Methionine biosynthetic genes and methionine sulfoxide reductase A are required for Rhizoctonia solani AG1-IA to cause sheath blight disease in rice.

Rhizoctonia solani is a polyphagous necrotrophic fungal pathogen that causes sheath blight disease in rice. It deploys effector molecules as well as carbohydrate-active enzymes and enhances the production of reactive oxygen species for killing host tissues. Understanding R. solani ability to sustain growth under an oxidative-stress-enriched environment is important for developing disease control strategies. Here, we demonstrate that R. solani upregulates methionine biosynthetic genes, including Rs_MET13 during infection in rice, and double-stranded RNA-mediated silencing of these genes impairs the pathogen's ability to cause disease. Exogenous treatment with methionine restores the disease-causing ability of Rs_MET13-silenced R. solani and facilitates its growth on 10 mM H2O2-containing minimal-media. Notably, the Rs_MsrA gene that encodes methionine sulfoxide reductase A, an antioxidant enzyme involved in the repair of oxidative damage of methionine, is upregulated upon H2O2 treatment and also during infection in rice. Rs_MsrA-silenced R. solani is unable to cause disease, suggesting that it is important for the repair of oxidative damage in methionine during host colonization. We propose that spray-induced gene silencing of Rs_MsrA and designing of antagonistic molecules that block MsrA activity can be exploited as a drug target for effective control of sheath blight disease in rice.

Comparative analysis reveals the trivial role of MsrP in defending oxidative stress and virulence of Salmonella Typhimurium in mice.

Sulphur containing amino acids, methionine and cysteine are highly prone to oxidation. Reduction of oxidized methionine (Met-SO) residues to methionine (Met) by methionine sulfoxide reductases (Msrs) enhances the survival of bacterial pathogens under oxidative stress conditions. S. Typhimurium encodes two types (cytoplasmic and periplasmic) of Msrs. Periplasmic proteins, due to their location are highly vulnerable to host-generated oxidants. Therefore, the periplasmic Msr (MsrP) mediated repair (as compared to the cytoplasmic counterpart) might play a more imperative role in defending host-generated oxidants. Contrary to this, we show that in comparison to the ΔmsrP strain, the mutant strains in the cytoplasmic Msrs (ΔmsrA and ΔmsrAC strains) showed many folds more susceptibility to chloramine-T and neutrophils. Further ΔmsrA and ΔmsrAC strains accumulated higher levels of ROS and showed compromised fitness in mice spleen and liver. Our data suggest the pivotal role of cytoplasmic Msrs in oxidative stress survival of S. Typhimurium.

Biochemical characterization of GAF domain of free-R-methionine sulfoxide reductase from Trypanosoma cruzi.

Trypanosoma cruzi is the causal agent of Chagas Disease and is a unicellular parasite that infects a wide variety of mammalian hosts. The parasite exhibits auxotrophy by L-Met; consequently, it must be acquired from the extracellular environment of the host, either mammalian or invertebrate. Methionine (Met) oxidation produces a racemic mixture (R and S forms) of methionine sulfoxide (MetSO). Reduction of L-MetSO (free or protein-bound) to L-Met is catalyzed by methionine sulfoxide reductases (MSRs). Bioinformatics analyses identified the coding sequence for a free-R-MSR (fRMSR) enzyme in the genome of T. cruzi Dm28c. Structurally, this enzyme is a modular protein with a putative N-terminal GAF domain linked to a C-terminal TIP41 motif. We performed detailed biochemical and kinetic characterization of the GAF domain of fRMSR in combination with mutant versions of specific cysteine residues, namely, Cys12, Cys98, Cys108, and Cys132. The isolated recombinant GAF domain and full-length fRMSR exhibited specific catalytic activity for the reduction of free L-Met(R)SO (non-protein bound), using tryparedoxins as reducing partners. We demonstrated that this process involves two Cys residues, Cys98 and Cys132. Cys132 is the essential catalytic residue on which a sulfenic acid intermediate is formed. Cys98 is the resolutive Cys, which forms a disulfide bond with Cys132 as a catalytic step. Overall, our results provide new insights into redox metabolism in T. cruzi, contributing to previous knowledge of L-Met metabolism in this parasite.

Response to oxidative stress of AML12 hepatocyte cells with knockout of methionine sulfoxide reductases.

