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 noncoding 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 ssrA 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 ssrA 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 ssrA 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 drugresistant pneumococci is a matter of concern and these results contribute to a better comprehension of the mechanisms underlying fluoroquinolones action.

A Small Non-Coding RNA Modulates Expression of Pilus-1 Type in Streptococcus pneumoniae

“…To investigate whether srn135 has a regulatory function, we cloned its coding sequence in a pROM plasmid [ 51 ] under the control of the constitutive vegT promoter (P vegT ) [ 49 ]. The resulting pveg135 plasmid was introduced in wild-type TIGR4 cells (wt(pveg135)) and accumulation of a band corresponding to srn135 was confirmed by Northern blot analysis ( Figure 4 a).…”

“…The srn135 coding sequence was cloned into the pROM plasmid previously constructed [ 51 ] under the control of the constitutive P vegT promoter to obtain the pveg135 plasmid. For this purpose, a fragment corresponding to the first 197 nucleotides (nt) of the rrgA gene was amplified from the TIGR4 genome using primers 135F-blunt and 135R-Bam ( Table 2 ).…”

“…Other previous and subsequent studies increased the battery of small RNAs expressed in the pneumococcus [ 45 , 46 , 47 , 48 ]. Inactivation of some of these sRNAs drastically affected pneumococcal virulence in a mouse model of infection, while others modulated competence or antibiotic susceptibilities [ 45 , 48 , 49 , 50 , 51 ], thus confirming that sRNAs are a major regulatory mechanism in the pneumococcus.…”