Design, expression and functional characterization of a synthetic gene encoding the Chlamydia trachomatis major outer membrane protein

Jones, Heath; Kubo, Aya; Stephens, Richard S.

doi:10.1016/s0378-1119(00)00367-x

Cited by 15 publications

(15 citation statements)

References 23 publications

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“…Another possibility is that A. phagocytophilum may have an additional porin. In the obligate intracellular pathogen Chlamydia, two porins, namely, MOMP (10,39) and PorB (12,13), have been identified. Further analysis of the A. phagocytophilum porin will help to delineate the physiology of the obligatory intracellular pathogen and illuminate a vaccine candidate to prevent HGA.…”

Section: Discussionmentioning

confidence: 99%

Porin Activity of Anaplasma phagocytophilum Outer Membrane Fraction and Purified P44

et al. 2007

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Anaplasma phagocytophilum, an obligatory intracellular bacterium that causes human granulocytic anaplasmosis, has significantly less coding capacity for biosynthesis and central intermediary metabolism than do free-living bacteria. Thus, A. phagocytophilum needs to usurp and acquire various compounds from its host. Here we demonstrate that the isolated outer membrane of A. phagocytophilum has porin activity, as measured by a liposome swelling assay. The activity allows the diffusion of L-glutamine, the monosaccharides arabinose and glucose, the disaccharide sucrose, and even the tetrasaccharide stachyose, and this diffusion could be inhibited with an anti-P44 monoclonal antibody. P44s are the most abundant outer membrane proteins and neutralizing targets of A. phagocytophilum. The P44 protein demonstrates characteristics consistent with porins of gram-negative bacteria, including detergent solubility, heat modifiability, a predicted structure of amphipathic and antiparallel ␤-strands, an abundance of polar residues, and a C-terminal phenylalanine. We purified native P44s under two different nondenaturing conditions. When reconstituted into proteoliposomes, both purified P44s exhibited porin activity. P44s are encoded by approximately 100 p44 paralogs and go through extensive antigenic variation. The 16-transmembrane-domain ␤-strands consist of conserved P44 Nand C-terminal regions. By looping out the hypervariable region, the porin structure is conserved among diverse P44 proteins yet enables antigenic variation for immunoevasion. The tricarboxylic acid (TCA) cycle of A. phagocytophilum is incomplete and requires the exogenous acquisition of L-glutamine or L-glutamate for function. Efficient diffusion of L-glutamine across the outer membrane suggests that the porin feeds the Anaplasma TCA cycle and that the relatively large pore size provides Anaplasma with the necessary metabolic intermediates from the host cytoplasm.

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Section: Discussionmentioning

confidence: 99%

Porin Activity of Anaplasma phagocytophilum Outer Membrane Fraction and Purified P44

et al. 2007

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“…Although a leadered version of recombinant chlamydial MOMP was expressed and functionally analysed previously [27], membranes containing the protein were co-reconstituted with endogenous E. coli porins for liposome-swelling studies. Although MOMP may have contributed additional porin-like activity, functional modification of endogenous porins could not be ruled out.…”

Section: Discussionmentioning

confidence: 99%

“…Jones et al . [27] co-reconstituted recombinant MOMP with endogenous E. coli porins, and showed altered solute permeabilities in liposome-swelling assays. Although attributed to novel porin activity, this could have reflected modification of endogenous porins.…”

Section: Introductionmentioning

confidence: 99%

Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

2005

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Background: 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 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.

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“…Several studies have confirmed that the C. trachomatis MOMP shares the characteristics of other gram-negative bacterial porins; it has a monomeric molecular weight of 39.5 kDa, a hydrophobic residue content of ∼40%, and migration patterns on SDS-page gels consistent with trimeric oligomerization [15]. Native MOMP also transports sugars at rates similar to Pseudomonas aeruginosa OprF [15]–[17].…”

Section: Introductionmentioning

confidence: 92%

A 3-Dimensional Trimeric β-Barrel Model for Chlamydia MOMP Contains Conserved and Novel Elements of Gram-Negative Bacterial Porins

et al. 2013

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Chlamydia trachomatis is the most prevalent cause of bacterial sexually transmitted diseases and the leading cause of preventable blindness worldwide. Global control of Chlamydia will best be achieved with a vaccine, a primary target for which is the major outer membrane protein, MOMP, which comprises ∼60% of the outer membrane protein mass of this bacterium. In the absence of experimental structural information on MOMP, three previously published topology models presumed a16-stranded barrel architecture. Here, we use the latest β-barrel prediction algorithms, previous 2D topology modeling results, and comparative modeling methodology to build a 3D model based on the 16-stranded, trimeric assumption. We find that while a 3D MOMP model captures many structural hallmarks of a trimeric 16-stranded β-barrel porin, and is consistent with most of the experimental evidence for MOMP, MOMP residues 320–334 cannot be modeled as β-strands that span the entire membrane, as is consistently observed in published 16-stranded β-barrel crystal structures. Given the ambiguous results for β-strand delineation found in this study, recent publications of membrane β-barrel structures breaking with the canonical rule for an even number of β-strands, findings of β-barrels with strand-exchanged oligomeric conformations, and alternate folds dependent upon the lifecycle of the bacterium, we suggest that although the MOMP porin structure incorporates canonical 16-stranded conformations, it may have novel oligomeric or dynamic structural changes accounting for the discrepancies observed.

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Design, expression and functional characterization of a synthetic gene encoding the Chlamydia trachomatis major outer membrane protein

Cited by 15 publications

References 23 publications

Porin Activity of Anaplasma phagocytophilum Outer Membrane Fraction and Purified P44

Porin Activity of Anaplasma phagocytophilum Outer Membrane Fraction and Purified P44

Surface expression, single-channel analysis and membrane topology of recombinant Chlamydia trachomatis Major Outer Membrane Protein

A 3-Dimensional Trimeric β-Barrel Model for Chlamydia MOMP Contains Conserved and Novel Elements of Gram-Negative Bacterial Porins

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