Methionine sulfoxide reductases are enzymes that reduce methionine oxidation in the cell. In mammals there are three B-type reductases that act on the R-diastereomer of methionine sulfoxide, and one A-type reductase (MSRA) that acts on the S-diastereomer. Unexpectedly, knocking out the four genes in the mouse protected from oxidative stresses such as ischemia-reperfusion injury and paraquat. To elucidate the mechanism by which lack of the reductases protects against oxidative stresses, we aimed to create a cell culture model with AML12 cells, a differentiated hepatocyte cell line. We employed CRISPR/Cas9 to create lines lacking the four individual reductases. All were viable and their susceptibility to oxidative stresses was the same as the parental strain. The triple knockout lacking all three methionine sulfoxide reductases B was also viable, but the quadruple knockout was lethal. We thus modeled the quadruple knockout mouse by creating an AML12 line lacking the three MSRB and heterozygous for the MSRA (Msrb3KO-Msra+/-). We measured the effect of ischemia-reperfusion on the various AML12 cell lines, using a protocol that modeled the ischemic phase by glucose and oxygen deprivation for 36 h followed by return of glucose and oxygen for 3 h as the reperfusion phase. This stress killed ∼50% of the parental line, an effect we chose to facilitate detection of either protective or deleterious changes in the knockout lines. Unlike the protection afforded the mouse, the knockout lines produced by CRISPR/Cas9 did not differ from the parental line in their response to ischemia-reperfusion injury or paraquat poisoning. In the mouse, inter-organ communication may be essential for protection induced by lack of methionine sulfoxide reductases.

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). Its pivotal role in the cellular processes has been well established by overexpressing, silencing, and knocking down MsrA or deleting the gene encoding MsrA in several species. We are specifically interested in understanding the role of secreted MsrA in 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 levels in MSM-infected BMDMs correlated with elevated necrotic cell death in this group. 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 the down-regulation of cancer-related signaling genes in MSM-infected cells, indicating that MsrA can potentially regulate the development and progression of cancer.

Recombinant Humanized IgG1 Antibody Protects against oxLDL-Induced Oxidative Stress and Apoptosis in Human Monocyte/Macrophage THP-1 Cells by Upregulation of MSRA via Sirt1-FOXO1 Axis.

Oxidized low-density lipoprotein (oxLDL)-induced oxidative stress and apoptosis are considered as critical contributors to cardiovascular diseases. Methionine sulfoxide reductase A (MSRA) is a potent intracellular oxidoreductase and serves as an essential factor that protects cells against oxidative damage. Here, we firstly provide evidence that recombinant humanized IgG1 antibody treatment upregulated the expression of MSRA in THP-1 cells to defend against oxLDL-induced oxidative stress and apoptosis. It was also observed that the upregulation of MSRA is regulated by the forkhead box O transcription factor (FOXO1), and the acetylation of FOXO1 increased when exposed to oxLDL but declined when treated with recombinant humanized IgG1 antibody. In addition, we identified that silent information regulator 1 (SIRT1) suppresses FOXO1 acetylation. Importantly, SIRT1 or FOXO1 deficiency impaired the anti-oxidative stress and anti-apoptotic effect of recombinant humanized IgG1 antibody. Together, our results suggest that recombinant humanized IgG1 antibody exerts its anti-oxidative stress and anti-apoptotic function by upregulation of MSRA via the Sirt1-FOXO1 axis.

The selenoprotein methionine sulfoxide reductase B1 (MSRB1).

Methionine (Met) can be oxidized to methionine sulfoxide (MetO), which exist as R- and S-diastereomers. Present in all three domains of life, methionine sulfoxide reductases (MSR) are the enzymes that reduce MetO back to Met. Most characterized among them are MSRA and MSRB, which are strictly stereospecific for the S- and R-diastereomers of MetO, respectively. While the majority of MSRs use a catalytic Cys to reduce their substrates, some employ selenocysteine. This is the case of mammalian MSRB1, which was initially discovered as selenoprotein SELR or SELX and later was found to exhibit an MSRB activity. Genomic analyses demonstrated its occurrence in most animal lineages, and biochemical and structural analyses uncovered its catalytic mechanism. The use of transgenic mice and mammalian cell culture revealed its physiological importance in the protection against oxidative stress, maintenance of neuronal cells, cognition, cancer cell proliferation, and the immune response. Coincident with the discovery of Met oxidizing MICAL enzymes, recent findings of MSRB1 regulating the innate immunity response through reversible stereospecific Met-R-oxidation of cytoskeletal actin opened up new avenues for biological importance of MSRB1 and its role in disease. In this review, we discuss the current state of research on MSRB1, compare it with other animal Msrs, and offer a perspective on further understanding of biological functions of this selenoprotein.

