Effects of Ribosomal Protein S10 Flexible Loop Mutations on Tetracycline and Tigecycline Susceptibility of Escherichia coli.

Norbert Izghirean,Brigitte Pertschy,Günther Koraimann,Gernot Zarfel,Clemens Kittinger,Claudia Waidacher,Miriam Chyba

doi:10.3389/fmicb.2021.663835

Abstract

Tigecycline is a tetracycline derivative that is being used as an antibiotic of last resort. Both tigecycline and tetracycline bind to the small (30S) ribosomal subunit and inhibit translation. Target mutations leading to resistance to these antibiotics have been identified both in the 16S ribosomal RNA and in ribosomal proteins S3 and S10 (encoded by the rpsJ gene). Several different mutations in the S10 flexible loop tip residue valine 57 (V57) have been observed in tigecycline-resistant Escherichia coli isolates. However, the role of these mutations in E. coli has not yet been characterized in a defined genetic background. In this study, we chromosomally integrated 10 different rpsJ mutations into E. coli, resulting in different exchanges or a deletion of S10 V57, and investigated the effects of the mutations on growth and tigecycline/tetracycline resistance. While one exchange, V57K, decreased the minimal inhibitory concentration (MIC) (Etest) to tetracycline to 0.75 μg/ml (compared to 2 μg/ml in the parent strain) and hence resulted in hypersensitivity to tetracycline, most exchanges, including the ones reported previously in resistant isolates (V57L, V57D, and V57I) resulted in slightly increased MICs to tigecycline and tetracycline. The strongest increase was observed for the V57L mutant, with a MIC (Etest) to tigecycline of 0.5 μg/ml (compared to 0.125 μg/ml in the parent strain) and a MIC to tetracycline of 4.0 μg/ml. Nevertheless, none of these exchanges increased the MIC to the extent observed in previously described clinical tigecycline-resistant isolates. We conclude that, next to S10 mutations, additional mutations are necessary in order to reach high-level tigecycline resistance in E. coli. In addition, our data reveal that mutants carrying S10 V57 exchanges or deletion display growth defects and, in most cases, also thermosensitivity. The defects are particularly strong in the V57 deletion mutant, which is additionally cold-sensitive. We hypothesize that the S10 loop tip residue is critical for the correct functioning of S10. Both the S10 flexible loop and tigecycline are in contact with helix h31 of the 16S rRNA. We speculate that exchanges or deletion of V57 alter the positioning of h31, thereby influencing both tigecycline binding and S10 function.

Highlights

Tigecycline is an antibiotic of last resort
Our data reveal for the first time that mutation of a single residue, valine 57 (V57), in the S10 flexible loop, is sufficient to cause altered tetracycline, minocycline, and tigecycline susceptibility in E. coli
The highest increase of minimal inhibitory concentration (MIC) to tetracycline, minocycline, and tigecycline was observed in the V57L mutant, with a fourfold increased MIC to tigecycline and a twofold increased MIC to tetracycline according to Etest data

Summary

Introduction

Tigecycline is an antibiotic of last resort. It is, beside colistin, far too often the last antibiotic that is effective, especially in infections with carbapenem-resistant Enterobacteriaceae (with some exceptions like Proteus spp., which is naturally resistant to tigecycline) (Gales and Jones, 2000; Meagher et al, 2005).Tigecycline is the first member of the glycylcycline class, which is derived from tetracycline, more precisely from the semisynthetic tetracycline minocycline. Tigecycline binds to the 30S ribosomal subunit, albeit with higher affinity, and inhibits translation elongation by preventing delivery of tRNA to the ribosomal A site (Bergeron et al, 1996). Structures of tigecycline bound to the 70S ribosome showed that the binding site of tigecycline comprises nucleotides of helices h31 and h34 of the 16S rRNA located at the base of the head domain of the 30S ribosomal subunit (Figure 1; Bergeron et al, 1996; Gales and Jones, 2000; Bauer et al, 2004; Jenner et al, 2013). Most tetracycline resistance mechanisms, like via ribosomal protection proteins (e.g., TetM in Escherichia coli) or specific tetracycline efflux pumps (e.g., TetL), are not or at least less effective against tigecycline (Jenner et al, 2013; Fiedler et al, 2016). Tigecycline-resistant mutations have been observed in RNA or protein components of the 30S ribosomal subunit (Yuhan et al, 2016; Argudin et al, 2017; Chen et al, 2020)

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