Molecular Imaging of Isolated Escherichia coli DH5α Peptidoglycan Sacculi Identifies the Mechanism of Action of Cell Wall-Inhibiting Antibiotics

Abstract

Antibiotic resistance of pathogenic bacteria needs to be urgently addressed by the development of new antibacterial entities. Although the prokaryotic cell wall comprises a valuable target for this purpose, development of novel cell wall-active antibiotics is mostly missing today. This is mainly caused by hindrances in the assessment of isolated enzymes of the co-dependent murein synthesis machineries, e.g., the elongasome and divisome. We therefore present imaging methodologies to evaluate inhibitors of bacterial cell wall synthesis by high-resolution atomic force microscopy on isolated Escherichia coli murein sacculi. With the ability to elucidate the peptidoglycan ultrastructure of E. coli cells, unprecedented molecular insights into the mechanisms of antibiotics were established. The nanoscopic impairments introduced by ampicillin, amoxicillin, and fosfomycin were not only identified by AFM but readily correlated with their known mechanism of action. These valuable in vitro capabilities will facilitate the identification and evaluation of new antibiotic leads in the future.

Relationship between the Rod complex and peptidoglycan structure in Escherichia coli

“…Although we do not know why these observations using AFM obtained different results, our results are consistent with those of Elsbroek et al (2023) and are therefore consistent with our conclusion that the RMR-producing strain probably has reduced peptidoglycan synthesis activity. Furthermore, Salamaga et al (2021) reported that peptidoglycan purified from S. aureus cells treated with methicillin or vancomycin had larger holes than those purified from nontreated cells, using AFM.…”

“…Turner et al (2018) reported that no change in the pore size of peptidoglycans was observed with the addition of A22. On the other hand, Elsbroek et al (2023) reported that the addition of β-lactam antibiotics made peptidoglycans less dense, that is, the pores became larger, in which the authors concluded that their results are consistent with the mechanism of action of β-lactam antibiotics to inhibit peptide cross-linking.…”

“…Peptidoglycan has been observed by EM and AFM, and its structures have been reported (de Pedro et al, 1997;Elsbroek et al, 2023;Gan et al, 2008;Pasquina-Lemonche et al, 2020;Salamaga et al, 2021;Tulum et al, 2019;Turner et al, 2018). In particular, QFDE-EM is an excellent tool for visualizing the structure of peptidoglycan with high resolution.…”

High molecular/low acetylated chitosans reduce adhesion of Campylobacter jejuni to host cells by blocking JlpA

“…Typically, similar types of elongation have been described for bacteria with damaged cell wall formation (e.g. by fosfomycin or β-lactam antibiotics (Elsbroek et al 2023 ). This finding indicates that low acetylated chitosans interact with the correct cell wall formation, which again could explain also changes in the outer membrane.…”

“…This finding indicates that low acetylated chitosans interact with the correct cell wall formation, which again could explain also changes in the outer membrane. As cell wall architecture can be monitored more specifically by isolation and preparation of the murein sacculi of the bacteria and subsequent AFM imaging (Elsbroek et al 2023 ), those were isolated and purified from untreated C. jejuni (Fig. 4 ).…”