Leishmania major Methionine Sulfoxide Reductase A Is Required for Resistance to Oxidative Stress and Efficient Replication in Macrophages

Abstract

Leishmania are protozoan parasites that proliferate within the phagolysome of mammalian macrophages. While a number of anti-oxidant systems in these parasites have been shown to protect against endogenous as well as host-generated reactive oxygen species, the potential role of enzymes involved in the repair of oxidatively damaged proteins remains uncharacterized. The Leishmania spp genomes encode a single putative methionine sulfoxide reductase (MsrA) that could have a role in reducing oxidized free and proteinogenic methionine residues. A GFP-fusion of L. major MsrA was shown to have a cytoplasmic localization by immunofluorescence microscopy and subcellular fractionation. An L. major msrA null mutant, generated by targeted replacement of both chromosomal allelles, was viable in rich medium but was unable to reduce exogenous methionine sulfoxide when cultivated in the presence of this amino acid, indicating that msrA encodes a functional MsrA. The ΔmsrA mutant exhibited increased sensitivity to H2O2 compared to wild type parasites and was unable to proliferate normally in macrophages. Wild type sensitivity to H2O2 and infectivity in macrophages was restored by complementation of the mutant with a plasmid encoding MsrA. Unexpectedly, the ΔmsrA mutant was able to induce normal lesions in susceptible BALB/c indicating that this protein is not essential for pathogenesis in vivo. Our results suggest that Leishmania MsrA contributes to the anti-oxidative defences of these parasites, but that complementary oxidative defence mechansims are up-regulated in lesion amastigotes.

Proteomic analysis of the proteins released from Staphylococcus aureus following exposure to Ag(I)

“…MsrA-specific methionine reductase activity in S. aureus plays a central role in the ability of the cell to tolerate oxidative stress (Singh and Moskovitz, 2003). Methionine sulfoxide reductase A has been shown to perform a critical role in the ability of Leishmania major to survive and replicate in macrophages thus highlighting a role for this enzyme in pathogenesis (Sanson et al, 2013). It was also found that this enzyme was induced upon exposure of S. aureus cells to the antibiotic, oxacillin .…”

Identification of Aeromonas hydrophila Genes Preferentially Expressed after Phagocytosis by Tetrahymena and Involvement of Methionine Sulfoxide Reductases

“…Methionine sulfoxide reductases (Msrs) are key enzymes in repairing ROS-mediated damage to proteins and include mainly MsrA and MsrB (Sansom et al, 2013). As the best characterized Msr, MsrA plays a role in resistance to oxidative stress and virulence in a number of bacteria, including Mycobacterium species (St. John et al, 2001; Douglas et al, 2004), S. aureus (Singh and Moskovitz, 2003), Salmonella typhimurium (Denkel et al, 2011), and E. coli (St. John et al, 2001).…”

“…These findings indicate that msrA and msrB were required for A. hydrophila to resist predatory protozoans. Moreover, the msr genes have previously been characterized as required for bacterial survival and replication within macrophages (Douglas et al, 2004; Sansom et al, 2013). These findings suggest that the mechanisms responsible for survival within the phagosomes of protozoa and macrophages may be similar.…”

Functional characterisation of the methionine sulfoxide reductase repertoire in Trypanosoma brucei

“…Alteration of MSR expression levels in other organisms has shown that they play an important role in protecting cells from exogenous oxidants [7,15,19,27,28]. Figure 6B).…”

“…One recipient of the electron flux via the T(SH)2/TXN cascade is MSRA, with analysis of the T. cruzi and Leishmania major homologues revealing its role as an anti-oxidant enzyme within the parasite [27,28]. Here, we report the dissection of two MetSO metabolising pathways expressed by T. brucei focusing on the biochemical properties, subcellular localisation, and functional importance of its MSRA and MSRB complement.…”