) ClpP 1 and ClpP 2 Function Together in Protein Degradation and are Required for Viability \ ( in \ ) \ ( vitro \ ) and During Infection

In most bacteria, Clp protease is a conserved, non-essential serine protease that regulates the response to various stresses. Mycobacteria, including Mycobacterium tuberculosis (Mtb) and Mycobacterium smegmatis, unlike most well studied prokaryotes, encode two ClpP homologs, ClpP1 and ClpP2, in a single operon. Here we demonstrate that the two proteins form a mixed complex (ClpP1P2) in mycobacteria. Using two different approaches, promoter replacement, and a novel system of inducible protein degradation, leading to inducible expression of clpP1 and clpP2, we demonstrate that both genes are essential for growth and that a marked depletion of either one results in rapid bacterial death. ClpP1P2 protease appears important in degrading missense and prematurely terminated peptides, as partial depletion of ClpP2 reduced growth specifically in the presence of antibiotics that increase errors in translation. We further show that the ClpP1P2 protease is required for the degradation of proteins tagged with the SsrA motif, a tag co-translationally added to incomplete protein products. Using active site mutants of ClpP1 and ClpP2, we show that the activity of each subunit is required for proteolysis, for normal growth of Mtb in vitro and during infection of mice. These observations suggest that the Clp protease plays an unusual and essential role in Mtb and may serve as an ideal target for antimycobacterial therapy. Citation: Raju RM, Unnikrishnan M, Rubin DHF, Krishnamoorthy V, Kandror O, et al. (2012) Mycobacterium tuberculosis ClpP1 and ClpP2 Function Together in Protein Degradation and Are Required for Viability in vitro and During Infection. PLoS Pathog 8(2): e1002511. doi:10.1371/journal.ppat.1002511 Editor: Sabine Ehrt, Weill Medical College of Cornell University, United States of America Received May 30, 2011; Accepted December 14, 2011; Published February 16, 2012 Copyright: 2012 Raju et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the National Institute of Allergy and Infectious Disease grant R01 AI071881 (EJR), the National Institute of General Medical Sciences grant R01 GM51923 (ALG), a Harvard Catalyst Pilot Grant, and a grant to Cornell University by the Bill and Melinda Gates Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: erubin@hsph.harvard.edu . These authors contributed equally to this work.