title: 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.
authors.affiliation: Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts, United States of America.
authors.forename: Ravikiran M
authors.initials: RM
authors.lastname: Raju
authors.forename: Meera
authors.initials: M
authors.lastname: Unnikrishnan
authors.forename: Daniel H F
authors.initials: DH
authors.lastname: Rubin
authors.forename: Vidhya
authors.initials: V
authors.lastname: Krishnamoorthy
authors.forename: Olga
authors.initials: O
authors.lastname: Kandror
authors.forename: Tatos N
authors.initials: TN
authors.lastname: Akopian
authors.forename: Alfred L
authors.initials: AL
authors.lastname: Goldberg
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Rubin
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D050296:Microbial Viability; D009169:Mycobacterium tuberculosis / Q000502:physiology*; D059748:Proteolysis; D012697:Serine Endopeptidases / Q000378:metabolism*; D014376:Tuberculosis / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; C119658:ClpP2 protein, bacteria; D012697:Serine Endopeptidases