Characterization of a new selenoprotein methionine sulfoxide reductase from Haematococcus pluvialis and its antioxidant activity in response to high light intensity, hydrogen peroxide, glyphosate, and cadmium exposure.

Selenium incorporates into selenocysteine (Sec) which is a key component of selenoproteins implicated in antioxidant defense and redox homeostasis. Methionine sulfoxide reductases (Msr) play crucial roles in cellular defense against environmental stress. Whereas mammals have the MsrB selenoprotein form, unicellular organisms have MsrA. The Sec residue at the conserved catalytic sites of selenoprotein MsrA confers a metabolic advantage over the non-selenoprotein type MsrA. In the present study, the novel selenoprotein HpMsrA from Haematococcus pluvialis was cloned by the rapid amplification of cDNA ends and transformed into the model green alga Chlamydomonas reinhardtii. Alignment of homologs revealed the presence of the conserved catalytic domain GUFW and showed that the HpMsrA protein comprises Sec (U) at the N-terminus but no recycled Cys at the C-terminus. We studied the response of HpMsrA expression to selenite, high light intensity, hydrogen peroxide, cadmium nitrate, and glyphosate exposure via real-time quantitative PCR and enzyme activity analysis. The results demonstrated that HpMsrA protects cellular proteins against oxidative and environmental stressors. Compared with wild type C. reinhardtii, the transformant exhibited a superior antioxidant ability. The discoveries made herein shed light on the antioxidant physiology and environmental stress resistance mechanisms of the selenoproteins in microalgae. This information may aid in conducting environmental risk assessments of aquatic ecosystems involving microalgae known to respond rapidly and quantitatively to abiotic stress factors promoting excessive reactive oxygen species generation.

Relevance of methionine sulfoxide reductase(s) (MSR) as candidate proteins in redox homeostasis-mediated resistance response to Helicoverpa armigera (Hübner) in the pigeonpea wild relative Cajanus platycarpus (Benth.) Maesen.

Pod borer, Helicoverpa armigera, a polyphagus herbivore causes extensive economic losses to crops, including pigeonpea. Exploitation of pod borer resistance in wild relatives is pertinent due to the absence of resistance sources in cultivated pigeonpea and crossing-incompatibility with the resistant wild relatives. We present leads obtained in deeper understanding of pod borer resistance mechanism in Cajanus platycarpus, a pigeonpea wild relative. Surge in cellular ROS during herbivory leads to redox-PTMs (post translational modifications) of methionine-rich proteins including antioxidant enzymes, causing oxidative damage. Plants then officiate methionine sulfoxide reductases (MSRs), that maintain the redox status of methionine and hence homeostasis. We demonstrate functionality of MSRs (MSRA and MSRB) in the resistance response of the wild relative to pod borer. Among 5 MSRA and 3 MSRB genes, CpMSRA2 and CpMSRB1 were herbivore-responsive based on expression during herbivory. Clues about the stress-responsiveness were obtained upon analyses of cis-elements and co-expressing genes. Apparently, the wild relative followed a non-canonical mode of redox management, as divulged by antioxidant enzymes and the scavenging capacity. Differential lipid peroxidation as an early response provided evidences for an effective redox management in the wild relative. This is the first report signifying redox homeostasis in the resistance response towards herbivory.

Methionine oxidation activates pyruvate kinase M2 to promote pancreatic cancer metastasis.

Cancer mortality is primarily a consequence of its metastatic spread. Here, we report that methionine sulfoxide reductase A (MSRA), which can reduce oxidized methionine residues, acts as a suppressor of pancreatic ductal adenocarcinoma (PDA) metastasis. MSRA expression is decreased in the metastatic tumors of PDA patients, whereas MSRA loss in primary PDA cells promotes migration and invasion. Chemoproteomic profiling of pancreatic organoids revealed that MSRA loss results in the selective oxidation of a methionine residue (M239) in pyruvate kinase M2 (PKM2). Moreover, M239 oxidation sustains PKM2 in an active tetrameric state to promote respiration, migration, and metastasis, whereas pharmacological activation of PKM2 increases cell migration and metastasis in vivo. These results demonstrate that methionine residues can act as reversible redox switches governing distinct signaling outcomes and that the MSRA-PKM2 axis serves as a regulatory nexus between redox biology and cancer metabolism to control tumor metastasis.

