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, 188 (16), 5839-50

Methionine Sulfoxide Reductase in Helicobacter Pylori: Interaction With Methionine-Rich Proteins and Stress-Induced Expression

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Methionine Sulfoxide Reductase in Helicobacter Pylori: Interaction With Methionine-Rich Proteins and Stress-Induced Expression

Praveen Alamuri et al. J Bacteriol.

Abstract

The reductive repair of oxidized methionine residues performed by methionine sulfoxide reductase is important for the gastric pathogen Helicobacter pylori to maintain persistent stomach colonization. Methionine-containing proteins that are targeted for repair by Msr were identified from whole-cell extracts (after cells were exposed to O(2) stress) by using a coimmunoprecipitation approach. Proteins identified as Msr-interacting included catalase, GroEL, thioredoxin-1 (Trx1), and site-specific recombinase; with one exception (Trx1, the reductant for Msr) all these proteins have approximately twofold higher methionine (Met) content than other proteins. These Met-rich proteins were purified and were shown to individually form a cross-linked adduct with Msr. Catalase-specific activity in an msr strain was one-half that of the parent strain; this difference was only observed under oxidative stress conditions, and the activity was restored to nearly wild-type levels by adding Msr plus dithiothreitol to msr strain extracts. In agreement with the cross-linking study, pure Msr used Trx1 but not Trx2 as a reductant. Comparative structure modeling classified the H. pylori Msr in class II within the MsrB family, like the Neisseria enzymes. Pure H. pylori enzyme reduced only the R isomer of methyl p-tolyl-sulfoxide with an apparent K(m) of 4.1 mM for the substrate. Stress conditions (peroxide, peroxynitrite, and iron starvation) all caused approximately 3- to 3.5-fold transcriptional up-regulation of msr. Neither the O(2) level during growth nor the use of background regulatory mutants had a significant effect on msr transcription. Late log and stationary phase cultures had the highest Msr protein levels and specific activity.

