Mutations in ribosomal protein L16 conferring reduced susceptibility to evernimicin (SCH27899): implications for mechanism of action

Abstract

A clinical isolate of Streptococcus pneumoniae (SP#5) that showed decreased susceptibility to evernimicin (MIC, 1.5 microgram/ml) was investigated. A 4,255-bp EcoRI fragment cloned from SP#5 was identified by its ability to transform evernimicin-susceptible S. pneumoniae R6 (MIC, 0.03 microgram/ml) such that the evernimicin MIC was 1.5 microgram/ml. Nucleotide sequence analysis of this fragment revealed that it contained portions of the S10-spc ribosomal protein operons. The nucleotide sequences of resistant and susceptible isolates were compared, and a point mutation (thymine to guanine) that causes an Ile52-Ser substitution in ribosomal protein L16 was identified. The role of this mutation in decreasing susceptibility to evernimicin was confirmed by direct transformation of the altered L16 gene. The presence of the L16 mutation in the resistant strain suggests that evernimicin is an inhibitor of protein synthesis. This was confirmed by inhibition studies using radiolabeled substrates, which showed that the addition of evernimicin at sub-MIC levels resulted in a rapid decrease in the incorporation of radiolabeled isoleucine in a susceptible isolate (SP#3) but was much less effective against SP#5. The incorporation of isoleucine showed a linear response to the dose level of evernimicin. The incorporation of other classes of labeled substrates was unaffected or much delayed, indicating that these were secondary effects.

FIG. 1

Gene organization of the 4,255-bp EcoRI fragment of the S10/spc ribosomal protein operon showing the nucleotide and amino acid sequence differences between the evernimicin-susceptible S. pneumoniae R6, the evernimicin-resistant transformant (ZR1), and the original clinical isolate (SP#5). The hatched lines indicate the putative points of crossing over during homologous recombination.

FIG. 2

In vivo labeling with [¹⁴C]isoleucine in SP#3 (A) and SP#5 (B). The results shown are from a single experiment that is representative of at least two additional experiments. Each point is the average value of triplicate samples that were taken at every time point. The peculiar sinusoidal response seen following the addition of drug or carrier alone occurred consistently in the labeling of S. pneumoniae and may be a response to the high pH or hydrophobic nature of the carrier agent. Symbols correspond to the evernimicin levels used in the experiment, which were added at the 90-min time point as follows: ⧫, 0.4 μg/ml; ■, 0.1 μg/ml; ▴, 0.025 μg/ml; ✖, 0.00625 μg/ml; ∗, 0.00156 μg/ml; ●, 0.0004 μg/ml; ✚, drug carrier, used at the same level used for 0.4 μg of evernimicin/ml. The growth curves shown in the insets are single OD₅₄₀ readings.

FIG. 3

The amino acid sequence of the S. pneumoniae L16 protein from residues 43 to 60 aligned with the same region of L16 from gram-positive bacteria (S. aureus, Enterococcus faecalis, and B. subtilis) and gram-negative bacteria (E. coli, Haemophilus influenzae, and Neisseria gonorrhoeae.