The essential M. tuberculosis Clp protease is functionally asymmetric in vivo

The Clp protease system is a promising, noncanonical drug target against Mycobacterium tuberculosis (Mtb). Unlike in Escherichia coli, the Mtb Clp protease consists of two distinct proteolytic subunits, ClpP1 and ClpP2, which hydrolyze substrates delivered by the chaperones ClpX and ClpC1. While biochemical approaches uncovered unique aspects of Mtb Clp enzymology, its essentiality complicates in vivo studies. To address this gap, we leveraged new genetic tools to mechanistically interrogate the in vivo essentiality of the Mtb Clp protease. While validating some aspects of the biochemical model, we unexpectedly found that only the proteolytic activity of ClpP1, but not of ClpP2, is essential for substrate degradation and Mtb growth and infection. Our observations not only support a revised model of Mtb Clp biology, where ClpP2 scaffolds chaperone binding while ClpP1 provides the essential proteolytic activity of the complex; they also have important implications for the ongoing development of inhibitors toward this emerging therapeutic target.