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

Abstract

Chlamydial major outer membrane protein (MOMP) is the major protein constituent of the bacterial pathogen Chlamydia trachomatis. Chlamydia trachomatis Serovars D–K are the leading cause of genital tract infections which can lead to infertility or ectopic pregnancies. A vaccine against Chlamydia is highly desirable but currently not available. MOMP accounts for ~ 60% of the chlamydial protein mass and is considered to be one of the lead vaccine candidates against C. trachomatis. We report on the spectroscopic analysis of C. trachomatis native MOMP Serovars D, E, F, and J as well as C. muridarum MOMP by size exclusion chromatography multi angle light scattering (SEC MALS), circular dichroism (CD) and attenuated total reflectance Fourier transform infrared spectroscopy (ATR‐FTIR). MOMP was purified from the native bacterium grown in either adherent HeLa cells or in different suspension cell lines. Our results confirm that MOMP forms homo‐trimers in detergent micelles. The secondary structure composition of C. trachomatis MOMP was conserved across serovars, but different from composition of C. muridarum MOMP with a 13% (CD) to 18% (ATR‐FTIR) reduction in β‐sheet conformation for C. trachomatis MOMP. When Serovar E MOMP was isolated from suspension cell lines the α‐helix content increased by 7% (CD) to 13% (ATIR‐FTIR). Maintenance of a native‐like tertiary and quaternary structure in subunit vaccines is important for the generation of protective antibodies. This biophysical characterization of MOMP presented here serves, in the absence of functional assays, as a method for monitoring the structural integrity of MOMP.

A Novel LNP-Based <i>Chlamydia</i> Subunit Vaccine Formulation That Induces Th1 Responses without Upregulating IL-17 Provides Equivalent Protection in Mice as Formulations That Induced IL-17 and Th1 Cytokines

“…The nMOMP materials used in these studies were analyzed by amino acid analysis to determine concentration and then by 1D/LC-MS-MS analysis (purity > 95%), size exclusion chromatography with multi-angle light scattering detection to determine structure (predominantly trimer with some monomer), circular dichroism to determine form (~40% Beta-sheet structure), and SDS-PAGE analysis to confirm purity and structural characteristics. Extensive analytical evaluation of purified native MOMP similar to the protein used in these studies was described previously [21]. As expected, potent IL-17 responses were detected from mice in groups that were vaccinated with DDA containing formulations (Figure 2(d)), and such responses were not induced by the LNP formulations or C. muridarum EB exposure.…”

Molecular pathogenesis of Chlamydia trachomatis

“…Chlamydia pneumoniae and CT are endemic to humans, they infect different anatomical sites, respiratory and oculogenital, respectively ( Whittum-Hudson and Hudson, 2005 ). CT is further divided into serovars based on the hypervariable region of the major outer membrane protein (MOMP) ( Hepler et al., 2018 ). CT serovars exhibit different tissue tropisms.…”

Overexpression of the Bam Complex Improves the Production of Chlamydia trachomatis MOMP in the E. coli Outer Membrane

“…These models placed the immunogenic VDs of Ct-MOMP at the cell surface. We have used the recent AlphaFold protein structure prediction method [53] to model the native Ct-MOMP. Interestingly, and in contrast to the previous predictions, it yielded a structural model of high confidence that includes only 10 very long antiparallel β-strands, while still placing all VDs at the cell surface (Figure S8A).…”