Split-GFP Reporter-Based Detection of Chlamydia trachomatis Inclusion Damage

Chlamydia trachomatis, an obligate intracellular pathogen responsible for trachoma and widespread urogenital infections, replicates exclusively within the confines of a membrane-bound vacuole known as the inclusion. This compartment shields the bacterium from host cellular immune detection; thus, clarifying how inclusion integrity is maintained and protected from vacuole-destabilizing host attacks may identify new therapeutic strategies. To enable such insights, we recently developed split-GFP-based microscopic reporters that can detect early forms of membrane damage and track individual bacteria released from compromised inclusions. These reporters rely on infecting GFP1-10-expressing cells with C. trachomatis strains engineered to express either a GFP11-tagged outer membrane protein, exposing GFP11 on the bacterial surface, or a GFP11-tagged inclusion membrane protein, exposing GFP11 to the inclusion lumen. Damage to the inclusion allows GFP1-10 to access the bacteria and inner surface of the inclusion membrane, leading to GFP reconstitution and fluorescence at either cytosol-exposed bacteria or damaged membrane sites. Here, we provide detailed protocols for applying these reporter systems, including the generation of the required bacterial strains, development of a GFP1-10-expressing cell line, and execution of a representative infection experiment.