The ClpX and ClpP2 Orthologs of Chlamydia trachomatis Perform Discrete and Essential Functions in Organism Growth and Development

Abstract: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, the EB and RB, each of which expresses its own specialized repertoire of proteins. Both primary (EB to RB) and secondary (RB to EB) differentiation require protein turnover, and we hypothesize that proteases are critical for mediating differentiation. The Clp protease system is well conserved in bacteria and important for protein turnover. Minimally, the system 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 3 to clpP2. We present evidence that the chlamydial ClpX and ClpP2 orthologs are essential to 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 with observable defects in EB morphology noted. Conversely, the overexpression of a catalytically inactive ClpP2 mutant significantly impacted developmental cycle progression by reducing the overall number of organisms. Blocking clpP2X transcription using CRISPR interference led to a decrease in bacterial growth, and this effect was complemented in trans by a plasmid copy of clpP2. Taken together, our data indicate that ClpX and the associated ClpP2 serve distinct functions in chlamydial developmental cycle progression and differentiation. Importance Chlamydia trachomatis is the leading cause of infectious blindness globally and the most reported bacterial sexually transmitted infection both domestically and internationally. Given the economic burden, the lack of an approved vaccine, and the use of broad-spectrum antibiotics for treatment of infections, an understanding of chlamydial growth and development is critical for the advancement of novel, targeted antibiotics. The Clp proteins comprise an important and conserved protease system in bacteria. Our work highlights the importance of the chlamydial Clp proteins to this clinically important bacterium. Additionally, our study implicates the Clp system playing an integral role in chlamydial developmental cycle progression, which may help establish models of how Chlamydia spp. and other bacteria progress through their respective developmental cycles. Our work also contributes to a growing body of Clp-specific research that underscores the importance and versatility of this system throughout bacterial evolution and further validates Clp proteins as drug targets.