ClpP1P2 peptidase activity promotes biofilm formation in Pseudomonas aeruginosa

Abstract

Caseinolytic proteases (Clp) are central to bacterial proteolysis and control cellular physiology and stress responses. They are composed of a double‐ring compartmentalized peptidase (ClpP) and a AAA+ unfoldase (ClpX or ClpA/ClpC). Unlike many bacteria, the opportunistic pathogen Pseudomonas aeruginosa contains two ClpP homologs: ClpP1 and ClpP2. The specific functions of these homologs, however, are largely elusive. Here, we report that the active form of PaClpP2 is a part of a heteromeric PaClpP17P27 tetradecamer that is required for proper biofilm development. PaClpP114 and PaClpP17P27 complexes exhibit distinct peptide cleavage specificities and interact differentially with P. aeruginosa ClpX and ClpA. Crystal structures reveal that PaClpP2 has non‐canonical features in its N‐ and C‐terminal regions that explain its poor interaction with unfoldases. However, experiments in vivo indicate that the PaClpP2 peptidase active site uniquely contributes to biofilm development. These data strongly suggest that the specificity of different classes of ClpP peptidase subunits contributes to the biological outcome of proteolysis. This specialized role of PaClpP2 highlights it as an attractive target for developing antimicrobial agents that interfere specifically with late‐stage P. aeruginosa development.

Acinetobacter baumannii Strains Deficient in the Clp Chaperone-Protease Genes Have Reduced Virulence in a Murine Model of Pneumonia

“…Our data show that the Clp chaperone-protease genes are required for the bacterium to cause disease and thrive within the mammalian host. Like what has been observed in other bacterial pathogens [35][36][37], we have uncovered several specific roles for the A. baumannii Clp chaperone-protease system, including intracellular survival and biofilm formation. We confirmed that the defects observed in A. baumannii clp mutants after interaction with phagocytes or biofilm formation are not caused by aberrant growth phenotypes under standard laboratory conditions.…”

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

“…The fact that Mtb ClpP1 proteolytic activity is essential while ClpP2 activity is not could be a result of their different substrate-binding pocket preferences ( 7 , 10 ) and/or cleavage kinetics between ClpP1 and ClpP2. For example, ClpP1 protease activity could be critical to producing a growth-promoting peptide or cleaving a growth-inhibitory peptide, analogous to the proposed role of ClpP2 in Pseudomonas biofilm formation ( 33 ). However, we favor a model whereby ClpP1 protease activity is critical to maintaining flux through the Clp system.…”

“…and Arabidopsis spp., which encode proteolytically inactive ClpR subunits whose function remains poorly understood (29,30). Given that other features of the Mtb Clp protease, for example, asymmetric binding of chaperones to one surface of the complex, are shared between Mtb and other multi-clpP allele-encoding bacteria (31)(32)(33)(34), it is possible that functional asymmetry in Clp proteolysis may exist in other bacterial species as well.…”

Listeria monocytogenes utilizes the ClpP1/2 proteolytic machinery for fine-tuned substrate degradation under heat stress

“…Some organisms have more than one clpP gene, but the role of multiple ClpPs in these organisms is not well understood. In bacteria, it is known that two different ClpPs are able to form heterocomplexes to become active, tune the cleavage specificity or enhance the activity of the homocomplexes (Dahmen et al, 2015; Li et al, 2016; Mawla et al, 2020; Pan et al, 2019). Here we examined the biological role of ClpP1/2 heterocomplex formation in L. monocytogenes and the specific physiological functions of both ClpPs.…”

“…ClpX is a hexameric ATP-dependent chaperone which recognizes protein substrates and directs unfolded peptide chains into the tetradecameric barrel of the ClpP serine protease for degradation (Baker & Sauer, 2012). Some bacteria such as L. monocytogenes encode two ClpP isoforms (ClpP1 and ClpP2) with yet largely unknown cellular roles (Dahmen et al, 2015; Hall et al, 2017; Mawla et al, 2020; Pan et al, 2019; Zeiler et al, 2011). In L. monocytogenes ClpP1 exhibits low sequence homology to ClpP isoforms from other bacteria and is expressed as an inactive heptamer with an impaired catalytic triad.…”