Figures

FIG. 1.
FIG. 1.
(A) Sypro-Ruby-stained gel showing Msr-interacting proteins precipitated using CIP procedure. Lanes M1, M2, and M3 are elutions from the msr strain; lanes S1, S2, S3, and S4 are serial elutions of the proteins captured from SS1 (parent strain). The molecular masses (lane MW) of the standards used are 200, 116.3, 97.4, 66.3, 55.4, 36.5, 31.0, 21.5, 14.4, 6.0, 3.5, and 2.5 kDa. Arrows indicate different proteins identified using MALDI-TOF MS. TolB precursor and seryl t-RNA synthetase are indicated by the two arrows immediately to the right of lane S2. The clarity of the molecular mass marker had to be compromised for a better resolution of the eluted proteins. (B) Gel-based analysis of purity of potential Msr-interacting proteins. Catalase (KatA) and GroEL were purified from cell extracts of SS1. TrxR, Trx1, Msr, Trx2, and SSR were purified as recombinant proteins from E. coli. CE, cell extract. UN and IN denote protein collected from cells not induced and induced with IPTG, respectively. Lanes Q, Q-Sepharose fractions; lanes M, molecular mass markers. The molecular masses (in kDa) of the markers are as follows: 97.4, 66.2, 45.0, 31.0, 21.5, and 14.4. Arrows in each panel identify the protein of interest.
FIG. 1.
FIG. 1.
(A) Sypro-Ruby-stained gel showing Msr-interacting proteins precipitated using CIP procedure. Lanes M1, M2, and M3 are elutions from the msr strain; lanes S1, S2, S3, and S4 are serial elutions of the proteins captured from SS1 (parent strain). The molecular masses (lane MW) of the standards used are 200, 116.3, 97.4, 66.3, 55.4, 36.5, 31.0, 21.5, 14.4, 6.0, 3.5, and 2.5 kDa. Arrows indicate different proteins identified using MALDI-TOF MS. TolB precursor and seryl t-RNA synthetase are indicated by the two arrows immediately to the right of lane S2. The clarity of the molecular mass marker had to be compromised for a better resolution of the eluted proteins. (B) Gel-based analysis of purity of potential Msr-interacting proteins. Catalase (KatA) and GroEL were purified from cell extracts of SS1. TrxR, Trx1, Msr, Trx2, and SSR were purified as recombinant proteins from E. coli. CE, cell extract. UN and IN denote protein collected from cells not induced and induced with IPTG, respectively. Lanes Q, Q-Sepharose fractions; lanes M, molecular mass markers. The molecular masses (in kDa) of the markers are as follows: 97.4, 66.2, 45.0, 31.0, 21.5, and 14.4. Arrows in each panel identify the protein of interest.
FIG. 2.
FIG. 2.
(A) Immunoblot using anti-Msr antibody. Protein-protein interactions identified using Msr and its substrates (1:1) were studied using the noncleavable cross-linker, dimethyl suberimidate. Oxidized Msr (and oxidized lysozyme) were mixed with Msr (Msr), Trx1, and Trx2 on the left side of the gel. Native Msr (and oxidized lysozyme) were mixed with individual oxidized substrates in lanes labeled SSR, KatA, GrL (GroEL), or both KatA and GrL. Oxidized HypB and oxidized UreE were used as controls. Arrows at right indicate the complexes Msr-KatA-GroEL, Msr-KatA and Msr-GroEL, Msr-SSR, and Msr-Trx1. (B) Immunoblot using anti-KatA antibody. The same reactions used in panel A (pertinent to KatA) were resolved on a separate gel and immunostained with anti-KatA antibody. Arrows identify the following complexes: KatA-GroEL-Msr, KatA-Msr, KatA-GroEL, and KatA alone. The molecular sizes (in kDa) of prestained markers are given. (C) Immunoblot using anti-GroEL antibody. The same mixtures used in panel B were resolved on a separate gel and immunostained with antibody against GroEL. The cross-linked adducts formed in each case are identified by an arrow. The prestained marker set as used in panel B was used here. GrL, GroEL.
FIG. 2.
FIG. 2.
(A) Immunoblot using anti-Msr antibody. Protein-protein interactions identified using Msr and its substrates (1:1) were studied using the noncleavable cross-linker, dimethyl suberimidate. Oxidized Msr (and oxidized lysozyme) were mixed with Msr (Msr), Trx1, and Trx2 on the left side of the gel. Native Msr (and oxidized lysozyme) were mixed with individual oxidized substrates in lanes labeled SSR, KatA, GrL (GroEL), or both KatA and GrL. Oxidized HypB and oxidized UreE were used as controls. Arrows at right indicate the complexes Msr-KatA-GroEL, Msr-KatA and Msr-GroEL, Msr-SSR, and Msr-Trx1. (B) Immunoblot using anti-KatA antibody. The same reactions used in panel A (pertinent to KatA) were resolved on a separate gel and immunostained with anti-KatA antibody. Arrows identify the following complexes: KatA-GroEL-Msr, KatA-Msr, KatA-GroEL, and KatA alone. The molecular sizes (in kDa) of prestained markers are given. (C) Immunoblot using anti-GroEL antibody. The same mixtures used in panel B were resolved on a separate gel and immunostained with antibody against GroEL. The cross-linked adducts formed in each case are identified by an arrow. The prestained marker set as used in panel B was used here. GrL, GroEL.
