RNA-sequencing Analysis Of Mycobacterium Smegmatis Response To Hydrogen Peroxide

Mycobacterium tuberculosis （M.tb） is a pathogen that caused tuberculosis （TB） which is a major public health threat. There are about a million new incident TB cases each year in China. The interactions between host and pathogen play an important role to the outcome of the disease development. Reactive oxygen species （ROS）, produced by NADPH phagocyte oxidase （phox）, is an important factor within innate Immunity response to defend the infection of Mtb. There are several types of ROS including O2produced by phox, H2O2produced by SOD and HO’produced by myeloperoxidase, respectively.The important role of the NADPH phagocyte oxidase in host defense is revealed though the clinical observations which the people carrying the mutated gene encoding the subunits of the NADPH phagocyte oxidase easily develop chronic granulomatous disease （CGD）, which is characterized by an increased susceptibility to infection by certain bacteria and fungi, and shorten life span. After co-evolution with host, M.tb has developed the successful detoxification systems to protect itself from host ROS attacks,, in the result that M.tb has the capacity of persist into the macrophage. In this study, we planed to explore the mechanisms how Mtb to maintain the redox balance under various hydrogen peroxide stress and identify the possible key candidates playing the important roles. The knowledge would lead to novel strategies to develop new type of vaccines. To avoid the use of BSL-3lab for handle the pathogen, Mtb, we chose the non-pathgoen, fast growth Mycobacterium smegmatis （M.smegmatis） as our model in this studying.First, we selected a mutant strain, named mc251, with high resistance ability to H2O2, using serial selection under continuous increased H2O2treatment in M.smegmatis me155. The minimum inhibitory concentration （MIC） of mc251to H2O2is80times higher than wild type me2155, which3.125mM in mc251compared to0.039mM in wild type strain me2155. Macrophage killing assay experiments suggested that mc251has growth advantage over wild type mc2155within the macrophage cells. To give an insight of the possible involved mechanisms, the RNA-sequencing was choose to analyze the transcriptome of the me2155and mc251at early log phase. The highly upregulated of Msmeg₃461, catalase/peroxidase in mc251, compared to its expression in me2155, was obtained and suggested that the mc251keeps high endogenous level of catalase/peroxidase enzyme activity and the high expression of Msmeg₃461was also shown in the in vitro characterization of enzyme activity using of native PAGE gel. In addition, the decreased level of ribosomal protein transcription hinted the reduced the translation and protein synthesis, which might be the reason of bacteria growth extremely, slow. Further experiments are to explore to study the correlation of H2O2resistance and slow growth.Take together in this study we selected a high H2O2resistant Msmegatis strain, named mc251. The systemic analysis suggested the resistance of H2O2is related to the higher endogenous level of catalase/peroxidase enzyme activity and the low level transcription of ribosome.