Methods for Identifying Type III Secretion System Effectors in Chlamydia trachomatis

To subvert host defenses and establish a replicative niche, obligate intracellular pathogens remodel the host cell using a discrete repertoire of effector proteins. For Chlamydia trachomatis, defining the repertoire of conventional type III secretion system effectors (cT3SS) has been exceedingly difficult as many of these proteins are present at low levels in the host cell and do not accumulate within a specific host compartment, necessitating signal amplification for accurate detection. Drawing on approaches widely used for assessing secretion of candidate T3SS and T4SS substrates, several enzymatic reporter tags have been adapted to evaluate secretion of putative C. trachomatis cT3SS substrates. Here we describe BlaM, CyaA, and GSK assays, which are three complementary assays that provide robust and reliable readouts of effector delivery into the host cell. This protocol describes each assay in detail, including the steps required to generate C. trachomatis transformants, confirm effector-reporter expression, and execute and analyze data for each assay.