Application of β-Lactamase Reporter Fusions as an Indicator of Effector Protein Secretion during Infections with the Obligate Intracellular Pathogen Chlamydia trachomatis

Abstract

Chlamydia spp. utilize multiple secretion systems, including the type III secretion system (T3SS), to deploy host-interactive effector proteins into infected host cells. Elucidation of secreted proteins has traditionally required ectopic expression in a surrogate T3SS followed by immunolocalization of endogenous candidate effectors to confirm secretion by chlamydiae. The ability to transform Chlamydia and achieve stable expression of recombinant gene products has enabled a more direct assessment of secretion. We adapted TEM-1 β-lactamase as a reporter system for assessment of chlamydial protein secretion. We provide evidence that this system facilitates visualization of secretion in the context of infection. Specifically, our findings provide definitive evidence that C. trachomatis CT695 is secreted during infection. Follow-up indirect immunofluorescence studies confirmed CT695 secretion and indicate that this effector can be secreted at multiple points during the chlamydial developmental cycle. Our results indicate that the BlaM-fusion reporter assay will allow efficacious identification of novel secreted proteins. Moreover, this approach can easily be adapted to enable more sophisticated studies of the secretion process in Chlamydia.

A working model for the type III secretion mechanism in Chlamydia

“…Purified chlamydial EBs can be stimulated to release effectors via in vitro treatment with T3S-inducing buffers containing BSA and EDTA [39, 49, 61]. Although inefficient, these data confirm that EBs contain a complete T3SA capable of secretion.…”

“…This function would be novel since corresponding gatekeepers in other T3SSs lack described effector activity. Now that an in vivo secretion assay is available for Chlamydia [49], it should be possible to directly assess whether CopN is translocated or remains within the inclusion lumen. Current evidence indicates that secreted CopB and CopD would form the invasion-related translocon enabling translocation of subsequently secreted effectors across the host membrane.…”

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

“…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).…”

A Coming of Age Story: Chlamydia in the Post-Genetic Era

“…These vectors used either the pSW2 or pL2 cryptic plasmid backbone reported by Wang et al (26) and increased the utility of the shuttle vector through the following: (i) development of an inducible TetR-based promoter system; (ii) introduction of multiple cloning sites with and without promoters to allow for constitutive expression or regulation by native promoters; (iii) expression of a variety of fluorescent proteins including green (23,(48)(49)(50)(51)(52)(53)(54). In addition, bsd (conferring resistance to blasticidin) was validated providing a third selection marker (55).…”

“…In addition, bsd (conferring resistance to blasticidin) was validated providing a third selection marker (55). These tools have enabled previously impossible research such as complementation of chromosomal mutants and analysis of type III effectors using C. trachomatis rather than surrogate bacterial systems (49,51,56). The ability to freely manipulate the cryptic plasmid backbone in shuttle vectors also led to a more thorough understanding of cryptic plasmid genes required for plasmid maintenance as well as virulence (24,(57)(58)(59).…”