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. Author manuscript; available in PMC: 2011 Nov 2.
Published in final edited form as: Tuberculosis (Edinb). 2009 Dec;89(Suppl 1):S26–S32. doi: 10.1016/S1472-9792(09)70008-3

Methionine sulfoxide reductase B (MsrB) of Mycobacterium smegmatis plays a limited role in resisting oxidative stress

Subramanian Dhandayuthapani a,*, Chinnaswamy Jagannath b, Celina Nino a, Sankaralingam Saikolappan a, Smitha J Sasindran a
PMCID: PMC3206628  NIHMSID: NIHMS212940  PMID: 20006300

summary

Pathogenic mycobacteria including Mycobacterium tuberculosis resists phagocyte generated reactive oxygen intermediates (ROI) and this constitutes an important virulence mechanism. We have previously reported, using Mycobacterium smegmatis as a model to identify the bacterial components that resist intracellular ROI, that an antioxidant methionine sulfoxide reductase A (MsrA) plays a critical role in this process. In this study, we report the role of methionine sulfoxide reductase B (MsrB) in resistance to ROI by constructing a msrB mutant (MSΔmsrB) and MsrA/B double mutant (MSΔmsrA/B) strains of M. smegmatis and testing their survival in unactivated and interferon gamma activated mouse macrophages. While msrB mutant exhibited significantly lower intracellular survival than its wild type counterpart, the survival rate seemed to be much higher than msrA mutant (MSΔmsrA) strain. Further, the msrB mutant showed no sensitivity to oxidants in vitro. The msrA/B double mutant (MSΔmsrA/B), on the other hand, exhibited a phenotype similar to that of msrA mutant in terms of both intracellular survival and sensitivity to oxidants. We conclude, therefore, that MsrB of M. smegmatis plays only a limited role in resisting intracellular and in vitro ROI.

Keywords: Macrophage, Reactive oxygen intermediates, Infection, Evasion, Mycobacteria

1. Introduction

Mycobacterium tuberculosis and other human pathogenic mycobacteria are well known for their ability to survive and replicate within mononuclear phagocytes, the host defense cells of the body1. This ability of pathogenic mycobacteria is primarily due to their potential to modulate and defend the antimicrobial responses of phagocytes that include reactive oxygen species (ROS) and reactive nitrogen species (RNS) (collectively called reactive oxygen intermediates [ROI]), generated by NADPH oxidase and iNOS, respectively. However, the mechanisms by which pathogenic mycobacteria and other intracellular bacteria evade ROI still remain elusive, although evidence suggests that bacterial antioxidants like Cu,Zn-superoxide dismutase (Cu,Zn-SOD)2, catalase-peroxidase3 and other related enzymes4 contribute to this process.

Methionine sulfoxide reductases (Msr) are antioxidant repair enzymes that have a role in the detoxification of ROI5,6. Msr catalyze the reduction of oxidized methionine (Met-O) to methionine (Met) in free and protein-bound forms5. Although two kinds of Msr namely MsrA and MsrB exist in both prokaryotes and eukaryotes, they share little identity between them either at primary sequence level or at structural level7–10. In the majority of organisms, the genes encoding MsrA and MsrB exist independently of each other. However, a single gene encoding MsrA/MsrB as a fused protein has also been noticed in a few organisms11. It has been reported that MsrA is specific to Met-S-O, while MsrB is specific to Met-R-O5,6,9,12. Further, it has been speculated that methionine residues in proteins serve as sinks for the ROI in the surroundings13. The redox cycle involving the chemical oxidation of Met to Met-O and enzymatic reduction of Met-O to Met is considered as an alternate mechanism to protect cells against oxidative stress. Interestingly, both prokaryotic and eukaryotic cells lacking Msr are sensitive to oxidative stress11,14,15. Further, msrA has been shown to be critical for the survival of Erwinia chrysanthemi16, Mycoplasma genitalium17 and Helicobacter pylori18 in their hosts.

We have earlier reported that an msrA deletion mutant (MSΔmsrA) of Mycobacterium smegmatis was less able to survive within unactivated and IFN-γ-activated mouse macrophage cell line19. We have also reported that phagosomes containing msrA mutant (MSΔmsrA) of M. smegmatis acquired p67phox component of phagocyte NADPH oxidase and inducible nitric oxide synthase (iNOS) much earlier than the phagosomes with wild-type M. smegmatis strain. Here, we show that MsrB has only a limited role in the intracellular survival of M. smegmatis. Further, we demonstrate that M. smegmatis deletion mutant lacking both MsrA and MsrB (MSΔmsrA/B) exhibits a phenotype similar to that of msrA mutant (MSΔmsrA) in terms of both intracellular survival and sensitivity towards in vitro oxidative stress. These results differ significantly from msrA/msrB mutant (MTΔmsrA/B) of M. tuberculosis which showed no resistance to hydroperoxides20.

2. Material and methods

2.1. Bacterial strains, media and growth conditions

Escherichia coli strain INV-α (invitrogen) was grown in LB broth or LB Agar plates. E. coli harboring plasmid was grown in the presence of ampicillin (100 μg/ml) or kanamycin (25 μg/ml) or hygromycin (100 μg/ml) depending upon the resistance gene in the plasmid. M. smegmatis mc2 155 (wild type) was grown in 7H9 broth or 7H10 agar supplemented with 10% albumin dextrose complex (ADC) and 0.05% Tween 80 (TW).

