The AAA+ ATPase ClpX Is Critical for Growth and Development of Chlamydia trachomatis

Chlamydia trachomatis (Ctr) is an obligate intracellular bacterium that undergoes a complex developmental cycle in which the bacterium differentiates between two functionally and morphologically distinct forms, each of which expresses its own specialized repertoire of proteins. The transitions between the infectious, non-dividing elementary body (EB) and the non-infectious, replicative reticulate body (RB) are not mediated by division events that re-distribute intracellular proteins. Rather, both primary (EB to RB) and secondary (RB to EB) differentiation require protein turnover. The Clp protease system is well conserved in bacteria and, minimally, relies on a serine protease subunit, ClpP, and a AAA+ ATPase, such as ClpX, that recognizes and unfolds substrates for ClpP degradation. In Chlamydia, clpX is encoded within an operon adjacent to clpP2. We present evidence that the chlamydial ClpX ortholog, and the co-transcribed ClpP2, play a key role in organism viability and development. We demonstrate here that chlamydial ClpX is a functional ATPase and forms the expected homohexamer in vitro. Overexpression of a ClpX mutant lacking ATPase activity had a limited impact on DNA replication or secondary differentiation but, nonetheless, reduced EB viability. Conversely, the overexpression of an inactive ClpP2 mutant significantly impacted later developmental cycle progression by reducing the overall number of organisms. Blocking clpP2X transcription using CRISPR interference led to a decrease in bacterial growth, which did not occur when the non-essential gene incA was targeted. Taken together, our data indicate that ClpX and the associated ClpP2 play a critical role in developmental cycle progression and differentiation.