A Novel Small RNA, DsrO, in Deinococcus radiodurans Promotes Methionine Sulfoxide Reductase (msrA) Expression for Oxidative Stress Adaptation.

Reactive oxygen species (ROS) can cause destructive damage to biological macromolecules and protein dysfunction in bacteria. Methionine sulfoxide reductase (Msr) with redox-active Cys and/or seleno-cysteine (Sec) residues can restore physiological functions of the proteome, which is essential for oxidative stress tolerance of the extremophile Deinococcus radiodurans. However, the underlying mechanism regulating MsrA enzyme activity in D. radiodurans under oxidative stress has remained elusive. Here, we identified the function of MsrA in response to oxidative stress. msrA expression in D. radiodurans was significantly upregulated under oxidative stress. The msrA mutant showed a deficiency in antioxidative capacity and an increased level of dabsyl-Met-S-SO, indicating increased sensitivity to oxidative stress. Moreover, msrA mRNA was posttranscriptionally regulated by a small RNA, DsrO. Analysis of the molecular interaction between DsrO and msrA mRNA demonstrated that DsrO increased the half-life of msrA mRNA and then upregulated MsrA enzyme activity under oxidative stress compared to the wild type. msrA expression was also transcriptionally regulated by the DNA-repairing regulator DrRRA, providing a connection for further analysis of protein restoration during DNA repair. Overall, our results provide direct evidence that DsrO and DrRRA regulate msrA expression at two levels to stabilize msrA mRNA and increase MsrA protein levels, revealing the protective roles of DsrO signaling in D. radiodurans against oxidative stress. IMPORTANCE The repair of oxidized proteins is an indispensable function allowing the extremophile D. radiodurans to grow in adverse environments. Msr proteins and various oxidoreductases can reduce oxidized Cys and Met amino acid residues of damaged proteins to recover protein function. Consequently, it is important to investigate the molecular mechanism maintaining the high reducing activity of MsrA protein in D. radiodurans during stresses. Here, we showed the protective roles of an sRNA, DsrO, in D. radiodurans against oxidative stress. DsrO interacts with msrA mRNA to improve msrA mRNA stability, and this increases the amount of MsrA protein. In addition, we also showed that DrRRA transcriptionally regulated msrA gene expression. Due to the importance of DrRRA in regulating DNA repair, this study provides a clue for further analysis of MsrA activity during DNA repair. This study indicates that protecting proteins from oxidation is an effective strategy for extremophiles to adapt to stress conditions.

An Intramolecular Charge Transfer-Förster Resonance Energy Transfer Integrated Unimolecular Platform for Two-Photon Ratiometric Fluorescence Sensing of Methionine Sulfoxide Reductases in Live-Neurons and Mouse Brain Tissues.

Oxidative stress in organisms is a factor leading to a series of diseases including tumors and neurological disorders, while methionine sulfoxide reductases (Msrs) may provide an antioxidant and self-repair mechanism through redox cycles of methionine residues in proteins. Thus, it is important to understand the crucial role of Msrs in maintaining the redox homeostasis. However, it remains a great challenge for real-time and quantitative monitoring of Msrs in live systems due to the lack of appropriate sensing tools. Herein, a novel unimolecular platform integrating the intramolecular charge transfer (ICT) and Förster resonance energy transfer (FRET) dual mechanisms was successfully developed. By employing the highly specific Msrs-catalyzed reduction from the electron-withdrawing sulfoxide moiety in the probe to an electron-donating sulfide group, a synergistic ICT-FRET activation process was achieved, leading to a ratiometric fluorescence response toward Msrs with high selectivity, sensitivity, and accuracy. Moreover, benefiting from the favorable features, including mitochondria-targeting, near-infrared two-photon excitation, low cytotoxicity, good stability, and biocompatibility, the probe was successfully used for monitoring mitochondrial Msrs levels in live-neurons, and a positively correlated up-regulation of endogenous Msrs levels under O2•- stimulation was observed for the first time, confirming a Msrs-involved adaptive antioxidant mechanism in neurons. Furthermore, two-photon microscopic imaging of various regions in Alzheimer's disease (AD) mice brains revealed a down-regulated Msrs levels compared with that in normal brains, especially in the cornuammonis of the hippocampus region, which may in turn lead to an aggravation of AD pathogenesis due to the weakened antioxidant and self-repair capability of neurons.

