A system biology approach to dissect superoxide signalling in Dictyostelium

Life of living organisms in oxidative environment leads to the formation of reactive oxygen species (ROS). These ROS are metabolic by-products and specially produced due to external factors. ROS can damage DNA, RNA, protein and lipid molecules of a cell. An excess of ROS may result into the cell damage and cell death.  To nullify these hazardous effects of ROS, organisms have evolutionarily developed systems of enzymes to sense and inactivate ROS [1, 2]. Most of the organisms have also acquired ROS dependent cellular signalling pathways. The ROS, hydrogen peroxide (H₂O₂) and the superoxide anion (O₂‾) play important role in cellular signalling in mammalian and plant cells by regulating their function [1, 2]. ROS are essential for the activity of many signals such as, mitogenic function of PDGF [3] and for NGF induced neuronal differentiation [4]. Plants produce ROS in response to wounding and infection, and acts as signal to induce genes expression involved that provide immunity to plants [5].

Generation of various ROS and their inactivation is depicted in Figure 1. The ROS, O₂‾ is generated by the reduction of oxygen and further reduction of O₂‾ with superoxide dismutase (SOD) converts it H₂O₂. H₂O₂ in presence of free transition elements forms highly reactive peroxide radical (OH‾). The enzymes, catalase (CAT) and glutathione peroxidases inactivates H₂O₂ to H₂O. Another radical nitrogen monoxide reacts with O₂‾ and forms highly reactive peroxinitrite (ONOO‾). These all ROS modify DNA, RNA, and lipid molecules.

Figure 1. Generation and inactivation of ROS

The signalling importance of H₂O₂ is well studied but signalling role of O₂‾ is limiting. The reported signalling roles of O₂‾ are limiting. The evidences that support the signalling roles of O₂‾ are; (i) Activation of Raf-1-MAPK pathway that leads to altered gene expression (Ref), (ii) Activation of ERK pathway [6]. O₂‾ also controls growth in Escherichia coli and Salmonella typhimurium[7]. In Dictystelium discoideum O₂‾ is developmentally regulated and controls development of this organism [8]. Here my goal is to investigate effects of O₂‾ on global gene expression and correlate the changes with the development using a genetically tractable model organism Dictystelium discoideum.

Gene expression profiling in response to superoxide radical

To determine the signaling roles of O₂‾ in D. discoideum, gene expression of cells having lower and higher levels O₂‾ than the physiological levels and wild type cells will be compared. To reduce the O₂‾ level, sodA will be over expressed, which is involved in the conversion of O₂‾ to H₂O_2. Mutants having high levels of O₂‾ will be generated by down regulating sodA. sodA will be silenced using proven protocols in D. discoideum for tetracycline (tet)-off regulatable long hairpin RNA (lhRNA) interference [18]. Testing a few different hairpins might be critical for success. A plasmid will be constructed to express ~700 bp hairpin of genes separated by ~300 bp of unpaired sequence [18]. This construct will be engineered into the response plasmid (MB38) of tetracycline regulated gene expression system (tet-off system) for D. discoideum [19] to make sodA-RNAi-MB38 vector. Prior to transfection of sodA-RNAi-MB38, wild type cells will be transfected with the transactivator plasmid (MB35) of tet-off system and the transfected cells (Wild type-MB35 cells) will be selected on 30 μg/ml of G418. The gene-RNAi-MB38 vector will be introduced into wild type-MB35 cells and double-transformed cells (RNAi cells) will ne selected in the presence of 30 μg/ml of G418 and 10 μg/ml of blasticidin S. To silence the gene expression RNAi cells will be grown in 10 μg/ml blasticidin S, 20 μg/ml of G418 and 30 μg/ml of tetracycline. Tet-regulated knock-down of sodA will be confirmed by qPCR, and the phenotypes of mutants will be characterized as described previously [20]. Empty vector (pMB38)-transformed cells will be used as controls. An elevation upon down regulation of sodA will be confirmed by assaying O₂‾ levels in wild type and mutant cells as described [8]. 500 mM 2,3-bis-[2-methoxy-4-nitro-5-sulphophenyl]-2H-tetrazolium-5-carboxanilide (XTT) will be added to developing cells. Reaction with superoxide converts the pale yellow XTT to bright orange-coloured formazan, and its accumulation of will be measured by monitoring its absorbance at 470 nm[21].

RNA sequencing (RNAseq) : Wild type, sodA over expressed and down regulated cells will be grown and allowed to develop, and RNA will be isolated from samples at 2-hour intervals during the entire developmental process. RNA will be processed as described [22]. Sequencing will be performed and the resulting FASTQ files will be analysed by PIPA, web-based software (http://pipa.biolab.si). The results – mapped reads, counts and transcript abundance -will be analyzed using software available [23].

The list of genes affected in the mutant and their aggregate annotations developed hypotheses as to the biological processes normally regulated by O₂‾ and provides further insights into the function of O₂‾ in development.

Significance

Formation of superoxide radicals is critical to the cellular function as this is involved in signalling process. What are the signalling processes controlled by superoxide radical? Where and how these molecules exert their effect on a cell? What are the genes that are regulated by superoxide radicals? Here, the goal is to determine dynamic changes in global transcripts resulted due to superoxide radical and to correlate such changes to the signalling pathways involved in development and differentiation. In addition to the better understanding of the superoxide radical function; this work might uncover new facets of the biogenesis of novel transcripts. Furthermore, this work will enlighten the antioxidant potential of D. discoideum which may be further utilized in the production of better anti-oxidants for human health.

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