The Molecular Mechanism of Peptide-mediated Erythromycin Resistance

Abstract

The macrolide antibiotic erythromycin binds at the entrance of the nascent peptide exit tunnel of the large ribosomal subunit and blocks synthesis of peptides longer than between six and eight amino acids. Expression of a short open reading frame in 23 S rRNA encoding five amino acids confers resistance to erythromycin by a mechanism that depends strongly on both the sequence and the length of the peptide. In this work we have used a cell-free system for protein synthesis with components of high purity to clarify the molecular basis of the mechanism. We have found that the nascent resistance peptide interacts with erythromycin and destabilizes its interaction with 23 S rRNA. It is, however, in the termination step when the pentapeptide is removed from the peptidyl-tRNA by a class 1 release factor that erythromycin is ejected from the ribosome with high probability. Synthesis of a hexa-or heptapeptide with the same five N-terminal amino acids neither leads to ejection of erythromycin nor to drug resistance. We propose a structural model for the resistance mechanism, which is supported by docking studies. The rate constants obtained from our biochemical experiments are also used to predict the degree of erythromycin resistance conferred by varying levels of resistance peptide expression in living Escherichia coli cells subjected to varying concentrations of erythromycin. These model predictions are compared with experimental observations from growing bacterial cultures, and excellent agreement is found between theoretical prediction and experimental observation.

Macrolide resistance mechanisms inEnterobacteriaceae: Focus on azithromycin

“…A translation of 4-6 amino acids can avoid the possibility of macrolides interacting with the ribosome, and the protein can successfully be translated (Tenson & Mankin, 2001;Tripathi et al, 1998). Lovmar et al (2006) reinforced this model by analyzing the erythromycin dissociation rates with the use of the E-peptide MRLFV. Moreover, the same study showed that alterations in the size of the peptide possess a strong effect on the resistance levels conferred, suggesting that the Leu residue plays a key role in the dissociation of erythromycin.…”

Transient Erythromycin Resistance Phenotype Associated with Peptidyl-tRNA Drop-Off on Early UGG and GGG Codons

“…The pentapeptide is removed from the peptidyl-tRNA by a class 1 release factor at the termination step, giving erythromycin ejection from the ribosome at a high probability. Synthesis of the longer heptapeptide with the same five N-terminal amino acids leads neither to ejection of erythromycin nor to drug resistance (14). Our data suggest that even in the absence of termination, an abortive translation event on early UGG or GGG codons can give rise to peptidyl-tRNA where the peptide moiety functions as an E peptide.…”

“…Thus, the Epeptide sequence does not show activity if it is part of a longer protein sequence (14,32,33). It must be in the form of a short peptide since a stop codon or a drop-off-prone NGG codon after this coding sequence is required to promote growth in the presence of erythromycin (Table 1) (14,32). The strains carrying constructs with UGG or GGG at ϩ5 gave a similar relative growth value in the presence of erythromycin, also suggesting E-peptide activity.…”

“…The drug has a high affinity for ribosomes with nascent peptides shorter than five amino acids, but it does not bind to ribosomes that carry long nascent peptide chains (6). It has been suggested that erythromycin sterically blocks elongation of the nascent peptide chain (35) and promotes dissociation of peptidyl-tRNA (16,17), thereby increasing the rate of peptidyl-tRNA dropoff (14).…”

“…A recent molecular model for peptide-mediated erythromycin resistance suggests that the E peptide interacts with erythromycin and destabilizes its interaction with 23S rRNA (14). The pentapeptide is removed from the peptidyl-tRNA by a class 1 release factor at the termination step, giving erythromycin ejection from the ribosome at a high probability.…”