The Fused Methionine Sulfoxide Reductase MsrAB Promotes Oxidative Stress Defense and Bacterial Virulence in Fusobacterium nucleatum.

Fusobacterium nucleatum, an anaerobic Gram-negative bacterium frequently found in the human oral cavity and some extra-oral sites, is implicated in several important diseases: periodontitis, adverse pregnancy outcomes, and colorectal cancer. To date, how this obligate anaerobe copes with oxidative stress and host immunity within multiple human tissues remains unknown. Here, we uncovered a critical role in this process of a multigene locus encoding a single, fused methionine sulfoxide reductase (MsrAB), a two-component signal transduction system (ModRS), and thioredoxin (Trx)- and cytochrome c (CcdA)-like proteins, which are induced when fusobacterial cells are exposed to hydrogen peroxide. Comparative transcriptome analysis revealed that the response regulator ModR regulates a large regulon that includes trx, ccdA, and many metabolic genes. Significantly, specific mutants of the msrAB locus, including msrAB, are sensitive to reactive oxygen species and defective in adherence/invasion of colorectal epithelial cells. Strikingly, the msrAB mutant is also defective in survival in macrophages, and it is severely attenuated in virulence in a mouse model of preterm birth, consistent with its failure to spread to the amniotic fluid and colonize the placenta. Clearly, the MsrAB system regulated by the two-component system ModRS represents a major oxidative stress defense pathway that protects fusobacteria against oxidative damage in immune cells and confers virulence by enabling attachment and invasion of multiple target tissues. IMPORTANCE F. nucleatum colonizes various human tissues, including oral cavity, placenta, and colon. How this obligate anaerobe withstands oxidative stress in host immune cells has not been described. We report here that F. nucleatum possesses a five-gene locus encoding a fused methionine sulfoxide reductase (MsrAB), a two-component signal transduction system (ModRS), and thioredoxin- and cytochrome c-like proteins. Regulated by ModRS, MsrAB is essential for resistance to reactive oxygen species, adherence/invasion of colorectal epithelial cells, and survival in macrophage. Unable to colonize placenta and spread to amniotic fluid, the msrAB mutant failed to induce preterm birth in a murine model.

Functional characterization of monothiol and dithiol glutaredoxins from Leptospira interrogans.

Thiol redox proteins and low molecular mass thiols have essential functions in maintaining cellular redox balance in almost all living organisms. In the pathogenic bacterium Leptospira interrogans, several redox components have been described, namely, typical 2-Cys peroxiredoxin, a functional thioredoxin system, glutathione synthesis pathway, and methionine sulfoxide reductases. However, until now, information about proteins linked to GSH metabolism has not been reported in this pathogen. Glutaredoxins (Grxs) are GSH-dependent oxidoreductases that regulate and maintain the cellular redox state together with thioredoxins. This work deals with recombinant production at a high purity level, biochemical characterization, and detailed kinetic and structural study of the two Grxs (Lin1CGrx and Lin2CGrx) identified in L. interrogans serovar Copenhageni strain Fiocruz L1-130. Both recombinant LinGrxs exhibited the classical in vitro GSH-dependent 2-hydroxyethyl disulfide and dehydroascorbate reductase activity. Strikingly, we found that Lin2CGrx could serve as a substrate of methionine sulfoxide reductases A1 and B from L. interrogans. Distinctively, only recombinant Lin1CGrx contained a [2Fe2S] cluster confirming a homodimeric structure. The functionality of both LinGrxs was assessed by yeast complementation in null grx mutants, and both isoforms were able to rescue the mutant phenotype. Finally, our data suggest that protein glutathionylation as a post-translational modification process is present in L. interrogans. As a whole, our results support the occurrence of two new redox actors linked to GSH metabolism and iron homeostasis in L. interrogans.

MsrB1 Promotes Proliferation and Invasion of Colorectal Cancer Cells via GSK-3ß/ß-catenin Signaling Axis.

