Nonessential tRNA and rRNA modifications impact the bacterial response to sub-MIC antibiotic stress

Abstract

Antimicrobial resistance develops as a major problem in infectious diseases treatment. While antibiotic resistance mechanisms are usually studied using lethal antibiotic doses, lower doses allowing bacterial growth are now considered as factors influencing the development and selection of resistance. Starting with a high-density Tn insertion library in Vibrio cholerae and following its evolution by TN-seq in the presence of subinhibitory concentrations of antibiotics, we discovered that RNA modification genes can have opposite fates, being selected or counter-selected. We, thus have undertaken the phenotypic characterization of 23 transfer RNA (tRNA) and ribosomal RNA (rRNA) modifications deletion mutants, for which growth is globally not affected in the absence of stress. We uncover a specific involvement of different RNA modification genes in the response to aminoglycosides (tobramycin and gentamicin), fluoroquinolones (ciprofloxacin), β-lactams (carbenicillin), chloramphenicol, and trimethoprim. Our results identify t/rRNA modification genes, not previously associated to any antibiotic resistance phenotype, as important factors affecting the bacterial response to low doses of antibiotics from different families. This suggests differential translation and codon decoding as critical factors involved in the bacterial response to stress.

Keywords: RNA modifications; Vibrio cholerae; antibiotic resistance; bacterial stress responses; differential translation; sub-MIC antibiotics.

Figure 1

TN-seq identifies rRNA and tRNA modification genes affecting fitness of V. cholerae in the presence of sub-MIC TOB and CIP. tRNA modification genes are indicated in red. rRNA modification genes are indicated in blue. rlmN modifies both tRNAs and rRNA. Volcano plots show genes for which the number of transposon inactivation is increased (beneficial) or decreased (detrimental) after 16 generations of growth, compared to growth without antibiotics. (A) TOB 50% of the MIC, (B) CIP 50% of the MIC. x-axis represents log2 fold change of the number of transposon reads associated with gene inactivations, detected after 16 generations in the indicated antibiotic versus nontreated condition. The y-axis represents the negative log₁₀P-value. Gene inactivations, which show the strongest antibiotic specific effects are highlighted in yellow. Dotted lines in the y-axis indicate P-values of .05 (lower line) and .01 (upper line). Dotted lines in the x-axis represent 2-fold decrease (left) and 2-fold increase (right).

Figure 2

GO analysis for TN-seq data. The analyzed lists were for each antibiotic (TOB/CIP), genes (plotted on Fig. 1) with at least 2-fold change in TN-seq data at 16 generations compared to nontreated condition, and with an adjusted (Bonferroni correction) P-value < .05. Red arrows indicate RNA modification processes. The total number of uploaded genes list to be analyzed were 449 genes for TOB and 1004 genes for CIP. The reference gene list was V. cholerae (all genes in database), 3782 genes. Annotation version: PANTHER Overrepresentation Test (Released 20220712). GO Ontology database DOI: 10.5281/zenodo.6399963 Released 2022-03-22. Only the results with the Fold Enrichment of the most specific categories are shown, the detailed results are shown in Table S2 (Supporting Information).

Figure 3

Impact of RNA modification gene deletions on fitness during growth in sub-MIC antibiotics. In vitro competition experiments of V. cholerae WT and mutant strains in the absence or presence of different antibiotics at sub-MICs (50% of the MIC). TCL: triclosan 0.01 mM; TOB: tobramycin 0.6 μg/ml; GEN: gentamicin 0.5 μg/ml; CIP: ciprofloxacin 0.002 µg/ml, CM: chloramphenicol 0.4 µg/ml, TRM: trimethoprim 0.4 µg/ml, and CRB: carbenicillin 2.5 µg/ml. The y-axis represents log₁₀ of competitive index value calculated as described in the methods. A competitive index of 1 indicates equal growth of both strains. NT: no antibiotic treatment. For multiple comparisons, we used one-way ANOVA on GraphPad Prism. **** means P< .0001, *** means P< .001, ** means P< .01, and * means P< .05. Only significant P-values are represented. Number of replicates for each experiment: 3 < n < 8.

Figure 4

Survival to lethal antibiotic treatment. Vibrio cholerae WT and deletion mutant cultures were grown without antibiotics up to early exponential phase. Total number of bacteria was determined by plating on MH plates before addition of the indicated antibiotic at >MIC, at time T0. After 20 h of incubation with the antibiotic, the number of surviving bacteria was determined by plating on MH plates (T20). Survival was calculated for each strain by dividing the number of surviving bacteria at T20 by the initial number of bacteria. The y-axis represents the log10 survival ratio of a given mutant over the survival of the WT strain. Antibiotic concentrations: TOB 10 µg/ml, CIP 0.04 µg/ml, TRM 50 µg/ml, and CRB 50 µg/ml. Means and geometric means for logarithmic values were calculated using GraphPad Prism. For multiple comparisons, we used one-way ANOVA on GraphPad Prism. **** means P< .0001, *** means P< .001, ** means P< .01, and * means P < .05. Only significant P-values are represented. Number of replicates for each experiment: 3 < n < 8.

Figure 5

Fitness versus survival. For each antibiotic, survival values from Fig. 3 (y-axis, log10 values) were plotted against competitive index values from Fig. 2 (x-axis, log10 values). TOB: tobramycin, CIP: ciprofloxacin, TRM: trimethoprim, and CRB: carbenicillin. Names of genes with significant changes are indicated.

Figure 6

Survival of V. cholerae WT and RNA modification deletion mutants after UV irradiation. Survival of indicated ∆-mutant relative to WT after UV irradiation at 60 Joules/m². For multiple comparisons, we used one-way ANOVA on GraphPad Prism. **** means P< .0001, *** means P< .001, and ** means P< .01.

Figure 7

Growth of E. coli MG1655 WT and derivatives deleted for selected RNA modification genes in sub-MIC TOB. Overnight cultures were diluted 100x in fresh MH medium, on 96-well plates. Plates were incubated with shaking in TECAN plate reader device at 37°C, OD 620 nm was measured every 15 min. Grey: no treatment. Blue: sub-MIC TOB, at 0.2 µg/ml (50% of the MIC for MG1655 in MH liquid culture). The y-axis represents the OD of the mutant divided by the OD of the WT strain in the same growth conditions, and thus reflects slower (below 1) or faster (above 1) growth. Standard deviation is shown.