Revisiting the mechanism of macrolide-antibiotic resistance mediated by ribosomal protein L22

Abstract

Bacterial antibiotic resistance can occur by many mechanisms. An intriguing class of mutants is resistant to macrolide antibiotics even though these drugs still bind to their targets. For example, a 3-residue deletion (⌬MKR) in ribosomal protein L22 distorts a loop that forms a constriction in the ribosome exit tunnel, apparently allowing nascent-chain egress and translation in the presence of bound macrolides. Here, however, we demonstrate that ⌬MKR and wild-type ribosomes show comparable macrolide sensitivity in vitro. In Escherichia coli, we find that this mutation reduces antibiotic occupancy of the target site on ribosomes in a manner largely dependent on the AcrAB-TolC efflux system. We propose a model for antibiotic resistance in which ⌬MKR ribosomes alter the translation of specific proteins, possibly via changes in programmed stalling, and modify the cell envelope in a manner that lowers steady-state macrolide levels.ermC ͉ erythromycin ͉ ribosome ͉ TolC M any antibiotics inhibit bacterial protein synthesis. Understanding how microbes become antibiotic resistant is important both for developing effective treatment regimens and designing new therapeutics. Macrolides consist of a 14-to 16-member lactone ring with different appended sugars and comprise a key group of inhibitors of bacterial translation (1, 2). The inhibitory activity of macrolides, including erythromycin, depends on binding to a site near the polypeptide exit tunnel of the large ribosomal subunit (3, 4). Because macrolides do not bind to ribosomes with an occupied exit tunnel and cause the synthesis of 2-10 residue peptides in translation assays in vitro, it has been proposed that drug binding physically blocks elongation of nascent proteins beyond this size (5-7).Some macrolide-resistance mutations alter the ribosomal target site and prevent binding (4,8). Intriguingly, other mutations confer resistance despite the fact that macrolides still bind the mutant ribosome well (8-10). For example, deletion of the M 82 K 83 R 84 sequence in Escherichia coli ribosomal protein L22 (⌬MKR) allows growth in the presence of high levels of erythromycin and other macrolides (11-13). The same mutation makes Haemophilus influenzae resistant to numerous macrolides (14); different L22 mutations also confer macrolide resistance in other bacterial species (2). When binding has been measured, ribosomes with macrolideresistant alterations in L22 bind erythromycin with near wild-type affinity (8,10,12,15).In a crystal structure of the E. coli ribosome, the MKR sequence is part of an extended L22 loop, which together with a similar loop in protein L4 forms a narrow constriction in the exit tunnel (Fig. 1A) (16, 17). Cryo-EM studies initially revealed a widened exit tunnel in E. coli ⌬MKR ribosomes (18). This loop is also displaced to create an expanded tunnel in structures of ⌬MKR ribosomes from Thermus thermophilus and Haloarcula marismortui (4, 19). These results explain the altered chemical reactivity in E. coli ⌬MKR ribosomes of 23S-RNA bases (13). Together,...

Drug efflux pump deficiency and drug target resistance masking in growing bacteria

“…In this, we use what we deem as realistic values for growth rate, drug permeability, drug affinities to target and target concentration. The experimental conditions mimic those of recently published observations (12,13), that will be discussed in the next section.…”

“…Very recently, it was demonstrated by more quantitative experiments that an amino acid deletion in protein L22 of an E. coli strain reduced its susceptibility to erythromycin in a drug efflux proficient, but not in a drug efflux deficient (⌬tolC) background (12,13). Interestingly, when the experiment was carried out in ⌬acrB strains, retaining residual drug efflux pump activity, the L22 mutation conferred a susceptibility reduction that was significant but much smaller than the reduction seen in the drug efflux pump proficient background (12,13).…”

“…Interestingly, when the experiment was carried out in ⌬acrB strains, retaining residual drug efflux pump activity, the L22 mutation conferred a susceptibility reduction that was significant but much smaller than the reduction seen in the drug efflux pump proficient background (12,13). Biochemical experiments showed that the L22 alteration reduced Ϸ50-fold the rate constant for erythromycin binding to and Ϸ10-fold the rate constant for erythromycin dissociation from the ribosome, corresponding to an Ϸ5-fold overall affinity loss (12).…”

“…This feature of drug efflux pump deficiency is intuitively obvious, but recent findings for the ribosome targeting macrolide antibiotic erythromycin suggest the existence of more subtle aspects of the interplay between drug efflux pump efficiency and drug susceptibility. An Escherichia coli mutant with an amino acid deletion in protein L22 of the large ribosomal subunit (10,11), causing reduced affinity of erythromycin to the ribosome (12), displayed reduced susceptibility to erythromycin in relation to a ribosome WT strain in a drug efflux pump proficient but the same susceptibility in a drug efflux pump deficient background (12,13). These experiments demonstrated how a mutation reducing drug affinity to a vital intracellular target may give a distinct growth advantage to drug exposed bacteria in a drug efflux pump proficient background but virtually no advantage in a drug efflux pump deficient background.…”

Role of the AraC–XylS family regulator YdeO in multi-drug resistance of Escherichia coli

“…[12][13][14][15] TolC is responsible for resistance to various antibiotics, including b-lactams, 12 quinolones 16 and macrolides. 17 Bacterial genome sequencing enables us to trace drug-resistance genes. [18][19][20] There are many putative and proven drug efflux pumps in the E. coli genome, and we have identified earlier 20 functional drug efflux pumps.…”