Ectopic overexpression and CRISPRi-based knockdown of <i>Chlamydia trachomatis</i> ObgE inhibits RB replication and EB reformation

<i>Chlamydia trachomatis</i> is an obligate intracellular bacterium of major clinical significance. While untreated sexually transmitted infections can result in pelvic inflammatory disease and infertility, ocular infections can cause the blinding disease trachoma. During infection of host cells, <i>C. trachomatis</i> transitions between the non-replicative, infectious elementary body (EB) and the replicative, non-infectious reticulate body (RB). ObgE is a GTPase that can promote morphological differentiation in some bacteria. In <i>C. trachomatis</i>, <i>obgE</i> is maximally expressed from 16 to 24hpi, a timeframe that is associated with logarithmic growth and the onset of production of infectious progeny; therefore, ObgE is predicted to have significance during <i>Chlamydia</i> replication and/or morphological transitions. To determine the role of ObgE during the <i>C. trachomatis</i> developmental cycle, we assessed the effects of ObgE ectopic overexpression and CRISPRi-based knockdown of <i>obgE</i> on RB replication and EB formation. When ectopic overexpression of ObgE was induced, we observed a significant decrease in infectious progeny but no changes in bacterial ultrastructure. These data suggest that during ectopic overexpression of ObgE, RBs can transition into EBs; however, EBs are diminished in their ability to establish new infections. CRISPRi-based knockdown of <i>obgE</i> resulted in a 2-log decrease in bacterial yield and infectious progeny. Ultrastructural analysis revealed that knockdown of <i>obgE</i> resulted in small, underdeveloped inclusions with few cells inside. In total, while ectopic overexpression of ObgE negatively affects production of infectious EBs, CRISPRi-based knockdown of <i>obgE</i> severely affects RB replication, inclusion development, and generation of EBs.IMPORTANCEThe pathogenesis of <i>C. trachomatis</i> is reliant on the transition between the non-replicative, infectious elementary body (EB) and the replicative, non-infectious reticulate body (RB). Therefore, understanding the molecular determinants of <i>Chlamydia</i> developmental transitions is of the utmost importance. ObgE has been shown to regulate morphological transitions in other bacteria and is thus predicted to have relevance during regulation of the <i>Chlamydia</i> developmental cycle. Using both ectopic overexpression and CRISPRi-based knockdown of ObgE/<i>obgE</i>, we identify the significance of balanced ObgE expression for RB replication and the formation of infectious EBs. Our findings further expand our knowledge of how developmental transitions in <i>Chlamydia</i> are regulated.