2.2. DNA manipulations and creation of plasmids

Isolation of genomic DNA from mycobacteria, Southern hybridization, radiolabelling and polymerase chain reaction (PCR) were performed as described by Ausubel et al.21. Qiaprep kit (Qiagen Inc) was used to isolate plasmid DNA. Oligonucleotides were synthesized at DNA core facility of University of Texas Health Science Center at San Antonio. To construct msrB disruption plasmid, we amplified 2.27 kb DNA containing msrB and adjacent region from M. smegmatis genomic DNA using primers MSMSRB1 (5-CTGCGGATCTTCGACTACAC-3) and MSMSRB2 (5-GCCGACTTCATGATCTGGAC-3). The PCR fragment was initially cloned in pCR2.1 vector, cleaved as EcoRI fragment and cloned into EcoRI site of pUCH (pUC18 plasmid lacking HindIII site). The resulting plasmid was cut with HindIII, blunt ended with klenow treatment and ligated to a blunt-ended 1.9 kb DNA, obtained from pIJ963 by BamHI/PstI digestion and klenow treated, that has the gene encoding hygromycin resistance. This plasmid pMSMSRBK, which has M. smegmatis msrB interrupted with hygromycin resistance gene, served as the disruption construct for msrB. In order to disrupt msrA in msrB mutant (MSΔmsrB) strain of M. smegmatis, we created another plasmid construct as follows. First, the plasmid pMSMSRA519 was cut with EcoRI to release the DNA fragment containing M. smegmatis msrA gene disrupted with kanamycin resistance gene. This fragment was ligated to similarly cut p1NIL plasmid22 to create p1MSMSRA1. A DNA fragment that contained lacZ and sacB genes, from pGOAL1722, was later added to this plasmid at PacI site to obtain plasmid p1MSMSRA2. This plasmid was used to disrupt msrA in the msrB mutant (MSΔmsrB) strain. We have earlier described the construction of pET16b based overexpression plasmid pTBMSRAEX for the expression of M. tuberculosis MsrA23. To generate MsrB overexpression construct, we amplified 585 bp msrB gene containing DNA fragment of M. tuberculosis by PCR with primers TBMSRBE1 (5′-GGACATATGACGCGCCCAAAGCTAGAACTG-3′) and TBMSRBE2 (5′-TGGAGCGGATCCGGGCGATTAAGCCGRGGC-3′). In these, primer TBMSRBE1 was designed to have an NdeI restriction site and primer TBMSRBE2 was designed to have a BamHI restriction site. The DNA fragment obtained was cut with NdeI and BamHI and ligated to a similarly cut pET16b vector to create plasmid pTBMSRBEX.

2.3. Construction of msr mutants

For the generation of msrB mutant (MSΔmsrB), wild-type M. smegmatis was electroporated with pMSMSRBK and transformants selected on 7H10-ADC-TW plates containing hygromycin (50 μg/ml). MSΔmsrB was identified by screening the transformants in Southern using msrB and hygromycin resistance gene specific probes. However, for the generation of msrA/msrB double mutant (MSΔmsrA/B), MSΔmsrB strain of M. smegmatis was electroporated with plasmid p1MSMSRA2 and transformants obtained were screened by a two-step selection method reported by Parish and Stoker22. In this method, transformants were initially selected in 7H10-ADC-TW plates containing the antibiotic kanamycin (25 μg/ml) and X-gal (40 μg/ml). Blue colonies representing single cross-overs were selected and streaked onto 7H10-ADC-TW plates. After growth, a loopful of bacteria was serially diluted and screened for white colonies on 7H10-ADC-TW plates containing 2% sucrose and X-gal. Chromosomal DNA from the white colonies was later screened for disruption of msrA by Southern using msrA gene as probe.

2.4. Intracellular survival

Mouse macrophage cell line J774A.1 grown in Dulbecco modified Eagle medium (DMEM) with 10% fetal bovine serum was used to determine the intracellular survival of M. smegmatis strains. Methods for macrophage infection and CFU determination are detailed in our previous paper17. Briefly, monolayer cultures were established in 24-well plates and infected with M. smegmatis strains by incubating the bacteria with macrophages at the ratio of 1:1 at 37°C. Each strain was infected in quadraduplicate wells. After 4 h of incubation, the wells were washed with 1 ml of warm DMEM medium to remove the non-phagocytosed bacteria. The plates were incubated at 37°C with 5% CO2. Cells were harvested at 0, 1 and 3 days of infection by lysing with 0.15% SDS for 15 min at room temperature. The lysed suspensions were serially diluted and plated on 7H10-ADC-TW plates, incubated for three days and colonies counted.

