The Fluoroquinolone Levofloxacin Triggers the Transcriptional Activation of Iron Transport Genes That Contribute to Cell Death in Streptococcus pneumoniae

Abstract

We studied the transcriptomic response of Streptococcus pneumoniae to levofloxacin (LVX) under conditions inhibiting topoisomerase IV but not gyrase. Although a complex transcriptomic response was observed, the most outstanding result was the upregulation of the genes of the fatDCEB operon, involved in iron (Fe 2؉ and Fe 3؉ ) uptake, which were the only genes varying under every condition tested. Although the inhibition of topoisomerase IV by levofloxacin did not have a detectable effect in the level of global supercoiling, increases in general supercoiling and fatD transcription were observed after topoisomerase I inhibition, while the opposite was observed after gyrase inhibition with novobiocin. Since fatDCEB is located in a topological chromosomal domain downregulated by DNA relaxation, we studied the transcription of a copy of the 422-bp (including the P fat promoter) region located upstream of fatDCEB fused to the cat reporter inserted into the chromosome 106 kb away from its native position: P fat fatD was upregulated in the presence of LVX in its native location, whereas no change was observed in the P fat cat construction. Results suggest that topological changes are indeed involved in P fat fatDCE transcription. Upregulation of fatDCEB would lead to an increase of intracellular iron and, in turn, to the activation of the Fenton reaction and the increase of reactive oxygen species. In accordance, we observed an attenuation of levofloxacin lethality in iron-deficient media and in a strain lacking the gene coding for SpxB, the main source of hydrogen peroxide. In addition, we observed an increase of reactive oxygen species that contributed to levofloxacin lethality.

Reactive Oxygen Species Contribute to the Bactericidal Effects of the Fluoroquinolone Moxifloxacin in Streptococcus pneumoniae

“…Group I represented Ͼ15% of the total affected genes, which included those coding for hypothetical proteins and membrane transport systems. These two classes were also predominant in the transcriptomic response to LVX (25). They formed the largest group of genes in the R6 genome (35).…”

“…Results indicated that fatDCEB transcription depends on its location in a topological domain. These results illustrated a link between the initial LVX interaction with its topo IV target and the production of ROS (25).…”

“…Microarrays results were validated by quantitative real-time reverse transcription (RT)-PCR (Chromo 4, Bio-Rad), as previously described (25). Genes selected to be amplified were the following: fatC, fatD, gyrA, gyrB, glnA, mutM, lmb, parE, psaB, pyrB, spr1680, and topA.…”

“…Genes selected to be amplified were the following: fatC, fatD, gyrA, gyrB, glnA, mutM, lmb, parE, psaB, pyrB, spr1680, and topA. For most genes, previously described oligonucleotides were used (24,25). For the rest of genes, oligonucleotides (forward/reverse) were as follows: mutM (5=-AGATGGTGGCACGCTTGTTTA-3=/5=-GTGTATCCATAGCACC GAGTCC-3=), lmB (5=-AAGGGGATGAAAATTGTGACCAG-3=/5=-GC CCAAGATTCGAGTGTATGAGA-3=) pyrB (5=-AACTCTTTTTCGCTG GACCTG-3=/5=-ACTATCATGGCGTTCGTGTTG-3=), psaB (5=-ATAC GTATCGAAAACCTCAGTGTC-3=/5=-CCCAGCATACCTTTTAATAG TGTT-3=).…”

“…Cells were grown to an optical density at 620 nm (OD 620 ) of 0.4 before MOX was added. Samples were collected and processed as previously described (25). Fluorescence was measured using a Tecan Infinite 2000 device and a filter with excitation and emission wavelengths of 485 nm and 535 nm, respectively.…”