Absence of tmRNA Has a Protective Effect against Fluoroquinolones in Streptococcus pneumoniae

Abstract

The transfer messenger RNA (tmRNA), encoded by the ssrA gene, is a small non-coding RNA involved in trans-translation that contributes to the recycling of ribosomes stalled on aberrant mRNAs. In most bacteria, its inactivation has been related to a decreased ability to respond to and recover from a variety of stress conditions. In this report, we investigated the role of tmRNA in stress adaptation in the human pathogen Streptococcus pneumoniae. We constructed a tmRNA deletion mutant and analyzed its response to several lethal stresses. The ΔssrA strain grew slower than the wild type, indicating that, although not essential, tmRNA is important for normal pneumococcal growth. Moreover, deletion of tmRNA increased susceptibility to UV irradiation, to exogenous hydrogen peroxide and to antibiotics that inhibit protein synthesis and transcription. However, the ΔssrA strain was more resistant to fluoroquinolones, showing twofold higher MIC values and up to 1000-fold higher survival rates than the wild type. Deletion of SmpB, the other partner in trans-translation, also reduced survival to levofloxacin in a similar extent. Accumulation of intracellular reactive oxygen species associated to moxifloxacin and levofloxacin treatment was also highly reduced (∼100-fold). Nevertheless, the ΔssrA strain showed higher intracellular accumulation of ethidium bromide and levofloxacin than the wild type, suggesting that tmRNA deficiency protects pneumococcal cells from fluoroquinolone-mediated killing. In fact, analysis of chromosome integrity revealed that deletion of tmRNA prevented the fragmentation of the chromosome associated to levofloxacin treatment. Moreover, such protective effect appears to relay mainly on inhibition of protein synthesis, since a similar effect was observed with antibiotics that inhibit that process. The emergence and spread of drug-resistant pneumococci is a matter of concern and these results contribute to a better comprehension of the mechanisms underlying fluoroquinolones action.

Keywords: Streptococcus pneumoniae; antibiotic resistance; chromosomal fragmentation; fluoroquinolones; reactive oxygen species; stress adaptation; tmRNA; trans-translation.

FIGURE 1

Transfer messenger RNA (tmRNA) deficiency is detrimental under certain stress conditions. (A) Inactivation of ssrA gene (black) by insertion/deletion of Km^r (light gray). Flanking ORFs (white) and tRNA gene (dark gray) are depicted. Promoters (curved arrows), terminators (stem-loop structures) and primers used (black arrows) are shown (B) Growth curves of wild type TIGR4 (wt) strain and its tmRNA deletion mutant (ΔssrA) in THY broth at 37°C. (C) Survival of wt and ΔssrA upon treatment with 30 mM paraquat at the indicated times, after UV irradiation at the intensities indicated, or after 30 min of exposure to 20 mM of hydrogen peroxide. Survival was determined as described in Section “Materials and Methods.” Values (mean ± SD) are the average of at least three independent experiments and the means were statistically compared using Student’s t-test (^∗P < 0.05; ^∗∗P < 0.01).

FIGURE 2

Deletion of tmRNA reduces lethality of FQs but not of penicillin. (A) Percentage of survival of wt, ΔssrA and TIGR4ΔsmpB (ΔsmpB) strains upon treatment with LVX, MOX and/or penicillin (PEN). Exponentially growing cells were treated with each antibiotic at the concentration and times indicated. Survival was determined as described in Section “Materials and Methods.” (B) Accumulation of ROS in wt and ΔssrA strains upon treatment with 2.5 and 5× MIC of LVX and MOX were measured in the same samples as described in Section “Materials and Methods.” RFU, relative fluorescence units; fluorescence units were divided by the number of viable cells and normalized to time zero and no antibiotic treatment condition. Values (mean ± SD) of at least three independent experiments are shown. The MIC values used were those determined in Table 4.

FIGURE 3

Transfer messenger RNA deficiency protects from FQs lethal action. (A) Wild type R6 strain (wt) and its tmRNA deletion mutant (ΔssrA) were grown under different LVX concentrations in A+Y medium. Growth was followed by turbidity (OD_{620 nm}) in a TECAN infinite F200 plate reader at 37°C. (B) Percentage of survival to different FQs of wt, ΔssrA, the complemented strain containing the pROM-TM (ΔssrA(ssrA⁺)) and the ΔssrA containing the empty vector (ROM) at 37°C, and of wt at 30°C. Exponentially growing cells were treated with 5× MIC (as determined in Table 4) of LVX, ciprofloxacin (CPX), norfloxacin (NFX), or MOX for the times indicated. After incubation, survival was determined as described in Section “Materials and Methods.” (C) Accumulation of ROS in wt and ΔssrA upon addition of 5× MIC of LVX was measured as described in Section “Materials and Methods.” RFU; fluorescence units were divided by the number of viable cells and normalized to time zero and no antibiotic treatment condition. (D) Northern-blot showing tmRNA expression in wt, ΔssrA and ΔssrA(ssrA⁺). Bands corresponding to tmRNA and the control 5S rRNA are indicated by arrows. Northern-blots were performed as described (Acebo et al., 2012). Values (mean ± SD) of at least three independent experiments are shown. MIC values were determined in Table 4.

FIGURE 4

Transfer messenger RNA deficiency leads to higher intracellular accumulation of drugs. (A) EtBr uptake (left panel) and efflux (right panel) of R6 (wt) and R6ΔssrA (ΔssrA) strains during 20 min was measured as described in Section “Materials and Methods.” Fluorescence intensity represents levels of intracellular EtBr. The change in fluorescence is normalized to the initial levels to allow for direct comparison. Fluorescence was directly recorded during 20 min after addition of EtBr in the presence (+Res) or in the absence of reserpine. (B) Fluorometric measurement of intracellular accumulation of levofloxacin (LVX) in wt and ΔssrA at different LVX concentrations (left panel) and time points (right panel), as described in Section “Materials and Methods.” Three to five independent experiments were conducted and the mean values were statistically compared using Student’s t-test (^∗P < 0.05).

FIGURE 5

Deficiency of tmRNA protects from chromosome fragmentation mediated by LVX. (A) Analysis of chromosome fragmentation upon LVX treatment by PFGE. Exponentially growing cultures of R6 (wt), the R6ΔssrA (ΔssrA) mutant, the complemented strain containing the pROM-TM (ΔssrA(ssrA⁺)) and the R6ΔssrA containing the empty vector (ROM), were treated with or without 10× MIC of LVX for 30 min and analyzed by PFGE as described in Section “Materials and Methods.” (B) Chromosomal fragmentation was estimated by quantification of the compression zone (CZ) relative to the intact chromosomal DNA retained in the well. Each value is the mean of at least three independent experiments and the mean values were statistically compared using Student’s t-test (^∗∗P < 0.01). (C) Percentage of survival to 1 h treatment of LVX at 5, 10, or 20× MIC of wt, ΔssrA strains previously incubated with 10 μ/ml of Chloramphenicol (CM) for 10 min. St stands for the MidRange PFG Marker (Biolabs) with size ranges from 15 to 300 Kb. (D) Percentage of survival along the time to treatment with 5× MIC of wt strains previously incubated with 10 μ/ml of CM or 0.3 μ/ml of erythromycin (EM) for 10 min. Survival was determined as described in Section “Materials and Methods.”