Methionine sulfoxide reductase B1 (MsrB1) can catalyze both free and protein-bound R-methionine sulfoxides (R-MetO) to methionine (Met). It has been reported that MsrB1 plays an important role in the development of HCC and human bone osteosarcoma. However, little is known about the functions of MsrB1 in human colorectal cancer (CRC). Herein, we detected MsrB1 expression level in CRC tissue and cell lines, and investigated the effect of MsrB1 knockdown on CRC phenotypes and possible mechanisms involved in. The results showed that MsrB1 was highly expressed in both CRC tissues and cell lines, and that cell proliferation, migration and invasion were significantly inhibited, but apoptosis was increased after MsrB1 knockdown in colorectal cancer HCT116 and RKO cell lines, compared to control siRNA group. In addition, E-cadherin protein level was increased, vimentin and Snail protein were greatly decreased after knockdown of MsrB1 in cells. Furthermore, pGSK-3ß (Ser9) and ß-catenin protein levels were reduced, the promoter activity of TCF/LEF construction was inhibited after MsrB1 knockdown in cells, suggesting that GSK-3ß/ß-catenin signaling axis was involved in the tumorigenesis of CRC. In conclusion, the oncogenic role and related mechanisms of MsrB1 in CRC discovered in our work determined the potential role of MsrB1 as a biomarker and may provide a new target for clinical therapy of CRC.

An ethylene-hypersensitive methionine sulfoxide reductase regulated by NAC transcription factors increases methionine pool size and ethylene production during kiwifruit ripening.

Ethylene plays an important role in regulating fruit ripening by triggering dynamic changes in expression of ripening-associated genes, but the functions of many of these genes are still unknown. Here, a methionine sulfoxide reductase gene (AdMsrB1) was identified by transcriptomics-based analysis as the gene most responsive to ethylene treatment in ripening kiwifruit. The AdMsrB1 protein exhibits a stereospecific activity toward the oxidative stress-induced R enantiomer of methionine sulfoxide (MetSO), reducing it to methionine (Met). Stable overexpression of AdMsrB1 in kiwifruit significantly increased the content of free Met and 1-aminocyclopropane-1-carboxylic acid (ACC), the immediate precursor of ethylene, and increased ethylene production. Dual-luciferase assays indicated that the AdMsrB1 promoter was not directly upregulated by ethylene treatment but was modulated by two ethylene-inducible NAM/ATAF/CUC transcription factors (AdNAC2 and AdNAC72) that bind directly to the AdMsrB1 promoter. Overexpression of AdNAC72 in kiwifruit not only enhanced AdMsrB1 expression, but also increased free Met and ACC content and ethylene production rates. This finding establishes an unexpected regulatory loop that enhances ethylene production and the concentration of its biosynthetic intermediates.

Deletion of both methionine sulfoxide reductase A and methionine sulfoxide reductase C genes renders Salmonella Typhimurium highly susceptible to hypochlorite stress and poultry macrophages.

Salmonella Typhimurium survives and replicates inside the oxidative environment of phagocytic cells. Proteins, because of their composition and location, are the foremost targets of host inflammatory response. Among others, Met-residues are highly prone to oxidation. Methionine sulfoxide reductase (Msr), with the help of thioredoxin-thioredoxin reductase, can repair oxidized methionine (Met-SO) residues to Met. There are four methionine sulfoxide reductases localized in the cytosol of S. Typhimurium, MsrA, MsrB, MsrC and BisC. MsrA repairs both protein-bound and free 'S' Met-SO, MsrB repairs protein-bound 'R' Met-SO, MsrC repairs free 'R' Met-SO and BisC repairs free 'S' Met-SO. To assess the role(s) of various Msrs in Salmonella, few studies have been conducted by utilizing ΔmsrA, ΔmsrB, ΔmsrC, ΔmsrAΔmsrB, ΔmsrBΔmsrC and ΔbisC mutant strains of S. Typhimurium. Out of the above-mentioned mutants, ΔmsrA and ΔmsrC were found to play important role in the stress survival of this bacterium; however, the combined roles of these two genes have not been determined. In the current study, we have generated msrAmsrC double gene deletion strain (ΔmsrAΔmsrC) of S. Typhimurium and evaluated the effect of gene deletions on the survival of Salmonella against hypochlorite stress and intramacrophage replication. In in vitro growth curve analysis, ΔmsrAΔmsrC mutant strain showed a longer lag phase during the initial stages of the growth; however, it attained similar growth as the wild type strain of S. Typhimurium after 5 h. The ΔmsrAΔmsrC mutant strain has been highly (~ 3000 folds more) sensitive (p < 0.001) to hypochlorite stress. Further, ΔmsrA and ΔmsrAΔmsrC mutant strains showed more than 8 and 26 folds more susceptibility to poultry macrophages, respectively. Our data suggest that the deletion of both msrA and msrC genes severely affect the oxidative stress survival and intramacrophage proliferation of S. Typhimurium.

