Structural and Functional Analyses of the Major Outer Membrane Protein ofChlamydia trachomatis

Abstract

Chlamydia trachomatis is a major pathogen throughout the world, and preventive measures have focused on the production of a vaccine using the major outer membrane protein (MOMP). Here, in elementary bodies and in preparations of the outer membrane, we identified native trimers of the MOMP. The trimers were stable under reducing conditions, although disulfide bonds appear to be present between the monomers of a trimer and between trimers. Cross-linking of the outer membrane complex demonstrated that the MOMP is most likely not in a close spatial relationship with the 60-and 12-kDa cysteine-rich proteins. Extraction of the MOMP from Chlamydia isolates under nondenaturing conditions yielded the trimeric conformation of this protein as shown by cross-linking and analysis by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis with different concentrations of acrylamide. Using circular dichroism spectroscopy, we determined that the trimers were formed mainly of ␤-pleated sheet structures in detergent micelles. Using a liposomal swelling assay, the MOMP was found to have porin activity, and the size of the pore was estimated to be approximately 2 nm in diameter. The trimers were found to be stable in SDS at temperatures ranging from 4 to 37°C and over a pH range of 5.0 to 8.0. In addition, the trimers of MOMP were found to be resistant to digestion with trypsin. In conclusion, these results show that the native conformation of the MOMP of C. trachomatis is a trimer with predominantly a ␤-sheet structure and porin function.Chlamydia strains are ubiquitous pathogens in nature (1,31,33,70). These bacteria have two distinct morphological and functional forms, the elementary bodies (EB) and the reticulate bodies (RB) (33). The EB measure approximately 300 nm in diameter and have a rigid cell membrane, while the RB measure from 1,000 to 1,200 nm in diameter and have a highly pliable membrane. The EB infect eukaryotic cells and become transformed into RB that replicate inside a cytoplasmic inclusion. As a result of replication, the cytoplasmic inclusion progressively enlarges. Following 1 to 3 days of replication, the RB transform again into EB, and cell lysis releases the infectious EB.Unlike other gram-negative bacteria, Chlamydia strains may lack a peptidoglycan layer (50). As a substitute for peptidoglycan, it has been postulated that the 60-kDa (OmcB) and the 12-kDa (OmcA) cysteine-rich proteins (CRPs), and maybe the major outer membrane protein (MOMP) and other membrane components, form a supramolecular structure that provides rigidity to the EB (16, 21, 37, 50, 63). The Chlamydia trachomatis MOMP belongs to a family of proteins found in the outer membranes of gram-negative bacteria that have a molecular mass of approximately 40 kDa and function as porins (40). Porins, such as OmpF and PhoE of Escherichia coli, form homotrimers held together by hydrophobic and electrostatic interactions (40,48). Due to the unusual binding of sodium dodecyl sulfate (SDS), the trimers migrate on 10% SDS-polyacrylamide gel elec...

Spectroscopic analysis of chlamydial major outer membrane protein in support of structure elucidation

“…MALS and RI detectors were directly connected to a size exclusion chromatography system to perform the analysis. nMOMP purifications obtained from adherent HeLa 229 cells infected with C. trachomatis Serovars D, E, F, or J were compared with C. muridarum nMOMP which has been reported to exist as a trimer in Zwittergent 3–14 detergent micelles 37, 39. The SEC UV chromatogram of C. muridarum nMOMP showed one major peak that accounted for approximately 95% of the area under curve.…”

“…The CD spectrum of C. muridarum nMOMP in Zwittergent has been published37 and a preparation of C. muridarum nMOMP obtained from adherent HeLa 229 cells was included in this analysis [Fig. 3(A)].…”

“…Chlamydia muridarum nMOMP has been extensively investigated and described in the literature24, 35, 36, 37, 38 and was, therefore, included in the analysis as a comparison. Size exclusion multi angle light scattering (SEC MALS) was applied to characterize protein‐detergent micelles of nMOMP solubilized in zwitterionic detergent.…”

Simkania negevensis, an insight into the biology and clinical importance of a novel member of the Chlamydiales order

“…These cysteine-rich proteins are sensitive to the redox state of the environment and they might provide some rigidity and resistance to osmotic pressure through the formation of disulfide bridges. Some authors suggested that they may replace the peptidoglycan layer present in the periplasm of other Gram-negative bacteria (Everett & Hatch, 1995;Sun et al, 2007). No homologs of OmcA and OmcB were identified in S. negevensis genome and none of the identified MOMP-like proteins could serve this function as their content in cysteine was extremely low (Aistleitner et al, 2015).…”

Toll-Like Receptor 2-Dependent Activity of Native Major Outer Membrane Protein Proteosomes of Chlamydia trachomatis

“…Bacterial porins, including the chlamydial major outer membrane porin protein (MOMP), constitute a high percentage of the total outer membrane protein content (over 60%) (38) and share structural and functional similarities among organisms. C. trachomatis MOMP is surface exposed, has a molecular mass of ϳ40 kDa in monomeric form, and is found in homotrimeric form in the bacterial outer membrane (39,40). It is immunogenic (41) and can induce protection in the mouse and monkey models (42,43).…”