Agents Targeting the Bacterial Cell Wall as Tools to Combat Gram-Positive Pathogens

Abstract

The cell wall is an indispensable element of bacterial cells and a long-known target of many antibiotics. Penicillin, the first discovered beta-lactam antibiotic inhibiting the synthesis of cell walls, was successfully used to cure many bacterial infections. Unfortunately, pathogens eventually developed resistance to it. This started an arms race, and while novel beta-lactams, either natural or (semi)synthetic, were discovered, soon upon their application, bacteria were developing resistance. Currently, we are facing the threat of losing the race since more and more multidrug-resistant (MDR) pathogens are emerging. Therefore, there is an urgent need for developing novel approaches to combat MDR bacteria. The cell wall is a reasonable candidate for a target as it differentiates not only bacterial and human cells but also has a specific composition unique to various groups of bacteria. This ensures the safety and specificity of novel antibacterial agents that target this structure. Due to the shortage of low-molecular-weight candidates for novel antibiotics, attention was focused on peptides and proteins that possess antibacterial activity. Here, we describe proteinaceous agents of various origins that target bacterial cell wall, including bacteriocins and phage and bacterial lysins, as alternatives to classic antibiotic candidates for antimicrobial drugs. Moreover, advancements in protein chemistry and engineering currently allow for the production of stable, specific, and effective drugs. Finally, we introduce the concept of selective targeting of dangerous pathogens, exemplified by staphylococci, by agents specifically disrupting their cell walls.

Keywords: Gram-positive bacteria; Staphylococcus aureus; antibiotic; bacterial cell wall; endolysin; lysostaphin; peptidoglycan; peptidoglycan hydrolase.

Figure 1

Antimicrobial agents acting on the bacterial cell wall. Classic antibiotics, low-molecular-weight compounds, and non-ribosomal peptides interfere with peptidoglycan synthesis, which is the main building material of the bacterial cell wall. The peptidoglycan layer in Gram-positive bacteria is thick and exposed to the outer environment. In contrast, in Gram-negative bacteria, the peptidoglycan layer is considerably thinner and covered by an outer membrane, which limits the penetration of antimicrobial agents, particularly those with high molecular weight. High-molecular-weight agents are proteinaceous and may be divided into two groups: lysins of phage origin (virion-associated lysins [VALs] and endolysins) and enzymes of bacterial origin (autolysins and bacteriocins). These antimicrobial agents are responsible for the enzymatic lysis of the bacterial cell wall.

Figure 2

Biosynthesis and disassembly of peptidoglycan (PG). The synthesis of PG begins in the cytoplasm, where GlcNAc–UDP is transformed into MurNAc–UDP by MurA and MurB enzymes. It is followed by a stepwise addition of five amino acids (the stem peptide) by MurC, MurD, MurE, and MurF. MurNAc–pentapeptide (MurNAc–pp) is anchored by two phosphate molecules to membrane-bound undecaprenyl lipid carrier (Und–P), creating Lipid I. MurG then adds GlcNAc to MurNAc–pp, which together form Lipid II. FemA, FemB, and FemX enzymes add an interpeptide bridge to the third amino acid in the stem peptide. The finished monomer is translocated to the outer edge of the membrane by MurJ flippase, where penicillin-binding proteins (PBPs) perform binding of Lipid II to the previous glycan strand by the transglycosylase (TG) domain. The transpeptidase (TP) domain crosslinks peptidoglycan by joining strands with interpeptide bridges. The activity of PBPs is inhibited by conventional antibiotics (penicillin, carbapenems, monobactams, cephalosporins). Acquisition of resistance results in mutated forms of PBPs; therefore, another approach is the use of glycopeptides such as vancomycin and dalbavancin (which interact with the stem peptide, preventing transglycosylation) or corbomycin and complestatin (which interfere with autolysins by preventing PG remodeling). Antibiotics and glycopeptides interfere with PG biosynthesis, whereas endolysins and lysostaphins activities affect mature cell walls.

Figure 3

The structural formulas of the cell-wall-targeting antibiotics. (A) The structures of the cores of β-lactam antibiotics. The β-lactam ring is colored in red. The bond hydrolyzed by β-lactamases is marked with an arrow. R, R1, and R2, variable functional groups. (B) The structural formulas of exemplary glycopeptide antibiotics.

Figure 4

Degradation of peptidoglycan is carried out by endolysins and lysostaphins. The singular monomer of PG contains several glycosidic, amide, and peptide bonds undergoing hydrolysis. Depending on enzymatic activity, endolysins can be divided into five groups. Muramidase (N-acetyl-β-d-muramidase) targets the N-acetylmuramoyl-β-1,4-N-glucosamine bond between MurNAc and GlcNAc (within a single monomer). The same bond is recognized by lytic transglycosylase, but this enzyme’s activity involves non-hydrolytic breakage of the β-1,4-glycosidic bond. Glucosaminidase (N-acetyl-glucosaminyl-β-d-glucosaminidase) disrupts glycan strands by cleaving the β-1,4-glysosidic bond between MurNAc and GlcNAc at the reducing end of GlcNAc. Amidase (N-acetylmuramoyl-l-alanine) cleaves the peptide bond linking MurNAc with the stem peptide, and endopeptidases also target peptide bonds, but between amino acids of the stem peptide. Interpeptide bridges can be targeted by either endopeptidases or lysostaphins.

Figure 5

The diversity of different groups of peptidoglycan hydrolases (PGHs). (A) Various architectures of PGHs with the names of their representatives. SP, signal peptide; EAD, enzymatically active domain; CBD, cell-wall-binding domain; SBD, spore-binding domain. (B) The molecular structure of PlyPSA and LysIME–EF1 endolysins. PDB ID: 8H1I and 6IST, respectively. The coloring corresponds to the first panel. For clarity, three of the four CBDs of LysIME–EF1 are colored yellow. The catalytic Ca²⁺ ions located within CHAP domains are depicted as gray spheres.

Figure 6

Structure and similarity of staphylococcal lysostaphins. (A) The most common lysostaphin architecture includes an N-terminal signal peptide (SP) followed by a propeptide (PP), which contains short 13-amino-acid-long repeats (R) or a putative MSCRAMM domain. The essential part of each lysostaphin comprises an enzymatically active domain and a cell-wall-binding domain, separated by a short linker (EAD–CBD). (B) The molecular structure of the mature form of Lys–Ss includes the EAD and CBD domains (M23-SH3b), with a Zn²⁺ ion located within the M23 metalloproteinase domain, depicted as a gray sphere. The coloring corresponds to the first panel. PDB ID: 4LXC. (C) Sequence identity and similarity of the EAD–CBD regions among known staphylococcal lysostaphins. Lys–Ss and ALE-1 sequences are highly similar, while sequence similarity among other lysostaphins is moderate. SP, signal peptide; PP, propeptide; R, repeat; EAD, enzymatically active domain; CBD, cell-wall-binding domain.