trachomatis and C. pneumoniae).
- Cysteine residue positions are highly conserved across Chlamydia spp. CRPs.
- The calculated molar ratio of C. psittaci 6BC elementary body envelope proteins is approximately 1:2:5 (large CRP:small CRP:MOMP).
Conclusions:
- The genes encoding the small and large CRPs of Chlamydia psittaci 6BC have been successfully sequenced and characterized.
- CRPs play a significant role in the structural integrity of the Chlamydia elementary body envelope, with conserved cysteine residues likely involved in disulfide cross-linking.
- The distinct molar ratios of MOMP and CRPs suggest a specific structural organization within the chlamydial envelope.