#PAGE_PARAMS# #ADS_HEAD_SCRIPTS# #MICRODATA#

ClpP1 and ClpP2 Function Together in Protein Degradation and Are Required for Viability 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.


Vyšlo v časopise: ClpP1 and ClpP2 Function Together in Protein Degradation and Are Required for Viability and During Infection. PLoS Pathog 8(2): e32767. doi:10.1371/journal.ppat.1002511
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.ppat.1002511

Souhrn

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.


Zdroje

1. IngmerHBrøndstedL 2009 Proteases in bacterial pathogenesis. Res Microbiol 160 704 710 doi:10.1016/j.resmic.2009.08.017

2. GoldbergAL 2003 Protein degradation and protection against misfolded or damaged proteins. Nature 426 895 899 doi:10.1038/nature02263

3. GlickmanMHCiechanoverA 2002 The ubiquitin-proteasome proteolytic pathway: destruction for the sake of construction. Physiol Rev 82 373 428 doi:10.1152/physrev.00027.2001

4. FreesDSavijokiKVarmanenPIngmerH 2007 Clp ATPases and ClpP proteolytic complexes regulate vital biological processes in low GC, Gram-positive bacteria. Mol Microbiol 63 1285 1295 doi:10.1111/j.1365-2958.2007.05598.x

5. BurnsKEPearceMJDarwinKH 2010 Prokaryotic ubiquitin-like protein provides a two-part degron to Mycobacterium proteasome substrates. J Bacteriol 192 2933 2935 doi:10.1128/JB.01639-09

6. Cerda-MairaFAPearceMJFuortesMBishaiWRHubbardSR 2010 Molecular analysis of the prokaryotic ubiquitin-like protein (Pup) conjugation pathway in Mycobacterium tuberculosis. Mol Microbiol 77 1123 1135 doi:10.1111/j.1365-2958.2010.07276.x

7. DarwinKH 2003 The Proteasome of Mycobacterium tuberculosis Is Required for Resistance to Nitric Oxide. Science 302 1963 1966 doi:10.1126/science.1091176

8. Katayama-FujimuraYGottesmanSMauriziMR 1987 A multiple-component, ATP-dependent protease from Escherichia coli. J Biol Chem 262 4477 4485

9. HwangBJParkWJChungCHGoldbergAL 1987 Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La. Proc Natl Acad Sci U S A 84 5550 5554

10. WangJHartlingJAFlanaganJM 1997 The structure of ClpP at 2.3 A resolution suggests a model for ATP-dependent proteolysis. Cell 91 447 456

11. MogkADouganDWeibezahnJSchliekerCTurgayK 2004 Broad yet high substrate specificity: the challenge of AAA+ proteins. J Struct Biol 146 90 98 doi:10.1016/j.jsb.2003.10.009

12. KennistonJABakerTAFernandezJMSauerRT 2003 Linkage between ATP Consumption and Mechanical Unfolding during the Protein Processing Reactions of an AAA+ Degradation Machine. Cell 114 511 520 doi:10.1016/S0092-8674(03)00612-3

13. FarrellCMGrossmanADSauerRT 2005 Cytoplasmic degradation of ssrA-tagged proteins. Mol Microbiol 57 1750 1761 doi:10.1111/j.1365-2958.2005.04798.x

14. PruteanuMBakerTA 2009 Controlled degradation by ClpXP protease tunes the levels of the excision repair protein UvrA to the extent of DNA damage. Mol Microbiol 71 912 924 doi:10.1111/j.1365-2958.2008.06574.x

15. GaillotOBregenholtSJaubertFDi SantoJPBercheP 2001 Stress-Induced ClpP Serine Protease of Listeria monocytogenes Is Essential for Induction of Listeriolysin O-Dependent Protective Immunity. Infect Immun 69 4938 4943 doi:10.1128/IAI.69.8.4938-4943.2001

16. GaillotOPellegriniEBregenholtSNairSBercheP 2000 The ClpP serine protease is essential for the intracellular parasitism and virulence of Listeria monocytogenes. Mol Microbiol 35 1286 1294

17. JenalUFuchsT 1998 An essential protease involved in bacterial cell-cycle control. EMBO J 17 5658 5669 doi:10.1093/emboj/17.19.5658

18. SassettiCMBoydDHRubinEJ 2003 Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48 77 84

19. CarrollPFaray-KeleM-CParishT 2011 Identifying vulnerable pathways in Mycobacterium tuberculosis using a knock-down approach. Appl Environ Microbiol: 77 5040 5043 doi:10.1128/AEM.02880-10