2.5. Survival under oxidative stress

The ability of msr mutant strains of M. smegmatis to defend against in vitro oxidative stress was tested as described previously17. Freshly grown M. smegmatis strains were diluted to 0.3 OD at 595 nm in 7H10-ADC-TW and 1 ml of each of diluted culture was incubated with either hydrogen peroxide (5 mM) or cumene hydroperoxide (5 mM) or t-butyl hydroperoxide (5 mM) or methyl viologen (5 mM) for 1 h at 37°C. Afterwards, cultures were serially diluted tenfold, plated onto 7H10-ADC-TW plates, incubated at 37°C and colonies counted after 3 days. Experiments with nitric oxide donors like GSNO (5 mM), sodium papanonate (1 mM) were also conducted similarly except the pH of the 7H10-ADC-TW medium was kept at 5.0.

2.6. Overexpression of M. tuberculosis Msr in E. coli and production of antisera

To overexpress MsrA and MsrB in E. coli, we used the T7 promoter-based pET16b overexpression system. This produces fusion proteins with 10 histidine amino acids (His10-tag) at the N-terminus. We have previously described methods to express and purify proteins using this system24, and the constructs for the expression of M. tuberculosis MsrA and MsrB are described in the previous section. The constructs were transformed into E. coli strain BL21(DE3) and log phase cultures of E. coli cells bearing the plasmid constructs were induced with 0.1 mM IPTG to overexpress the MsrA and MsrB proteins. Analysis of E. coli proteins extracts in SDS-PAGE 3 h post induction revealed overexpression of 22 kDa and 15 kDa proteins for constructs pTBMSRAEX and pTBMSRBEX, respectively (Fig. 1). The sizes of these proteins corresponded to the expected sizes of 20.4 kDa and 13.3 kDa for MsrA and MsrB, respectively. Using Ni-NTA agarose column, we purified the His10-MsrA and His10-MsrB proteins and these proteins were used to immunize rabbits for the production of antisera according to standard protocols. The antisera reacted with MsrA and MsrB from a wide variety of mycobacteria including M. smegmatis (Fig. 2).

Fig. 1.

Fig. 1

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SDS-PAGE protein profile showing overexpression and purification of M. tuberculosis MsrA and MsrB. Lane 1, Molecular marker. Lanes 2 and 3, extracts of E. coli strain BL21 carrying overexpression plasmid pTBMSRAEX in the absence (lane 2) and presence (lane 3) of 0.1 mM IPTG. Lanes 5 and 6, extracts of E. coli strain BL21 carrying overexpression plasmid pTBMSRBEX in the absence (lane 5) and presence (lane 6) of 0.1 mM IPTG. Lanes 4 and 7, His10-MsrA and His10-MsrB, respectively after Ni-NTA chromatography.

Fig. 2.

Fig. 2

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Immunoblot analysis of MsrA and MsrB from different mycobacteria. Whole extracts were prepared from mycobacteria grown in 7H9-ADC-TW medium. Each lane contained about 150 μg of protein. Anti-MsrA antiserum and anti-MsrB antiserum were used at 1:500 dilutions. Peroxidase conjugated anti-rabbit IgG was used as secondary antibody (1:10,000 dilution; Sigma). Bands were visualized by chemiluminescence method (Amersham).

2.7. SDS-PAGE and immunoblotting

Standard methods described by Ausubel et al.21 were followed for SDS-PAGE and immunoblotting. Protein extracts of M. smegmatis strains were obtained by bead-beating technique and total protein content of the extracts was determined by using BCA (bicinchoninic acid) method. Soluble and membrane fractions of M. smegmatis were prepared as described previously25. Fifteen percent SDS-PAGE was used to separate the proteins and each lane had 150 μg total protein. SDS-PAGE-separated proteins were transferred to nitrocellulose membranes and probed with anti-M. tuberculosis MsrA (anti-MsrA) and anti-M. tuberculosis MsrB (anti-MsrB) antisera.

3. Results

3.1. Organization of msrB in the chromosome of M. smegmatis

A blast search with Neisseria gonnorrhoeae MsrB sequence26 led to the identification of gene MSMEG2784 in the M. smegmatis genome sequence (J. Craig Venter Institute [JCVI], earlier known as The Institute for Genome Research [TIGR], database) as the gene encoding MsrB of M. smegmatis. The product of this gene exhibits extensive homology and identity with a variety of prokaryotic organisms including msrB gene of M. tuberculosis,for which it showed 87% identity. msrB gene of M. smegmatis shows complementary strand-based transcription, and analysis of the flanking regions revealed that it is the terminal gene of a cluster of genes showing transcription towards this direction (Fig. 3A). Gene MSMEG2783 which is upstream to msrB shows transcriptional direction that is opposite to msrB, hence disruption of msrB in M. smegmatis is not expected to have any polar effect on adjacent genes.

Fig. 3.

Fig. 3

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(A) Organization of msrA and msrB in the genome of M. smegmatis. Black arrows in the upper and lower panels represent the genes in the flanking regions of msrA and msrB, respectively. The genes are named according to JVCI (TIGR) annotations. Open arrows represent msrA (MSMEG6477) and msrB (MSMEG2784). (B) Immunoblot showing the expression of MsrA and MsrB in M. smegmatis. W, whole extract; S, soluble fraction; M, membrane fraction. Anti-MsrA antiserum and anti-MsrB antiserum were used at 1:500 dilutions. Peroxidase conjugated anti rabbit IgG was used as secondary antibody (1:10,000 dilution; Sigma). Bands were visualized by chemiluminescence method (Amersham).

