Abstract
BackgroundChlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP) with important structural and, in many species, immunity-related roles. MOMP forms extensive disulphide bonds with other chlamydial proteins, and is difficult to purify. Leaderless, recombinant MOMPs expressed in E. coli have yet to be refolded from inclusion bodies, and although leadered MOMP can be expressed in E. coli cells, it often misfolds and aggregates. We aimed to improve the surface expression of correctly folded MOMP to investigate the membrane topology of the protein, and provide a system to display native and modified MOMP epitopes.ResultsC. trachomatis MOMP was expressed on the surface of E. coli cells (including "porin knockout" cells) after optimizing leader sequence, temperature and medium composition, and the protein was functionally reconstituted at the single-channel level to confirm it was folded correctly. Recombinant MOMP formed oligomers even in the absence of its 9 cysteine residues, and the unmodified protein also formed inter- and intra-subunit disulphide bonds. Its topology was modeled as a (16-stranded) β-barrel, and specific structural predictions were tested by removing each of the four putative surface-exposed loops corresponding to highly immunogenic variable sequence (VS) domains, and one or two of the putative transmembrane strands. The deletion of predicted external loops did not prevent folding and incorporation of MOMP into the E. coli outer membrane, in contrast to the removal of predicted transmembrane strands.ConclusionsC. trachomatis MOMP was functionally expressed on the surface of E. coli cells under newly optimized conditions. Tests of its predicted membrane topology were consistent with β-barrel oligomers in which major immunogenic regions are displayed on surface-exposed loops. Functional surface expression, coupled with improved understanding of MOMP's topology, could provide modified antigens for immunological studies and vaccination, including live subunit vaccines, and might be useful to co-express MOMP with other chlamydial membrane proteins.
Highlights
Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP) with important structural and, in many species, immunity-related roles
We showed that MOMP can insert into the outer membrane of E. coli cells and form SDS-sensitive oligomers in the absence of cysteine residues, and generated a "working model" of the topology of MOMP to provide structural hypotheses that could be tested by engineering the recombinant protein
Building on previous work (e.g. [26]), BL21(DE3) cells were transformed with pET-ompA or pETporB constructs, and expression was induced by 1 mM IPTG at 37°C after growth to an OD600 of 0.6
Summary
Chlamydial bacteria are obligate intracellular pathogens containing a cysteine-rich porin (Major Outer Membrane Protein, MOMP) with important structural and, in many species, immunity-related roles. MOMP (~40 kDa) is expressed in both EBs and RBs [4] It contains extensive β-sheet secondary structure and forms large pores [5,6], similar to β-barrel porins found in other outer bacterial membranes (e.g. E_coli OmpF). MOMP's pore-forming ability is enhanced by reduction [5], compatible with a link between reversible disulphide bond formation and the developmental stage of the bacteria Supporting this idea, DTT-reduced EBs tend to resemble RBs [5], and native MOMP is monomeric when solubilised in SDS under reducing conditions, but forms monomers, dimers, trimers, tetramers and even larger complexes [e.g. Supporting this idea, DTT-reduced EBs tend to resemble RBs [5], and native MOMP is monomeric when solubilised in SDS under reducing conditions, but forms monomers, dimers, trimers, tetramers and even larger complexes [e.g. [6,10,11]] under oxidising conditions
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