Eukaryotic Cell Permeabilisation to Identify New Putative Chlamydial Type III Secretion System Effectors Secreted within Host Cell Cytoplasm

<i>Chlamydia trachomatis</i> and <i>Waddlia chondrophila</i> are strict intracellular bacteria belonging to the <i>Chlamydiales</i> order<i>. C. trachomatis</i> is the most frequent bacterial cause of genital and ocular infections whereas <i>W. chondrophila</i> is an opportunistic pathogen associated with adverse pregnancy outcomes and respiratory infections. Being strictly intracellular, these bacteria are engaged in a complex interplay with their hosts to modulate their environment and create optimal conditions for completing their life cycle. For this purpose, they possess several secretion pathways and, in particular, a Type III Secretion System (T3SS) devoted to the delivery of effector proteins in the host cell cytosol. Identifying these effectors is a crucial step in understanding the molecular basis of bacterial pathogenesis. Following incubation of infected cells with perfringolysin O, a pore-forming toxin that binds cholesterol present in plasma membranes, we analysed by mass spectrometry the protein content of the host cell cytoplasm. We identified 13 putative effectors secreted by <i>C. trachomatis</i> and 19 secreted by <i>W. chondrophila</i>. Using <i>Y. enterocolitica</i> as a heterologous expression and secretion system, we confirmed that four of these identified proteins are secreted by the T3SS. Two <i>W. chondrophila</i> T3SS effectors (hypothetical proteins Wcw_0499 and Wcw_1706) were further characterised and demonstrated to be early/mid-cycle effectors. In addition, Wcw_1706 is associated with a tetratricopeptide domain-containing protein homologous to <i>C. trachomatis</i> class II chaperone. Furthermore, we identified a novel <i>C. trachomatis</i> effector, CT460 that localises in the eukaryotic nucleus when ectopically expressed in 293 T cells.