3.2. Expression of MsrB and its localization

In order to understand the expression of MsrB in M. smegmatis and its localization within the cell, we analyzed the whole, soluble and membrane fractions of M. smegmatis lysates in immunoblot. Although MsrB protein was readily detectable with anti-MsrB antibodies in the whole extract and soluble fraction, there was no detectable MsrB protein in the membrane fraction. We also probed these fractions against anti-MsrA antiserum to understand its localization. This also showed a pattern similar to that of MsrB (Fig. 3B). In contrast, M. tuberculosis has been reported to have MsrA in both soluble and membrane fractions and MsrB only in the soluble fraction27. The non-membrane association of Msr enzymes in M. smegmatis may suggest that they have limited role in the reduction of Met-O on the membrane/surface of the bacterium.

3.3. Construction of msrB and msrA/B mutants

A msrB mutant (MSΔmsrB; MS20) strain was constructed through allelic replacement by electroporating the plasmid pMSMSRBK into wild-type M. smegmatis and subsequent screening of the hygromycin-resistant transformants by Southern analysis (Figs. 4A,B). The disruption of expression of MsrB in this strain was confirmed by the absence of MsrB using anti-MsrB antiserum (Fig. 5A).

Fig. 4.

Fig. 4

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(A) Southern analysis of M. smegmatis strains. Genomic DNA of M. smegmatis wild-type strain (MSWt) and msrB mutant (MS20; MSΔmsrB) strains were cut with SalI and probed with 1.75 kb hygromycin-resistance gene (hyg) and 1 kb M. smegmatis msrB gene (msrB). (B) Schematic explaining msrB locus in the wild-type (WT) and MSΔmsrB (MS20) strain. White boxes represent the flanking region of msrB, stippled boxes represent the msrB gene, black box represents the hygromycin resistance gene. Arrows indicate the direction of transcription of genes. The sites for restriction enzymes in and around msrB gene are indicated. The lines below the boxes indicate the expected size of fragments from SalI-cut genomic DNA of WT and MS20 when probed with 1 kb msrB gene containing fragment.

Fig. 5.

Fig. 5

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(A) Immunoblot analysis of M. smegmatis strains. MSWt, wild-type M. smegmatis; MS97, msrA mutant (MSΔmsrA); MS20, msrB mutant (MSΔmsrB); MS441, msrA/B double mutant (MSΔmsrA/B). Note the absence of MsrA, MsrB and both MsrA and MsrB in MSΔmsrA,MSΔmsrB and MSΔmsrA/B strains, respectively. (B) Graph showing the growth of M. smegmatis strains in broth culture. MSWt, wild-type M. smegmatis; MS97, msrA mutant (MSΔmsrA); MS20, msrB mutant (MSΔmsrB); MS441, msrA/B double mutant (MSΔmsrA/B). Cultures were grown in 7H9-ADC-TW broth for forty hours and their growth was assessed at different time points.

We have earlier reported the disruption of msrA (MSMEG6477) gene in M. smegmatis through homologous recombination. A msrA/B double mutant (MSΔmsrA/B) was created by electroporating the plasmid p1MSMSRA2 in MSΔmsrB (MS20) and subsequent screening for sucrose-resistant white colonies. Transformant MS441 was identified by Southern analysis as a double mutant that had disruption in both msrA and msrB (data not shown). Immunoblot analysis showed that this strain lacked the expression of both MsrA and MsrB (Fig. 5A).

In vitro growth of msr mutant strains indicated that the growth of MSΔmsrA/B strain was slightly lower than MSΔmsrA,MSΔmsrB and M. smegmatis wild-type (MSWt) strains only during the exponential phase (Fig. 5B).

3.4. Intracellular survival of msr mutants

Since our goal was to determine the role of MsrB in the intracellular survival, we infected J774A.1 cell line with MSWt, MSΔmsrA, MSΔmsrB and MSΔmsrA/B strains of M. smegmatis and examined the survival of these strains within macrophages for 3 days. In the unactivated macrophages the survival of MSΔmsrB was considerably lower than that of MSWt 1 day post infection but higher than that of MSΔmsrA and MSΔmsrA/B strains (Fig. 6). This trend continued even 3 days post infection and the survival of MSΔmsrB was noticed to be significantly lower than MSWt. In contrast, MSΔmsrA and MSΔmsrA/B strains showed almost no viability 3 days post infection. In the IFN-γ-activated macrophages, MSWt and MSΔmsrB showed a moderate but more or less similar decline 1 day post infection, while MSΔmsrA and MSΔmsrA/B strains exhibited no survival at this time point. Although a further decline was noticed with MSWt and MSΔmsrB 3 days post infection, they still had some survival at this time point. These results indicate that MsrB protects only moderately against killing by macrophages, although MsrA plays a major role19 . Further, there seems to be no synergistic effect with regard to protection against macrophage killing by MsrA and MsrB, since MSΔmsrA/B mutant exhibited a phenotype very much similar to that of MSΔmsrA strain. In order to understand whether the observed moderate protection by MSΔmsrB strain was really due to lack of MsrB, we determined the survival of msrB merodiploid strain (MS19) in macrophages. This showed a phenotype (data not shown) similar to that of MSWt in both unactivated and activated macrophages, indicating that MsrB does play a role in the intracellular survival. Although it would be of interest to check the intracellular survival of complemented MSΔmsrA/B strain, the similarity of its phenotype to that of MSΔmsrA strain discouraged such an effort. We have earlier reported that complemented MSΔmsrA strain had a phenotype similar to that of MSWt19.

