Cellular functions of the ClpP protease impacting bacterial virulence

Abstract

Proteostasis mechanisms significantly contribute to the sculpting of the proteomes of all living organisms. ClpXP is a central AAA+ chaperone-protease complex present in both prokaryotes and eukaryotes that facilitates the unfolding and subsequent degradation of target substrates. ClpX is a hexameric unfoldase ATPase, while ClpP is a tetradecameric serine protease. Substrates of ClpXP belong to many cellular pathways such as DNA damage response, metabolism, and transcriptional regulation. Crucially, disruption of this proteolytic complex in microbes has been shown to impact the virulence and infectivity of various human pathogenic bacteria. Loss of ClpXP impacts stress responses, biofilm formation, and virulence effector protein production, leading to decreased pathogenicity in cell and animal infection models. Here, we provide an overview of the multiple critical functions of ClpXP and its substrates that modulate bacterial virulence with examples from several important human pathogens.

Keywords: ATP-dependent proteases; ClpP protease; pathogenesis; substrates; virulence.

FIGURE 1

ClpP and Clp ATPases general architecture. (A) Top view of the E. coli ClpP cylinder (PDB ID: 1YG6). In the illustration on the right, the N-terminal loop (silver), head domain (green), handle region (blue) and catalytic triad (S, H, D) of the ClpP monomer are indicated (PDB ID 1YG6). (B) The association of a Clp ATPase hexamer (PDB ID 6SFW) with a ClpP tetradecamer (PDB ID 6SFX). Clp ATPase subunits (blue and grey) with their corresponding IGF loops (orange) are shown. ClpP subunits (green) and their hydrophobic pockets are denoted as brown circles. (C) General schematic of the proteolytic degradation cycle mediated by ClpP with its cognate Clp ATPase. Substrates are unfolded by the Clp ATPase and then translocated in an ATP-dependent manner into the ClpP catalytic chamber for proteolysis.

FIGURE 2

The diversity of ClpP substrate regulation. (A) The Clp ATPases and ClpPs of a select group of bacterial pathogens are shown. The tetradecameric organization for the active form(s) of the ClpP protease for each of these species is also shown. (B) Shown is the mechanism of SsrA tagging of nascent chains as means of targeting them for ClpP-mediated degradation. During translation of aberrant mRNAs lacking a stop codon, the ribosome stalls, resulting in the recruitment of a charged alanine-SsrA RNA (tmRNA) to the A site of the ribosome. Following transpeptidation of the charged alanine-residue to the nascent chain, the aberrant mRNA open reading frame is replaced with the RNA carried by the tmRNA, resulting in the translation of the SsrA RNA coding sequence (yellow). This C-terminal tag placed on the stalled nascent chains acts as a degron that is recognized by the Clp ATPase (e.g., ClpX or ClpA) enabling ClpP-mediated degradation.

FIGURE 3

ClpP regulates the type II toxin-antitoxin system. (A) The MazEF toxin-antitoxin module. The MazE antitoxin (green) binds and inhibits the MazF toxin (red). During stress, ClpAP (E. coli) or ClpCP in association with TrfA (S. aureus) degrades the MazE antitoxin. Free MazF, which is an mRNA endoribonuclease, cleaves various intracellular targets leading to bacterial growth arrest. (B) The parDE toxin-antitoxin module. The RK2 plasmid encodes for the ParD antitoxin (yellow) and the ParE toxin (purple) and harbors an antibiotic resistance cassette (orange). Following asymmetrical cell division with unequal plasmid distribution to daughter cells, one of the progenies may not inherit any RK2 plasmid copies. Without the ability to synthesize the ParD antitoxin de novo, the antitoxin is degraded by ClpAP leading to free ParE toxin, which causes growth arrest. (C) The toxin-antitoxin-chaperone (TAC) module in M. tuberculosis. The SecB-like chaperone (grey) binds the C-terminal region (yellow) of the HigA1 antitoxin (blue) and assists in its folding and stabilization. HigA1 in turn binds and inhibits the HigB1 toxin (light brown). During stress, the SecB-like chaperone dissociates from HigA1 via an unknown signalling mechanism causing M. tuberculosis ClpXP to degrade the HigA1 antitoxin via the recognition of the now unmasked C-terminal degron. Free HigB1 toxin induces growth arrest causing the bacteria to enter a state of dormancy.

FIGURE 4

ClpP regulates toxin production. (A) Listeria monoctyogenes ClpXP2 transcriptionally increases the production of Listeriolysin O toxin for phagolysosomal escape and propagation. (B) S. aureus ClpXP regulates the production of toxic shock syndrome toxin (TSST), enterotoxin D (SED), enterotoxin C (SEC) and hemolysin alpha-toxin (Hla) through transcriptional control of the global master regulator mgrA and the agr quorum sensor. Through an unknown target, S. aureus ClpXP stimulates the transcription of mgrA and agr leading to RNAIII upregulation, which in turn inhibits the repressor of toxins (Rot). The inhibition of Rot enables the production of Tsst, Sed, Sec, and Hla toxins. In addition, MgrA and RNAIII can directly activate the production of Hla.

FIGURE 5

ClpP regulates the assembly of the Type III secretion system as well as flagellum synthesis in various pathogens. Degradation of the repressor GrlR by ClpXP removes the inhibition of transcription of the Locus of the Enterocyte Effacement (LEE) genes, which encode for the T3SS apparatus in E. coli. In addition, degradation of GrlR prevents repression of FlhD and FlhC, the master regulators of flagellar genes, thereby causing flagellum synthesis to occur. Furthermore, RpoS turnover by ClpXP prevents upregulation of CsrA, thereby causing expression of LEE in E. coli and Salmonella Pathogenicity Islands 1 and 2 in S. typhimurium. Additionally, CsrA positively regulates FlhD and FlhC expression, therefore, turnover of RpoS by ClpXP ultimately results in FlhD and FlhC downregulation through CsrA, leading to decreased flagellar synthesis. In Y. pestis, degradation of the repressor YmoA enables LcrF to stimulate production of the T3SS apparatus.