20. van KesselJCHatfullGF 2008 Mycobacterial recombineering. Methods Mol Biol 435 203 215 doi:10.1007/978-1-59745-232-8_15

21. WeiJ-RKrishnamoorthyVMurphyKKimJ-HSchnappingerD 2011 Depletion of antibiotic targets has widely varying effects on growth. Proc Natl Acad Sci USA 108 4176 4181 doi:10.1073/pnas.1018301108

22. FreesDIngmerH 1999 ClpP participates in the degradation of misfolded protein in Lactococcus lactis. Mol Microbiol 31 79 87

23. HAHNFEWISSEMANCLHOPPSHE 1955 Mode of action of chloramphenicol. III. Action of chloramphenicol on bacterial energy metabolism. J Bacteriol 69 215 223

24. WykaMASt JohnAC 1990 Effects of production of abnormal proteins on the rate of killing of Escherichia coli by streptomycin. Antimicrob Agents Chemother 34 534 538

25. SmithCKBakerTASauerRT 1999 Lon and Clp family proteases and chaperones share homologous substrate-recognition domains. Proc Natl Acad Sci U S A 96 6678 6682

26. KiranMChauhanADziedzicRMaloneyEMukherjiSK 2009 Mycobacterium tuberculosis ftsH expression in response to stress and viability. Tuberculosis (Edinb) 89 S70 S73 doi:10.1016/S1472-9792(09)70016-2

27. IngvarssonHMatéMJHögbomMPortnoïDBenaroudjN 2007 Insights into the inter-ring plasticity of caseinolytic proteases from the X-ray structure of Mycobacterium tuberculosis ClpP1. Acta Crystallogr D Biol Crystallogr 63 249 259 doi:10.1107/S0907444906050530

28. QuonKCMarczynskiGTShapiroL 1996 Cell cycle control by an essential bacterial two-component signal transduction protein. Cell 84 83 93

29. GoldbergAL 1972 Degradation of abnormal proteins in Escherichia coli (protein breakdown-protein structure-mistranslation-amino acid analogs-puromycin). Proc Natl Acad Sci U S A 69 422 426

30. EstorninhoMSmithHTholeJHarders-WesterveenJKierzekA 2010 ClgR regulation of chaperone and protease systems is essential for Mycobacterium tuberculosis parasitism of the macrophage. Microbiology 156 3445 3455 doi:10.1099/mic.0.042275-0

31. SherridAMRustadTRCangelosiGAShermanDR 2010 Characterization of a Clp Protease Gene Regulator and the Reaeration Response in Mycobacterium tuberculosis. PLoS ONE 5 e11622 doi:10.1371/journal.pone.0011622

32. DragMSalvesenGS 2010 Emerging principles in protease-based drug discovery. Nat Rev Drug Discov 9 690 701 doi:10.1038/nrd3053

33. KirsteinJHoffmannALilieHSchmidtRRübsamen-WaigmannH 2009 The antibiotic ADEP reprogrammes ClpP, switching it from a regulated to an uncontrolled protease. EMBO Mol Med 1 37 49 doi:10.1002/emmm.200900002

34. SchmittEKRiwantoMSambandamurthyVRoggoSMiaultC 2011 The Natural Product Cyclomarin Kills Mycobacterium Tuberculosis by Targeting the ClpC1 Subunit of the Caseinolytic Protease. Angew Chem Int Ed Engl 50 5889 5891 doi:10.1002/anie.201101740

35. BöttcherTSieberSA 2008 Beta-lactones as specific inhibitors of ClpP attenuate the production of extracellular virulence factors of Staphylococcus aureus. J Am Chem Soc 130 14400 14401 doi:10.1021/ja8051365

Štítky
Hygiena a epidemiológia Infekčné lekárstvo Laboratórium

Článok vyšiel v časopise

PLOS Pathogens


2012 Číslo 2
Najčítanejšie tento týždeň
Najčítanejšie v tomto čísle
Kurzy

Zvýšte si kvalifikáciu online z pohodlia domova

Získaná hemofilie - Povědomí o nemoci a její diagnostika
nový kurz

Eozinofilní granulomatóza s polyangiitidou
Autori: doc. MUDr. Martina Doubková, Ph.D.

Všetky kurzy
Prihlásenie
Zabudnuté heslo

Zadajte e-mailovú adresu, s ktorou ste vytvárali účet. Budú Vám na ňu zasielané informácie k nastaveniu nového hesla.

Prihlásenie

Nemáte účet?  Registrujte sa

#ADS_BOTTOM_SCRIPTS#