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

Abstract

A licensed Chlamydia trachomatis (Ct) vaccine is not yet available. Recombinant Chlamydia trachomatis major outer membrane protein (Ct-MOMP), the most abundant constituent of the chlamydial outer membrane complex, is considered the most attractive candidate for subunit-based vaccine formulations. Unfortunately, Ct-MOMP is difficult to express in its native structure in the E. coli outer membrane (OM). Here, by co-expression of the Bam complex, we improved the expression and localization of recombinant Ct-MOMP in the E. coli OM. Under these conditions, recombinant Ct-MOMP appeared to assemble into a β-barrel conformation and express domains at the cell surface indicative of correct folding. The data indicate that limited availability of the Bam complex can be a bottleneck for the production of heterologous OM vaccine antigens, information that is also relevant for strategies aimed at producing recombinant OMV-based vaccines.

Heterologous Display of Chlamydia trachomatis PmpD Passenger at the Surface of Salmonella OMVs

“…Indeed, we achieved efficient expression and OM assembly in E. coli of C. trachomatis AT PmpD (FL), in which the C-terminal β-signal was optimized for recognition by the E. coli Bam complex. Expression did not increase in the BL21(DE3)omp8 strain, which lacks multiple highlevel OMPs, indicating that the availability of the E. coli Bam complex is not a bottleneck for PmpD (FL) localization, or some other heterologous OMPs [40,58,59]. Surprisingly, however, PmpD (FL) was not included in OMVs pinching off from the BL21(DE3)omp8 surface (Figure 1E).…”

“…Cultures of E. coli BL21(DE3) and BL21(DE3)omp8 strains harboring pLemo-PmpD (FL) were grown to an OD 600 of 0.4-0.5, induced for expression by addition of 8 mM L-rhamnose and then further incubated for 2 h [40]. Cultures of E. coli BL21(DE3) and BL21(DE3)omp8 cells harboring pEH3-derivatives were grown until an OD 600 of 0.4-0.5 and then induced with 0.1 mM IPTG for 2 h for expression of the HbpD-fusions [23].…”

“…Cell envelopes (CEs) were collected using a subcellular fractionation procedure as described previously [40]. In short, ~200 OD 600 units of BL21(DE3)omp8 cells expressing PmpD (FL) were resuspended in lysis buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris HCl, pH 8.0) in the presence of protease inhibitor (Roche, Mannheim, Germany).…”

Intranasal delivery of Salmonella OMVs decorated with Chlamydia trachomatis antigens induces specific local and systemic immune responses

“…In a previous study, we successfully achieved high-level expression of wt-MOMP in an attenuated Salmonella strain. 37 However, when we transferred the construct to an isogenic hypervesiculating Salmonella vaccine strain, the overproduced wt-MOMP protein was not incorporated into OMVs (data not shown). Similarly, our attempts to express other C. trachomatis outer membrane protein antigens, such as wt-PmpD, 50 in OMVs were unsuccessful.…”

“…Briefly, ClearColi (DE3) cells containing pET22-pelB-HtrA-H6 were grown at 30°C before protein expression was induced with 0.1 mM IPTG for 2 h. Cells were harvested by centrifugation (6,000 × g , 10 min, 4°C), and a cleared lysate containing soluble wt-HtrA was obtained through subcellular fractionation, as described previously. 37 wt-HtrA-H6 was purified by affinity chromatography using Talon Superflow medium. The eluate was dialyzed against the TG buffer (pH 7.4) overnight at 4°C.…”