Staphylococcus aureus Peptide Methionine Sulfoxide Reductases Protect from Human Whole-Blood Killing.

The generation of oxidative stress is a host strategy used to control Staphylococcus aureus infections. Sulfur-containing amino acids, cysteine and methionine, are particularly susceptible to oxidation because of the inherent reactivity of sulfur. Due to the constant threat of protein oxidation, many systems evolved to protect S. aureus from protein oxidation or to repair protein oxidation after it occurs. The S. aureus peptide methionine sulfoxide reductase (Msr) system reduces methionine sulfoxide to methionine. Staphylococci have four Msr enzymes, which all perform this reaction. Deleting all four msr genes in USA300 LAC (Δmsr) sensitizes S. aureus to hypochlorous acid (HOCl) killing; however, the Δmsr strain does not exhibit increased sensitivity to H2O2 stress or superoxide anion stress generated by paraquat or pyocyanin. Consistent with increased susceptibility to HOCl killing, the Δmsr strain is slower to recover following coculture with both murine and human neutrophils than USA300 wild type. The Δmsr strain is attenuated for dissemination to the spleen following murine intraperitoneal infection and exhibits reduced bacterial burdens in a murine skin infection model. Notably, no differences in bacterial burdens were observed in any organ following murine intravenous infection. Consistent with these observations, USA300 wild-type and Δmsr strains have similar survival phenotypes when incubated with murine whole blood. However, the Δmsr strain is killed more efficiently by human whole blood. These findings indicate that species-specific immune cell composition of the blood may influence the importance of Msr enzymes during S. aureus infection of the human host.

Seven novel genetic variants in a North Indian cohort with classical homocystinuria.

Classical homocystinuria is the most common cause of isolated homocystinuria. The variants of the CBS gene remain unidentified in Indian children with this disorder. Based on the hallmark clinical features, family history, and/or biochemical clues for classical homocystinuria, 16 children below the age of 18 years were evaluated by Sanger sequencing of the coding exons of CBS gene with flanking intronic regions. The common C677T variant of the MTHFR gene was also screened by restriction fragment length polymorphism. Fifteen children were clinically suspected of having classical homocystinuria and one asymptomatic child with positive family history. Only seven children had biochemical features of classical homocystinuria. Sanger sequencing of the CBS gene confirmed 15 different pathogenic or likely pathogenic variants in 14 cases. Of these, seven variants were novel (three frameshift deletions, two nonsense, one missense, one splice site variant) and were predicted to be deleterious by Mutation Taster software. Seven cases were homozygous, another six were compound heterozygous, and one case was single heterozygous in the study. None of the three most frequent mutations reported worldwide viz., I278T, G307S, and IVS 11-2A>C were found in our cohort. No variants were detected in the exons 2, 8, 12, and 14 as compared to reported literature. Eleven out of 15 variants were associated with the conserved catalytic domain of the CBS polypeptide. The MTHFR polymorphism C677T was observed in heterozygous state in six cases. Our study reports the detailed genotype and seven novel variants in the CBS gene, causing classical homocystinuria in Indian children. The genetic analysis will help to offer accurate genetic counseling, prenatal diagnosis, and development of mutation-based novel therapeutic strategies.

Implication of Staphylococcus aureus MsrB dimerization upon oxidation.

Oxidative modification of protein structure has been shown to play a significant role in bacterial virulence and metabolism. The sulfur-containing residues are susceptible to oxidation and the enzymatic reversal of oxidized cysteine or methionine is detected in many organisms. Methionine sulfoxide reductases (Msr) are responsible for reducing oxidized methionine. The two different Msrs, MsrA and MsrB, reduce methionine R-sulfoxide and methionine S-sulfoxide, respectively through self-oxidation. This study elucidated the structure of MsrB from Staphylococcus aureus Mu50 and its changes upon oxidation. The active site shows two reduced cysteines in a close contact, implying disulfide bond would form without major structural rearrangement. When the protein is exposed to an oxidative condition, a dimeric state is observed. The dimerization of SAMsrB creates a valley structure for accepting peptidyl substrates. To the best of our knowledge, oxidation induced dimerization of SAMsrB would help to understand mechanism behind redox control that has not been well characterized.