Distinct transcriptomic response of S. coelicolor to ciprofloxacin in a nutrient-rich environment.

With the rising threat of anti-microbial resistance (AMR), there is an urgent need to enhance efficacy of existing antibiotics. Understanding the myriad mechanisms through which bacteria evade these drugs would be of immense value to designing novel strategies against them. Streptomyces coelicolor A3(2) M145 belongs to the actinomyctes species that are responsible for more than two-thirds of antibiotics. This group of bacteria therefore encodes for various mechanisms that can resist both endogenous and non-endogenous antibiotics. In an earlier study, we had studied the transcriptomic response of these bacteria to ciprofloxacin, when cultured in a minimal media. In this work, we investigate why the minimum inhibitory concentration of the drug increases by fourfold when the bacteria are grown in a nutrient-rich media. Through transcriptomic, biochemical, and microscopic studies, we show that S. coelicolor responds to ciprofloxacin in a concentration-dependent manner. While, sub-inhibitory concentration of the drug primarily causes oxidative stress, the inhibitory concentration of ciprofloxacin evokes a more severe genome-wide response in the cell, which ranges from the familiar upregulation of the SOS response and DNA repair pathways to the widespread alterations in the central metabolism pathway to accommodate the increased needs of nucleotides and other precursors. Further, the upregulation of peptidoglycan synthesis genes, along with microscopy images, suggest alterations in the cell morphology to increase fitness of the bacteria during the antibiotic stress. The data also points to the enhanced efflux activity in cells cultured in rich media that contributes significantly towards reducing intracellular drug concentration and thus promotes survival.

Proteomic analysis of Streptomyces coelicolor in response to Ciprofloxacin challenge.

Multi-drug tolerance is an important phenotypic property that complicates treatment of infectious diseases and reshapes drug discovery. Hence a systematic study of the origins and mechanisms of resistance shown by microorganisms is imperative. Since soil-dwelling bacteria are constantly challenged with a myriad of antibiotics, they are potential reservoirs of resistance determinants that can be mobilized into pathogens over a period of time. Elucidating the resistance mechanisms in such bacteria could help future antibiotic discoveries. This research is a preliminary study conducted to determine the effects of ciprofloxacin (CIP) on the intrinsically resistant Gram-positive soil bacterium Streptomyces coelicolor. The effect was investigated by performing 2-DE on total protein extracts of cells exposed to sub-lethal concentrations of ciprofloxacin as compared to the controls. Protein identification by MALDI-TOF/TOF revealed 24 unique differentially expressed proteins, which were statistically significant. The down-regulation of proteins involved in carbohydrate metabolism indicated a shift in the cell physiology towards a state of metabolic shutdown. Furthermore, the observed decline in protein levels involved in transcription and translation machinery, along with depletion of enzymes involved in amino acid biosynthesis and protein folding could be a cellular response to DNA damage caused by CIP, thereby minimizing the effect of defective and energetically wasteful metabolic processes. This could be crucial for the initial survival of the cells before gene level changes could come into play to ensure survival under prolonged adverse conditions. These results are a first attempt towards profiling the proteome of S. coelicolor in response to antibiotic stress. This article is part of a Special Issue entitled: Trends in Microbial Proteomics. BIOLOGICAL SIGNIFICANCE: Soil-dwelling bacteria could serve as a reservoir of resistance determinants for clinically important bacteria. In this work, we investigated, for the first time, the differential proteomic profile of S. coelicolor cells in response to sub-inhibitory concentrations of Ciprofloxacin using 2-DE. Results indicate a shift in the cell physiology towards a state of metabolic shutdown, possibly to counter the DNA damage by ciprofloxacin. Further, up-regulation of GAPDH, RNA pol mRNA and Translation IF2 protein indicates a reprogramming of the cell for long-term survival. This study could serve as a basis for further investigations to elucidate the general mechanism by which soil bacteria exhibit resistance to fluroquinolones. This may help in developing new drug protocols and inventing novel drugs to counter resistance to this class of antibiotics in pathogenic bacteria.

Transcriptomic study of ciprofloxacin resistance in Streptomyces coelicolor A3(2).

Soil organisms exhibit resistance to a wide range of antibiotics as they either need to protect themselves from endogenous antibiotics or from those present in their soil environment. The soil could serve as a reservoir for resistance mechanisms that have already emerged or have the potential to emerge in clinically important bacteria. Streptomyces coelicolor, a non-pathogenic soil-dwelling organism, is thus used as a model for the study of intrinsic resistance. Preliminary screening of several compounds showed that S. coelicolor had high intrinsic resistance for the fluoroquinolone group of antibiotics. We subjected the bacteria to sub-inhibitory concentrations of ciprofloxacin and studied the transcriptomic response using microarrays. The data were supported with various biochemical and phenotypic assays. Ciprofloxacin treatment leads to differential expression of many genes with enhanced mRNA expression of its target, DNA gyrase gene. High induction of DNA repair pathways was also observed and many transporters were upregulated. Ciprofloxacin was found to induce ROS formation in a dose dependent manner. Reduction of ROS via anti-oxidants increased the effective MIC of the drug in the bacteria. The regulation of antibiotic resistance in S. coelicolor was studied systematically and contribution of different mechanisms in the development of resistance was assessed. Our data suggest that multiple mechanisms work in coordination to facilitate the cell to combat the stress due to ciprofloxacin.

Existence of a distinct concentration window governing daunorubicin-induced mammalian liver mitotoxicity--implication for determining therapeutic window.

Daunorubicin (DNR) is a well known anticancer drug believed to act mainly by topoisomerase II inhibition and mitochondria-mediated free radical generation. Though several studies were dedicated to elucidate the mechanism of action of DNR, however the mechanism still remains illusive. DNR is reported to affect mitochondrial respiration. However, there are contradictory reports regarding DNR effect on oxygen consumption. Interestingly, DNR at low concentration (<10 microM) dose-dependently augments respiration but at higher concentration inhibits respiration. To investigate, if a concentration window exists in which the effect of DNR on mitochondria is optimum, dose-dependent effect of DNR on mitochondria was studied. DNR inhibited electron transfer and generates reactive oxygen species (ROS) at complex I and III but not at complex II. DNR-induced ROS generation was found instrumental in mitochondrial membrane potential collapse and mitochondrial permeability transition (MPT) opening. MPT closure reduced the observed respiratory burst. Thus, at lower DNR concentration, MPT opening leads to a sudden burst of respiration while at higher concentration electron transfer gets inhibited, therefore respiration gets repressed. We for the first time, provide a possible explanation for the reports regarding the differential regulation of respiration by DNR. Thus, further establishing the concept of concentration window and justifying the need for dose optimization for maximal therapeutic effect.