Methionine Sulfoxide Reductase A (MsrA) Deficiency Affects the Survival ofMycobacterium smegmatiswithin Macrophages

Abstract

Methionine sulfoxide reductase A (MsrA) is an antioxidant repair enzyme which reduces oxidized methionine to methionine. Since oxidation of methionine in proteins impairs their function, an absence of MsrA leads to abnormalities in different organisms, including alterations in the adherence patterns and in vivo survival of certain pathogenic bacteria. To understand the role of MsrA in intracellular survival of bacteria, we disrupted the gene encoding MsrA in Mycobacterium smegmatis through homologous recombination. The msrA mutant strain of M. smegmatis exhibited significantly reduced intracellular survival in murine J774A.1 macrophages compared to the survival of its wild-type counterpart. Furthermore, immunofluorescence and immnunoblotting of phagosomes containing M. smegmatis strains revealed that the phagosomes with the msrA mutant strain acquired both p67 phox of phagocyte NADPH oxidase and inducible nitric oxide synthase much earlier than the phagosomes with the wild-type strain. In addition, the msrA mutant strain of M. smegmatis was observed to be more sensitive to hydroperoxides than the wild-type strain was in vitro. These results suggest that MsrA plays an important role in both extracellular and intracellular survival of M. smegmatis.Although mononuclear phagocytes are specially equipped to produce superoxide (O 2 Ϫ˙) radicals through the NADPH oxidase (phox) complex (32, 35), most living cells also generate superoxide as a by-product of normal aerobic metabolism. This occurs due to incomplete reduction of oxygen at the level of the cytochrome oxidase system, which accounts for approximately 4% of the oxygen that enters into this system (50). Superoxide reacts with a variety of organic and inorganic compounds and generates toxic reactive oxygen intermediates (ROI), such as H 2 O 2 , HO˙Ϫ, HOCl, and OONO˙Ϫ (25, 52). Toxic ROI oxidize cellular macromolecules like proteins, lipids, and nucleic acids, which accelerates aging processes and disease in living organisms (4).In proteins, oxidation occurs mainly at the sulfur-containing amino acids cysteine and methionine (40). Oxidation of methionine leads to the formation of methionine sulfoxide, and methionine sulfoxide reductase A (MsrA), which was first identified in Escherichia coli (5), has long been implicated in the reduction of methionine sulfoxide in proteins. However, recent studies have demonstrated that oxidation of methionine leads to two stereomeric forms of methionine sulfoxide, methionione S-sulfoxide and methionine R-sulfoxide (21, 50); the reduction of these compounds is catalyzed by MsrA and MsrB, respectively, (21, 30). Analysis of the sequences of different genomes has revealed that the genes coding for both MsrA and MsrB are present in almost all living organisms, from prokaryotes to humans (11,18,21). Interestingly, however, MsrB from different organisms shows no sequence identity with MsrA (17). Also, in contrast to MsrA, MsrB requires the metal cofactor selenium or zinc (18) for reactivity.Since the oxidation of methionine in protein...

Extra- and intracellular antimycobacterial activity of Arbutus unedo L.

“…This showed that the strain persist within phagocytes during the initial hours of post-infection. Afterward, this value is gradually decreasing with the incubation period and reached 701 ± 52 UFC/well after 24 h, 387 ± 11 UFC/well after 48 h and finally 287 ± 14 UFC/well after 72 h. These results showed that M. smegmatis is unable to multiply inside macrophages, as reported in several previous works (Douglas et al, 2004;Sharbati-Tehrani et al, 2005). For the macrophages treated with the extract (Figure 1), we observed a significant decrease in the number of mycobacteria compared with mycobacterial load in untreated rat peritoneal macrophages.…”

The Reduced Bactericidal Function of Complement C5-Deficient Murine Macrophages Is Associated with Defects in the Synthesis and Delivery of Reactive Oxygen Radicals to Mycobacterial Phagosomes

“…Mechanisms of phox targeting were investigated, and initial studies showed that live virulent MTB excluded phox complex in naive macrophages. Because the nonpathogen Mycobacterium smegmatis readily acquired phox in macrophages (43,44), this suggested that MTB is able to prevent the acquisition of phox. Separate studies in progress have indicated that MTB inhibits PKC activation in macrophages, and this effect could be overcome with IFN-␥ in macrophages (our unpublished observations).…”

“…The latter studies used immunostains to localize p47 phox and p67 phox proteins of the phox complex. Our own recent studies showed that phox proteins assembled around M. smegmatis phagosomes, and their assembly was enhanced on mutant phagosomes that were more susceptible to oxidants (44). We used a similar strategy to colocalize phox in and around MTB phagosomes.…”

Dual Sites of Protein Initiation Control the Localization and Myristoylation of Methionine Sulfoxide Reductase A

“…Those studies provide strong evidence supporting the physiological importance of cyclic oxidation and reduction of Met residues. Knocking out MsrA caused increased susceptibility to oxidative stress in mice (5), yeast (6), and bacteria (7)(8)(9). Conversely, overexpressing MsrA conferred increased resistance to Drosophila (10), Saccharomyces (11), Arabidopsis (12), PC-12 cells (13), and human T cells (11).…”