The Chlamydia trachomatis type III secretion substrates CT142, CT143, and CT144 are secreted into the lumen of the inclusion

<div><p><i>Chlamydia trachomatis</i> is a human bacterial pathogen causing ocular and genital infections. It multiplies exclusively within an intracellular membrane-bound vacuole, the inclusion, and uses a type III secretion system to manipulate host cells by injecting them with bacterially-encoded effector proteins. In this work, we characterized the expression and subcellular localization in infected host cells of the <i>C</i>. <i>trachomatis</i> CT142, CT143, and CT144 proteins, which we previously showed to be type III secretion substrates. Transcriptional analyses in <i>C</i>. <i>trachomatis</i> confirmed the prediction that <i>ct142</i>, <i>ct143</i> and <i>ct144</i> are organized in an operon and revealed that their expression is likely driven by the main σ factor, σ<sup>66</sup>. In host cells infected by <i>C</i>. <i>trachomatis</i>, production of CT142 and CT143 could be detected by immunoblotting from 20–26 h post-infection. Immunofluorescence microscopy of infected cells revealed that from 20 h post-infection CT143 appeared mostly as globular structures outside of the bacterial cells but within the lumen of the inclusion. Furthermore, immunofluorescence microscopy of cells infected by <i>C</i>. <i>trachomatis</i> strains carrying plasmids producing CT142, CT143, or CT144 under the control of the <i>ct142</i> promoter and with a C-terminal double hemagglutinin (2HA) epitope tag revealed that CT142-2HA, CT143-2HA or CT144-2HA showed an identical localization to chromosomally-encoded CT143. Moreover, CT142-2HA or CT144-2HA and CT143 produced by the same bacteria co-localized in the lumen of the inclusion. Overall, these data suggest that the CT142, CT143, and CT144 type III secretion substrates are secreted into the lumen of the inclusion where they might form a protein complex.</p></div>