ESKAPE Pathogens: Looking at Clp ATPases as Potential Drug Targets

Abstract

Bacterial antibiotic resistance is rapidly growing globally and poses a severe health threat as the number of multidrug resistant (MDR) and extensively drug-resistant (XDR) bacteria increases. The observed resistance is partially due to natural evolution and to a large extent is attributed to antibiotic misuse and overuse. As the rate of antibiotic resistance increases, it is crucial to develop new drugs to address the emergence of MDR and XDR pathogens. A variety of strategies are employed to address issues pertaining to bacterial antibiotic resistance and these strategies include: (1) the anti-virulence approach, which ultimately targets virulence factors instead of killing the bacterium, (2) employing antimicrobial peptides that target key proteins for bacterial survival and, (3) phage therapy, which uses bacteriophages to treat infectious diseases. In this review, we take a renewed look at a group of ESKAPE pathogens which are known to cause nosocomial infections and are able to escape the bactericidal actions of antibiotics by reducing the efficacy of several known antibiotics. We discuss previously observed escape mechanisms and new possible therapeutic measures to combat these pathogens and further suggest caseinolytic proteins (Clp) as possible therapeutic targets to combat ESKAPE pathogens. These proteins have displayed unmatched significance in bacterial growth, viability and virulence upon chronic infection and under stressful conditions. Furthermore, several studies have showed promising results with targeting Clp proteins in bacterial species, such as Mycobacterium tuberculosis, Staphylococcus aureus and Bacillus subtilis.

Keywords: Clp ATPases; ESKAPE pathogens; antibiotic resistance; caseinolytic proteins.

Figure 1

General characteristics ESKAPE pathogens. The ESKAPE group of pathogens consists of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter [1,2,3,10,11,12,13,14,15,16,17,18,19,20,21].

Figure 2

ClpP in its unbound and bound state. (A) Top view of the unbound ClpP tetradecamer (PDB—1YG6). (B) Top view of the active form of ClpP (PDB—5E0S). The Ser⁹⁷, His¹²² and Asp¹⁷¹ catalytic residues are shown up-close and coloured orange, pink and blue, respectively. Upon Clp ATPase binding, ClpP assumes an opened/active conformation resulting in the ordering of the axial pore (represented by grey dotted lines) and the alignment of the catalytic residues. The structures were visualised using PyMol [58].

Figure 3

Schematic representation of the general structural features of Class I and Class II Clp ATPases of prokaryotes. (A) Class I Clp ATPases contain two nucleotide-binding domains (NBD) referred to as domain 1 and domain 2. A middle domain has been identified to be present in certain Clp ATPases and plays a role in separating the two NBDs. (B) Class II Clp ATPases contain one NBD. Each domain contains Walker A and Walker B motifs. Certain Clp ATPases contain a P-loop, a tripeptide (represented in pink) required for interaction with ClpP. n represents the number of amino acids in the sequence [60,61].

Figure 4

Two Clp ATPase subfamilies. A tagged protein is recognised by a Clp ATPase. (a) Members of the ClpB/Hsp104 subfamily lack the tripeptide for ClpP interaction and function along with the DnaK system to unfold and refold the protein into its functional conformation. (b) Members of the ClpA subfamily contain the tripeptide for ClpP interaction and therefore redirect proteins which cannot be unfolded and reactivated to ClpP for degradation. Adapted from Maurizi and Xia [61].

Figure 5

The binding site of Ecumicin, Cyclomarin-A and Rufomycin in the ClpC1 N-terminal domain. (A) The crystal structure of ClpC1-NTD-Ecumicin (PDB-6pbs), ClpC1-NTD-Rufomycin (PDB-6cn8) and ClpC1-NTD-Cyclomarin A (PDB-3wdc) are shown in blue, green and pink, respectively, and were superimposed to compare the local environment of the three ligands. (B) The binding of two ecumicin molecules (ecumicin 1-light green, ecumicin 2-dark green) per ClpC1 N-terminal domain shows that these molecules bind adjacent to each other. The binding stochiometric ratio of rufomycin (pink) or cyclomarin A (green) to ClpC1 N-terminal domain is 1:1, represented in (C) and (D), respectively. The binding sites for these anti-TB peptides are predominantly located on residues in α-helices 1 and 5 and the loop region connecting α-helices 4 and 5. The interacting residues are coloured light pink on their respective secondary structural elements. The structures and interactions were visualised using PyMol [58].