Studies on Aminoglycoside Susceptibility Identify a Novel Function of KsgA To Secure Translational Fidelity during Antibiotic Stress

Abstract

Antibiotic resistance has become a global crisis. Studies on the mechanism of bacterial tolerance to antibiotics will not only increase our conceptual understanding of bacterial death but also provide potential targets for novel inhibitors. We screened a mutant library containing a full set of in-frame deletion mutants of K-12 and identified 140 genes that possibly contribute to gentamicin tolerance. The deletion of increased the inhibition and killing potency against mid-log-phase bacteria by aminoglycosides. Initially identified as a 16S rRNA methyltransferase, KsgA also has additional functions as a ribosomal biogenesis factor and a DNA glycosylase. We found that the methyltransferase activity of KsgA is responsible for the tolerance, as demonstrated by a site-directed mutagenesis analysis. In contrast to the mechanism for cold sensitivity, the decreased tolerance to aminoglycoside is not related to the failure of ribosomal biogenesis. Furthermore, the DNA glycosylase activity of KsgA contributes minimally to kanamycin tolerance. Importantly, we discovered that KsgA secures protein translational fidelity upon kanamycin killing, in contrast to its role during cold stress and kasugamycin treatment. The results suggest that the compromise in protein translational fidelity in the absence of KsgA is the root cause of an increased sensitivity to a bactericidal aminoglycoside. In addition, KsgA in the pathogenic contributes not only to the tolerance against aminoglycoside killing but also to virulence in the host, warranting its potential application as a target for inhibitors that potentiate aminoglycoside therapeutic killing as well as disarm bacterial virulence simultaneously.

Non-walled spherical Acinetobacter baumannii is an important type of persister upon β -lactam antibiotic treatment

“…Thus, colistin might be a good candidate for combinational therapy with β-lactam antibiotics to treat A. baumannii infection. This result is also consistent with our previous studies that targeting bacterial persistence or tolerance could highly enhance the antibiotic treatment [27,57].…”

“…The bacterial killing was performed as described previously [27] and a detailed protocol is provided in supplementary experimental procedures.…”

Structure of the Bacterial Ribosome at 2 Å Resolution

“…By contrast, bacteria lacking KsgA, the methyltransferase responsible for dimethylation of both nucleotides, become highly susceptible to other antibiotics including aminoglycosides and macrolides Phunpruch et al, 2013;Zou et al, 2018). In the present structure, m 6 2A1519 is singly-methylated whereas m 6 2A1518 is fully methylated (Figure 1-figure supplement 5).…”

Antibiotic Resistance Patterns of Pseudomonas spp. Isolated From Raw Milk Revealed by Whole Genome Sequencing

“…The most prevalent aminoglycoside resistance gene, aph(6)-Ic, is one of three resistance genes present in the composite transposon Tn5, which is found in Gram-negative bacteria (Steiniger-White et al, 2004). The ksgA gene, which confers resistance to kasugamycin (Zou et al, 2018), was also found in seven isolates. Genes ant(3")-Ia, aph(6)-Id and aph(3')-IIc as well as rpsL mutations, known to be responsible for streptomycin resistance (Ramirez and Tolmasky, 2010), were also found in these isolates ( Supplementary Table S3).…”