Fig. 6.

Fig. 6

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Survival of M. smegmatis strains in murine macrophage-like J774A.1 cells. Naive (unactivated) IFN-γ-activated macrophages were infected at an MOI of 1:1 for 4 h, washed, lysed at intervals and plated onto 7H10-ADC-TW plates for CFUs. Solid squares, wild-type (MSWt); open squares, msrA mutant (MS97; MSΔmsrA); solid circles, msrB mutant (MS20; MSΔmsrB); open circles msrA/B double mutant (MS441; MSΔmsrA/B). Error bars are not seen for some time points because of the small SE.

3.5. Sensitivity of msr mutant strains for in vitro oxidative stress

Absence of MsrA in bacteria has been reported to lead to hypersensitivity to peroxide, nitric oxide and hydroperoxide stress16–19,28–32. We have previously reported that MSΔmsrA strain of M. smegmatis was resistant to hydrogen peroxide, superoxide donors and nitric oxide donors but sensitive to hydroperoxides. To understand the role of MsrB in protection against oxidative stress, we determined the susceptibility of M. smegmatis mutant strains to different oxidants. MSΔmsrB strain showed no sensitivity to hydrogen peroxide, superoxide and nitric oxide stress as noticed for MSΔmsrA strain, hence the data are not shown here. Surprisingly, however, MSΔmsrB strain also did not exhibit any sensitivity to hydroperoxides (t-butyl hydroperoxide and cumene hydroperoxide) for which MSΔmsrA strain was highly susceptible (Fig. 7). MSΔmsrA/B strain, on the other hand, showed sensitivity similar to that of MSΔmsrA strain19, indicating that sensitivity to hydroperoxides in MSΔmsrA/B strain was primarily due to the absence of MsrA.

Fig. 7.

Fig. 7

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Survival of M. smegmatis after exposure to hydroperoxides. MSWt, wild-type M. smegmatis; MS20, msrB mutant (MSΔmsrB); MS441, msrA/B double mutant (MSΔmsrA/B). Aliquots (1 ml) of culture were exposed to 5 mM H2O2,5mM t-butyl hydroperoxide or 5 mM cumene hydroperoxide for 1 h at 37°C. Cultures were serially diluted and plated on 7H10-ADC-TW plates.

3.6. Accumulation of oxidized proteins in msr mutant strains

Since lack of Msr makes bacterial proteins more susceptible to oxidation at methionine residue18,33, we determined, qualitatively by oxyblot method, if oxidized proteins accumulate in M. smegmatis msr mutant strains. Oxidized protein profiles of exponentially growing mutant strains showed only negligible difference from that of oxidized protein profile of MSWt strain (data not shown). However, the intensities of oxidized proteins in msr mutant strains were distinctly different from that of MSWt during their stationary phase (Fig. 8). MSΔmsrA and MSΔmsrA/B displayed strongly oxidized polypeptides in the range of 36–79 kDa. Although MSΔmsrB strain displayed a profile similar to that of MSΔmsrA and MSΔmsrA/B strains, the intensity of the bands in this strain is relatively lower than for other msr mutants, indicating indirectly that MsrB plays only a limited role in protecting M. smegmatis against oxidative stress.

Fig. 8.

Fig. 8

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Oxyblot of M. smegmatis strains. MSWt, wild-type M. smegmatis; MS97, msrA mutant (MSΔmsrA); MS20, msrB mutant (MSΔmsrB); MS441, msrA/B double mutant (MSΔmsrA/B). 7 μg of protein extract was loaded in each lane. Protein extracts were treated with DNPH (dinitrophenyl hydrazine) and then with DTT (dithiothreitol) and mercaptoethanol to avoid further oxidation of proteins. Proteins separated on SDS-PAGE and transferred to nitrocellulose were detected with oxyblot protein oxidation detection kit (Chemicon).

4. Discussion

Msr is a new family of antioxidant-related enzymes for which the role in defense against oxidative stress has been elucidated in few species16–19,28–32. As mentioned previously, Msr can only detoxify Met residues that are already oxidized by ROI. Thus, Msr is distinct from conventional antioxidants like SOD, peroxidase and alkyl hydroperoxide reductase, which can directly detoxify the ROI. Nevertheless, the fact that absence of Msr makes prokaryotes and eukaryotes hypersensitive to ROI tends to indicate that the Met-O/Msr oxidoreduction mechanism is one of the major pathways to detoxify ROI in living cells.

In this study, we have shown that both MsrA and MsrB of M. smegmatis are localized only in the soluble fraction. This is contrary to the observations that were made in M. tuberculosis, N. gonnorrhoeae26 and H. pylori18. Whereas MsrA alone is reported to be present in both soluble and membrane fractions of M. tuberculosis, both MsrA and MsrB have been shown to be located in the membrane fractions of N. gonnorrhoeae26 and H. pylori18 as fused proteins. Whether or not the membrane-bound Msr in these species provides any additional advantages in combating ROI is not clear at present. However, it may be presumed that Msr in the soluble fraction can still be associated with the detoxification of ROI, since ROI from the environment can freely diffuse into cells as evident from in vitro studies19.

