Expression and Targeting of Secreted Proteins from Chlamydia trachomatis

Abstract

Chlamydia trachomatis is an obligate intracellular pathogen that replicates in a vacuole termed the inclusion. Many of the interactions of chlamydiae with the host cell are dependent upon bacterial protein synthesis and presumably exposure of these proteins to the cytosol. Because of the dearth of genetic tools for chlamydiae, previous studies examining secreted proteins required the use of heterologous bacterial systems. Recent advances in genetic manipulation of chlamydia now allow for transformation of the bacteria with plasmids. We describe here a shuttle vector system, pBOMB4, that permits expression of recombinant proteins under constitutive or conditional promoter control. We show that the inclusion membrane protein IncD is secreted in a type III-dependent manner from Yersinia pseudotuberculosis and also secreted from C. trachomatis in infected cells where it localizes appropriately to the inclusion membrane. IncD truncated of the first 30 amino acids containing the secretion signal is no longer secreted and is retained by the bacteria. Cytosolic exposure of secreted proteins can be confirmed by using CyaA, GSK, or microinjection assays. A protein predicted to be retained within the bacteria, NrdB is indeed localized to the chlamydia. In addition, we have shown that the chlamydial effector protein, CPAF, which is secreted into the host cell cytosol by a Sec-dependent pathway, also accesses the cytosol when expressed from this system. These assays should prove useful to assess the secretion of other chlamydial proteins that are potentially exposed to the cytosol of the host cell.

Emancipating Chlamydia: Advances in the Genetic Manipulation of a Recalcitrant Intracellular Pathogen

“…These vectors are well suited for expressing epitope-tagged or untagged gene products under the control of native promoters. pBOMB4 and its derivative pBOMB4-MCI also express GFP and mCherry, respectively, from the Neisseria promoter used in pSW2: GFP (19), providing a convenient marker to confirm the presence and maintenance of recombinant plasmids in transformants (21). Genes can also be expressed by using constitutive promoters, such as the rpoB promoter, located upstream of the MCS in pBOMB4R (GFP ϩ ) and pBOMB4R-MCI (mCherry ϩ ) (21).…”

“…pBOMB4 and its derivative pBOMB4-MCI also express GFP and mCherry, respectively, from the Neisseria promoter used in pSW2: GFP (19), providing a convenient marker to confirm the presence and maintenance of recombinant plasmids in transformants (21). Genes can also be expressed by using constitutive promoters, such as the rpoB promoter, located upstream of the MCS in pBOMB4R (GFP ϩ ) and pBOMB4R-MCI (mCherry ϩ ) (21). If precise control of gene expression at different times in the Chlamydia developmental cycle is required, several vectors in which gene expression is controlled by anhydrotetracycline via the tetracycline repressor are available.…”

“…A system based on FRT/FLP recombination would permit in vivo gene ablation by introducing flanking FRT sites into a locus of interest and promoting gene excision by inducing trans-expression of the FLP recombinase. Plasmids in which expression is controlled by the Tet-inducible operon have already been developed and can easily be coopted for such approaches (21,26). An inducible FRT/FLP system could also be harnessed for genome editing and selectable marker recycling during the engineering of strains with multiple gene knockouts.…”

“…Fluorophore-encoding genes can be substituted with a gene of interest for expression under the control of the incD promoter. (B) pBOMB4 vectors (21) are derived from an intact thase kinase), and TEM-1 ␤-lactamase, can be used to monitor effector secretion and translocation (20,21,29,(149)(150)(151)(152).…”

“…Transformation of exogenous DNA into Chlamydia has been achieved via several methods, including electroporation, dendrimer-based delivery, and a calcium chloride-based treatment (14)(15)(16)(17)(18)(19). Successful and stable transformation of C. trachomatis LGV L2 with an Escherichia coli-C. trachomatis L2 shuttle plasmid conferring ␤-lactam resistance led to the development of expression vectors for expressing Chlamydia open reading frames (ORFs), fluorescent proteins (green fluorescent protein [GFP], cyan fluorescent protein [CFP], mCherry, and mKate2), and reporter proteins (␤-galactosidase, adenylate cyclase, and glycogen synthase kinase [GSK]-tagged proteins) (19)(20)(21)(22)(23)(24)(25)(26). A conditional expression vector was also developed using a tetracycline-inducible system as well as vectors conferring chloramphenicol and blasticidin resistance (21,(26)(27)(28)(29).…”