Mutations in the Bacterial Ribosomal Protein L3 and Their Association with Antibiotic Resistance

Abstract

b Different groups of antibiotics bind to the peptidyl transferase center (PTC) in the large subunit of the bacterial ribosome. Resistance to these groups of antibiotics has often been linked with mutations or methylations of the 23S rRNA. In recent years, there has been a rise in the number of studies where mutations have been found in the ribosomal protein L3 in bacterial strains resistant to PTC-targeting antibiotics but there is often no evidence that these mutations actually confer antibiotic resistance. In this study, a plasmid exchange system was used to replace plasmid-carried wild-type genes with mutated L3 genes in a chromosomal L3 deletion strain. In this way, the essential L3 gene is available for the bacteria while allowing replacement of the wild type with mutated L3 genes. This enables investigation of the effect of single mutations in Escherichia coli without a wild-type L3 background. Ten plasmid-carried mutated L3 genes were constructed, and their effect on growth and antibiotic susceptibility was investigated. Additionally, computational modeling of the impact of L3 mutations in E. coli was used to assess changes in 50S structure and antibiotic binding. All mutations are placed in the loops of L3 near the PTC. Growth data show that 9 of the 10 mutations were well accepted in E. coli, although some of them came with a fitness cost. Only one of the mutants exhibited reduced susceptibility to linezolid, while five exhibited reduced susceptibility to tiamulin. During the last 10 years, mutations in the L3 ribosomal protein have been associated with bacterial resistance to linezolid (an oxazolidinone) and tiamulin (a pleuromutilin). These drugs bind to the ribosomal peptidyl transferase center (PTC), where specific 23S rRNA mutations and methylation at 2503 also cause resistance to oxazolidinones and pleuromutilins (reviewed in references 1, 2, and 3). The main part of L3 is positioned on the surface of the 50S ribosomal subunit, but a branched loop extends close to the PTC (Fig. 1), the binding site for many different ribosomal antibiotics. The first L3 resistance mutation in bacteria was detected in 2003 in Escherichia coli selected with tiamulin, and its role in resistance was verified by genetic evidence (4). Since then, L3 mutations have been associated with resistance to linezolid, tiamulin or valnemulin, and anisomycin, as reviewed in reference 1. L3 mutations related to antibiotic resistance have been reported in Brachyspira spp., Staphylococcus spp., E. coli, and Mycobacterium tuberculosis. Most of the studies have lacked proof that these mutations actually cause antibiotic resistance, except a few (4-7). It is not evident exactly which L3 mutations have a relation to reduced antibiotic susceptibility, but only those in the part of the L3 protein nearest to the PTC are assumed to confer a resistance effect. Most of the known L3 mutations are found together with other resistance determinants, mainly mutations in the domain V region of 23S rRNA or mutations in ribosomal protein L4 and/or th...

Combined Effect of the Cfr Methyltransferase and Ribosomal Protein L3 Mutations on Resistance to Ribosome-Targeting Antibiotics

“…These data are in accordance with our previous study (6). The effect is retained in the presence of Cfr.…”

“…Various clinical data point to the coexistence of L3 mutations and either 23S RNA mutations or the presence of the cfr gene in strains resistant to PTC antibiotics (6,19). This raises the question of a synergistic effect of L3 mutations and Cfr on antibiotic resistance.…”

“…The mutations correspond to mutations observed in pathogenic bacteria, and that study thus supports, in general, the presumed or proven effects from other studies. For example, an LZD resistance effect from L3 N149R in E. coli (6) correlates well with a resistance effect of the L3 C154R substitution in M. tuberculosis at the corresponding position (7)(8)(9).…”

“…For this, we constructed a cfr-encoding plasmid termed pKPCfr that is compatible with the plasmids encoding the mutated L3 genes constructed by Klitgaard et al (6). We measured the doubling times of the L3 mutant strains with and without Cfr to assay the effect of the L3 mutations and Cfr on growth rates.…”

A Bright Future for Antibiotics?

“…Prominent processes acquiring resistance include modifications of the antibiotic binding pockets by mutations (e.g., macrolide resistance by modification of a component crucial for their binding, A2058G). Other frequently used mechanisms include activation of key enzymatic processes (e.g., methylation of the binding components of macrolide and aminoglycosides by methylases); enzymatic inactivation of the drug, such as the macrolide molecule by esterases (61); removal of the antibiotic drug from its target (i.e., resistance to tetracycline by disturbing the ribosomal protection proteins) (62)(63)(64)(65); and modification of ribosomal proteins essential for ribosomal functionality at the PTC and tunnel entrance, such as rpL3, which is associated with resistance to linezolid, tiamulin, and anisomycin (66,67), or disruption of the interactions between proteins that play key roles in protein biosynthesis (68).…”