The observation that MsrB plays only a modest role in the survival of M. smegmatis within macrophages may indicate that MsrB is not very critical for the detoxification of ROI within macrophages. This is in sharp contrast to the reports that both MsrA and MsrB contribute to the in vivo survival of H. pylori18 and that Lactobacillus reuteri34 lacking MsrB is less efficient in survival inside the mouse gut. Although the reason for this discrepancy is not immediately apparent, one possibility could be the differences in their substrate specificity. Several studies5,6,9,12, including studies on M. tuberculosis20, have demonstrated that MsrA is very specific to the reduction of Met-S-O and MsrB is specific to the reduction of Met-R-O. It is, therefore, possible that oxidation of Met with R-epimers in M. smegmatis is more infrequent or insignificant as compared to oxidation of Met with S-epimers, thus requiring less or no MsrB to defend intracellular ROI. Since very limited studies describe the significance of msrB in the intracellular and in vivo survival, an extensive comparison seems to be difficult at this point. Recently, generation of a msrB mutant of M. tuberculosis has been reported but its role has been determined neither in intracellular nor in in vivo survival, although a msrA/B mutant has been shown to have a log less survival as compared to wild-type strain in a mouse model of infection20.

Our in vitro studies demonstrate that msrB mutant (MSΔmsrB) is insensitive to any of the oxidants tested and msrA/B double mutant (MSΔmsrA/B) is sensitive only to hydroperoxides, similar to msrA mutant (MSΔmsrA) strain. This is consistent with our hypothesis that M. smegmatis is relatively resistant to oxidation with Met-R epimer and possibly explains the better survival of this species in the intracellular environment. Similarly, a M. tuberculosis strain deficient in MsrB has also been reported to show no sensitivity to ROI. Interestingly, however, msrA mutant of M. tuberculosis has also been shown to be insensitive to oxidants-mediated ROI. Further, a msrA/B double mutant of M. tuberculosis has been shown to be sensitive to nitrite- and hypochlorite-mediated ROI. These results are not only in contrast to the observations that were made in msrA (MSΔmsrA) and msrA/B (MSΔmsrA/B) mutants of M. smegmatis but also with several other bacterial species deficient in MsrA. Existence of redundant pathways to defend against ROI in different species and diversity between species to adapt to different levels of ROI are considered the possible reason for these differences20. This notion draws support from the fact that MsrB of H. pylori18 and Campylobacter jejuni35 resist in vitro oxidative stress, although its shows little resistance in mycobacteria.

Moreover, our study reveals that msrB mutant (MSΔmsrB) strain accumulates relatively lower levels of oxidized protein carbonyls than msrA (MSΔmsrA) and msrA/B (MSΔmsrA/B) mutant strains during stationary phase with no added oxidants to the medium. This again reinforces our hypothesis that oxidation of Met with R-epimers is relatively lower. Further, this also provides additional evidence as to why MsrA is more critical in protecting M. smegmatis against intracellular ROI than MsrB. Protein carbonyl accumulation has also been noticed in the msr mutant strains of H. pylori that were exposed to H2O2 and GSNO (NO donor) and it has been demonstrated that proteins like catalase, GroEL chaperone and site-specific recombinase are the prominent proteins that undergo oxidation in the msr mutant strains of this species18,33. Further, accumulation of oxidized proteins has also been reported from eukaryotic cells deficient in MsrA36,37. It appears, therefore, that some of the oxidized proteins in msr mutant strains of M. smegmatis may be associated with important physiological functions, and identification of these proteins may provide additional insights into the role of Msr in protection against ROI.

Acknowledgements

This study was partly supported by San Antonio Area foundation.

Footnotes

Competing interests: No competing interests declared.

