Mycobacterium smegmatis secreting methionine sulfoxide reductase A (MsrA) modulates cellular processes in mouse macrophages

Methionine sulfoxide reductase A (MsrA) is an antioxidant repair enzyme that reduces the oxidized methionine (Met-O) in proteins to methionine (Met). It plays a pivotal role in the cellular processes has been well established by overexpressing, silencing and knocking down of MsrA or by deleting the gene encoding MsrA in several species. We are specifically interested in understanding the role of secreted MsrA of bacterial pathogens. To elucidate this, we infected mouse bone marrow derived macrophages (BMDMs) with recombinant Mycobacterium smegmatis strain (MSM) secreting a bacterial MsrA or M. smegmatis strain (MSC) carrying only the control vector. BMDMs infected with MSM induced higher levels of ROS and TNF-α than BMDMs infected with MSC. The increased ROS and TNF-α in BMDMs infected with MSM correlated with elevated necrotic cell death. Further, RNA-Seq transcriptome analysis of BMDMs infected with MSC and MSM revealed differential expression of protein and RNA coding genes, suggesting that bacterial delivered MsrA could modulate the host cellular processes. Finally, KEGG pathway enrichment analysis identified down-regulation of cancer related signaling genes in MSM infected cells, indicating that MsrA has the potential to regulate the development and progression of cancer. BMDMs (1 x10^6 cells/well) in each well of a 6-well culture plate were infected with MSC or MSM at an MOI of 1:5 or uninfected (control cells) and incubated for 2 h at 37°C in a CO2 incubator. After the incubation, the cells were washed with 1xPBS to remove the extracellular bacteria. Then the cells were lifted using cell scraper and collected via centrifugation at 200xg for 10 minutes. Total RNA was isolated from the infected BMDMs using EZ-10 DNAaway RNA Mini-Preps Kit (Bio Basic, Canada). TapeStation (Agilent Technologies 4200) was used to analyze RNA integrity of the preparation, and RNA quantification was done using Nanodrop (Thermo Scientific, USA). 4 ug of total RNA with >9 RIN (RNA Integrity Number) was used for RNA-Seq library preparation.