Mycobacterium tuberculosis ClpP1 and ClpP2 Function Together in Protein Degradation and Are Required for Viability in vitro and During Infection

Abstract

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.

Crystal structure of Mycobacterium tuberculosis ClpP1P2 suggests a model for peptidase activation by AAA+ partner binding and substrate delivery

“…Because we find that ADEPs both open the pores and activate M. tuberculosis ClpP1P2, it is plausible that rogue degradation of cellular proteins is also responsible for ADEP killing of M. tuberculosis (30). However, given that the M. tuberculosis clpX and clpC1 genes are essential (26)(27)(28), it also is possible that a loss-of-function mechanism contributes to ADEP killing. Specifically, ADEP disruption of the interaction between ClpP1P2 and an accessory AAA+ partner could lead to the accumulation of toxic proteins that are normally substrates of these Clp proteases (2).…”

“…For example, Mycobacterium tuberculosis, a pathogenic actinobacterium, encodes cotranscribed clpP1 and clpP2 genes (24,25). The importance of Clp-family proteolysis in M. tuberculosis is highlighted by the facts that the clpP1, clpP2, clpX, and clpC1 genes are all essential and that mechanism-based ClpP inhibitors suppress growth (24,(26)(27)(28). Recent studies indicate that M. tuberculosis ClpP1 and ClpP2 form discrete heptameric rings that assemble into an active ClpP1P2 tetradecamer only in the presence of a ClpX or ClpC1 AAA+ partner and one additional factor, either protein substrates being actively translocated into the degradation chamber or N-blocked peptide agonists (23,29).…”

Mycobacterium tuberculosis ESAT-6 Exhibits a Unique Membrane-interacting Activity That Is Not Found in Its Ortholog from Non-pathogenic Mycobacterium smegmatis

“…For instance, MtbCFP-10 has been shown to interact with several other proteins, including ClpC1, an AAA-ATPase chaperone involved in protein translocation and quality control, and ClpC1 associates with the proteolytic component ClpP2 (33). It has recently been shown that ClpP1 and ClpP2 form a single proteolytic complex and function together in protein degradation, and both are required for bacterial viability in vitro and during infection (34,35). Thus, further studies of the roles of PTMs and other interacting factors in the heterodimer dissociation will facilitate our understanding of the MtbESAT-6-mediated pathogenesis.…”

Structural and functional insights into caseinolytic proteases reveal an unprecedented regulation principle of their catalytic triad

“…Inhibition of LmClpP2 with lactonebased inhibitors led to down-regulation of virulence without affecting viability (27). In contrast, both mycobacterial ClpP subunits are essential for bacterial survival, emphasizing defined functional roles of ClpP proteins among species (26,28).…”