References

  • 1.Cosma CL, Sherman DR, Ramakrishnan L. The secret lives of the pathogenic mycobacteria. Annu Rev Microbiol. 2003;57:641–76. doi: 10.1146/annurev.micro.57.030502.091033. [DOI] [PubMed] [Google Scholar]
  • 2.Piddington DL, Fang FC, Laessig T, Cooper AM, Orme IM, Buchmeier NA. Cu,Zn superoxide dismutase of Mycobacterium tuberculosis contributes to survival in activated macrophages that are generating an oxidative burst. Infect Immun. 2001;69:4980–7. doi: 10.1128/IAI.69.8.4980-4987.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 3.Ng VH, Cox JS, Sousa AO, MacMicking JD, McKinney JD. Role of KatG catalase-peroxidase in mycobacterial pathogenesis: countering the phagocyte oxidative burst. Mol Microbiol. 2004;52:1291–302. doi: 10.1111/j.1365-2958.2004.04078.x. [DOI] [PubMed] [Google Scholar]
  • 4.Bryk R, Griffin P, Nathan C. Peroxynitrite reductase activity of bacterial peroxiredoxins. Nature. 2000;407:211–5. doi: 10.1038/35025109. [DOI] [PubMed] [Google Scholar]
  • 5.Weissbach H, Etienne F, Hoshi T, Heinemann SH, Lowther WT, Matthews B, et al. Peptide methionine sulfoxide reductase: structure, mechanism of action, and biological function. Arch Biochem Biophys. 2002;397:172–8. doi: 10.1006/abbi.2001.2664. [DOI] [PubMed] [Google Scholar]
  • 6.Weissbach H, Resnick L, Brot N. Methionine sulfoxide reductases: history and cellular role in protecting against oxidative damage. Biochim Biophys Acta. 2005;1703:203–12. doi: 10.1016/j.bbapap.2004.10.004. [DOI] [PubMed] [Google Scholar]
  • 7.Grimaud R, Ezraty B, Mitchell JK, Lafitte D, Briand C, Derrick PJ, et al. Repair of oxidized proteins. Identification of a new methionine sulfoxide reductase. J Biol Chem. 2001;276:48915–20. doi: 10.1074/jbc.M105509200. [DOI] [PubMed] [Google Scholar]
  • 8.Kumar RA, Koc A, Cerny RL, Gladyshev VN. Reaction mechanism, evolutionary analysis, and role of zinc in Drosophila methionine-R-sulfoxide reductase. J Biol Chem. 2002;277:37527–35. doi: 10.1074/jbc.M203496200. [DOI] [PubMed] [Google Scholar]
  • 9.Lowther WT, Weissbach H, Etienne F, Brot N, Matthews BW. The mirrored methionine sulfoxide reductases of Neisseria gonorrhoeae pilB. Nat Struct Biol. 2002;9:348–52. doi: 10.1038/nsb783. [DOI] [PubMed] [Google Scholar]
  • 10.Rodrigo MJ, Moskovitz J, Salamini F, Bartels D. Reverse genetic approaches in plants and yeast suggest a role for novel, evolutionarily conserved, selenoprotein-related genes in oxidative stress defense. Mol Genet Genomics. 2002;267:613–21. doi: 10.1007/s00438-002-0692-3. [DOI] [PubMed] [Google Scholar]
  • 11.Sasindran SJ, Saikolappan S, Dhandayuthapani S. Methionine sulfoxide reductases and virulence of bacterial pathogens. Future Microbiol. 2007;2:619–30. doi: 10.2217/17460913.2.6.619. [DOI] [PubMed] [Google Scholar]
  • 12.Boschi-Muller S, Olry A, Antoine M, Branlant G. The enzymology and biochemistry of methionine sulfoxide reductases. Biochim Biophys Acta. 2005;1703:231–8. doi: 10.1016/j.bbapap.2004.09.016. [DOI] [PubMed] [Google Scholar]
  • 13.Levine RL, Mosoni L, Berlett BS, Stadtman ER. Methionine residues as endogenous antioxidants in proteins. Proc Natl Acad Sci USA. 1996;93:15036–40. doi: 10.1073/pnas.93.26.15036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Ezraty B, Aussel L, Barras F. Methionine sulfoxide reductases in prokaryotes. Biochim Biophys Acta. 2005;1703:221–9. doi: 10.1016/j.bbapap.2004.08.017. [DOI] [PubMed] [Google Scholar]
  • 15.Moskovitz J. Methionine sulfoxide reductases: ubiquitous enzymes involved in antioxidant defense, protein regulation, and prevention of aging-associated diseases. Biochim Biophys Acta. 2005;1703:213–9. doi: 10.1016/j.bbapap.2004.09.003. [DOI] [PubMed] [Google Scholar]
  • 16.Hassouni ME, Chambost JP, Expert D, Van Gijsegem F, Barras F. The minimal gene set member msrA, encoding peptide methionine sulfoxide reductase, is a virulence determinant of the plant pathogen Erwinia chrysanthemi. Proc Natl Acad Sci USA. 1999;96:887–92. doi: 10.1073/pnas.96.3.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Dhandayuthapani S, Blaylock MW, Bebear CM, Rasmussen WG, Baseman JB. Peptide methionine sulfoxide reductase (MsrA) is a virulence determinant in Mycoplasma genitalium. J Bacteriol. 2001;183:5645–50. doi: 10.1128/JB.183.19.5645-5650.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Alamuri P, Maier RJ. Methionine sulphoxide reductase is an important antioxidant enzyme in the gastric pathogen Helicobacter pylori. Mol Microbiol. 2004;53:1397–406. doi: 10.1111/j.1365-2958.2004.04190.x. [DOI] [PubMed] [Google Scholar]
  • 19.Douglas T, Daniel DS, Parida BK, Jagannath C, Dhandayuthapani S. Methionine sulfoxide reductase A (MsrA) deficiency affects the survival of Mycobacterium smegmatis within macrophages. J Bacteriol. 2004;186:3590–8. doi: 10.1128/JB.186.11.3590-3598.2004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Lee WL, Gold B, Darby C, Brot N, Jiang X, de Carvalho LP, et al. Mycobacterium tuberculosis expresses methionine sulphoxide reductases A and B that protect from killing by nitrite and hypochlorite. Mol Microbiol. 2009;71:583–93. doi: 10.1111/j.1365-2958.2008.06548.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Ausubel F, Brent R, Kingston R, Moore D, Seidman J, Smith J, et al. Current Prtocols in Molecular Biology. Wiley; New York: 1989. [Google Scholar]
  • 22.Parish T, Stoker NG. Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology. 2000;146(Pt 8):1969–75. doi: 10.1099/00221287-146-8-1969. [DOI] [PubMed] [Google Scholar]
  • 23.Taylor AB, Benglis DM, Jr, Dhandayuthapani S, Hart PJ. Structure of Mycobacterium tuberculosis methionine sulfoxide reductase A in complex with protein-bound methionine. J Bacteriol. 2003;185:4119–26. doi: 10.1128/JB.185.14.4119-4126.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Dhandayuthapani S, Mudd M, Deretic V. Interactions of OxyR with the promoter region of the oxyR and ahpC genes from Mycobacterium leprae and Mycobacterium tuberculosis. J Bacteriol. 1997;179:2401–9. doi: 10.1128/jb.179.7.2401-2409.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Mueller-Ortiz SL, Wanger AR, Norris SJ. Mycobacterial protein HbhA binds human complement component C3. Infect Immun. 2001;69:7501–11. doi: 10.1128/IAI.69.12.7501-7511.2001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 26.Skaar EP, Tobiason DM, Quick J, Judd RC, Weissbach H, Etienne F, et al. The outer membrane localization of the Neisseria gonorrhoeae MsrA/B is involved in survival against reactive oxygen species. Proc Natl Acad Sci USA. 2002;99:10108–13. doi: 10.1073/pnas.152334799. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Stalford SA, Fascione MA, Sasindran SJ, Chatterjee D, Dhandayuthapani S, Turnbull WB. A natural carbohydrate substrate for Mycobacterium tuberculosis methionine sulfoxide reductase A. Chem Commun (Camb) 2009:110–2. doi: 10.1039/b817483k. [DOI] [PubMed] [Google Scholar]
  • 28.Moskovitz J, Rahman MA, Strassman J, Yancey SO, Kushner SR, Brot N, et al. Escherichia coli peptide methionine sulfoxide reductase gene: regulation of expression and role in protecting against oxidative damage. J Bacteriol. 1995;177:502–7. doi: 10.1128/jb.177.3.502-507.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Singh VK, Moskovitz J, Wilkinson BJ, Jayaswal RK. Molecular characterization of a chromosomal locus in Staphylococcus aureus that contributes to oxidative defence and is highly induced by the cell-wall-active antibiotic oxacillin. Microbiology. 2001;147:3037–45. doi: 10.1099/00221287-147-11-3037. [DOI] [PubMed] [Google Scholar]
  • 30.Singh VK, Moskovitz J. Multiple methionine sulfoxide reductase genes in Staphylococcus aureus: expression of activity and roles in tolerance of oxidative stress. Microbiology. 2003;149:2739–47. doi: 10.1099/mic.0.26442-0. [DOI] [PubMed] [Google Scholar]
  • 31.Vattanaviboon P, Seeanukun C, Whangsuk W, Utamapongchai S, Mongkolsuk S. Important role for methionine sulfoxide reductase in the oxidative stress response of Xanthomonas campestris pv. phaseoli. J Bacteriol. 2005;187:5831–6. doi: 10.1128/JB.187.16.5831-5836.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Vriesema AJ, Dankert J, Zaat SA. A shift from oral to blood pH is a stimulus for adaptive gene expression of Streptococcus gordonii CH1 and induces protection against oxidative stress and enhanced bacterial growth by expression of msrA. Infect Immun. 2000;68:1061–8. doi: 10.1128/iai.68.3.1061-1068.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Alamuri P, Maier RJ. Methionine sulfoxide reductase in Helicobacter pylori: interaction with methionine-rich proteins and stress-induced expression. J Bacteriol. 2006;188:5839–50. doi: 10.1128/JB.00430-06. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Walter J, Chagnaud P, Tannock GW, Loach DM, Dal Bello F, Jenkinson HF, et al. A high-molecular-mass surface protein (Lsp) and methionine sulfoxide reductase B (MsrB) contribute to the ecological performance of Lactobacillus reuteri in the murine gut. Appl Environ Microbiol. 2005;71:979–86. doi: 10.1128/AEM.71.2.979-986.2005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Atack JM, Kelly DJ. Contribution of the stereospecific methionine sulphoxide reductases MsrA and MsrB to oxidative and nitrosative stress resistance in the food-borne pathogen Campylobacter jejuni. Microbiology. 2008;154:2219–30. doi: 10.1099/mic.0.2008/019711-0. [DOI] [PubMed] [Google Scholar]
  • 36.Oien D, Moskovitz J. Protein-carbonyl accumulation in the non-replicative senescence of the methionine sulfoxide reductase A (msrA) knockout yeast strain. Amino Acids. 2007;32:603–6. doi: 10.1007/s00726-006-0448-1. [DOI] [PubMed] [Google Scholar]
  • 37.Moskovitz J. Prolonged selenium deficient diet in MsrA knockout mice causes enhanced oxidative modification to proteins and affects the levels of antioxidant enzymes in a tissue-specific manner. Free Radic Res. 2007;41:162–71. doi: 10.1080/10715760600978823. [DOI] [PubMed] [Google Scholar]

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