FIG. 3.
FIG. 3.
(A) Relative catalase expression in SS1 and msr mutant. Cell extracts (5 μg) from the described conditions were run on a 12.5% SDS-PAGE gel, and immunoblotting was performed using anti-catalase antibody. The arrow indicates the immunostained catalase protein in all lanes. 4% O2, cells grown in 4% oxygen, 10% O2, cells grown in 4% oxygen and exposed for 3 h to 10% oxygen (see Materials and Methods); WT, SS1 strain; msr, msr mutant in SS1; M, molecular mass marker. The immunostained catalase (∼55 kDa) is identified by an arrow. (B) Catalase activities in SS1 and msr. Specific activities of catalase are determined from extracts of cells grown in 4% oxygen or grown in 4% oxygen and then exposed to 10% oxygen for 3 h. One unit of activity is equivalent to the number of micromoles of H2O2 decomposed/min/mg of protein. Gray bars indicate the SS1 parent strain, and black bars indicate the msr mutant. The mean ± SD from 12 individual samples taken from four separate experiments (three replicates each) is shown here. The mutant results are significantly less than the wild type when both strains are in 10% O2 (P < 0.01).
FIG. 4.
FIG. 4.
Homology based 3-D model of Msr in H. pylori. The amino acid sequence of H. pylori Msr was aligned with known Msr proteins from different bacteria using the Swiss-Prot protein database. MsrAB from Neisseria (N. gonorrhoeae and N. meningitidis) shares a high percentage homology with H. pylori Msr and was used as a model to understand the folding of this protein. Surface-exposed cysteines and proximal histidines are labeled.
FIG. 5.
FIG. 5.
In vitro Msr activity using R- and S-isomers of sulfoxide. The ability of Msr to reduce methyl p-tolyl R-sulfoxide and p-tolyl-S-sulfoxide was tested using purified reaction components (Msr, Trx1, and TrxR). Oxidation of NADPH was monitored at 340 nm as a measure of substrate reduction by Msr. V, nmoles of NADPH oxidized/min; [s] concentration (mM) of substrate used in this assay. Values are the means ± SD from four independent experiments, each performed in triplicate (total of 12 samples for each mean).
FIG. 6.
FIG. 6.
xylE activities to monitor msr expression in H. pylori. Whole cells collected at 6 h postexposure to stress conditions were assayed for xylE activity. Gray bars indicate genomic fusions (Pmsr-xylE) and black bars indicate fusions on the shuttle vector pHel3 (see Materials and Methods). Values are the means ± SD from 15 samples taken from five separate experiments (three replicates each) for each condition. One unit of xylE activity is equivalent to the number of micromoles of catechol oxidized/min/109 cells. The S-nitrosoglutathione (GSNO), peroxide, and iron-chelated conditions were all significantly greater (P < 0.05) for both the plasmid and the genomic expression than the non-stress-treated (4% O2) samples, based on a Student's t test. Basal xylE activity (under 4% O2 on the plasmid) is indicated by the dashed line.
FIG. 7.
FIG. 7.
Growth-phase-dependent expression of Msr. H. pylori strain SS1 was grown in Mueller-Hinton broth supplemented with 5% calf serum; 7% partial pressure oxygen was maintained in the atmosphere of the (sealed) bottles. Cells were collected from each time point, extracts were prepared identically for all time points, and cell protein from each sample was run on two separate 12.5% SDS-PAGE gels. Immunoblotting was performed using anti-Msr antibody and anti-UreB (as a control). The arrows identify immunostained UreB (∼66 kDa) and Msr (∼43 kDa) in the cell extracts (A). Membrane protein fractions were simultaneously collected from the cell extracts, and specific Msr activity was determined using MetSO or methyl p-tolyl sulfoxide as substrates. Values are the means ± SD from nine samples (from three separate experiments, each assay performed in triplicate) (B).Units of Msr activity are plotted against time of growth in hours. One unit of activity is equivalent to the number of nanomoles of NADPH oxidized/min/mg of membrane protein. Time samples at 56 and 72 h correspond to late log and stationary phases of growth, respectively. White bars indicate the assay using (equal mixture of R and S isomers) methyl p-tolyl sulfoxide, and black bars indicate (equal mixture of R and S isomers) MetSO as a substrate. The 56- and 72-h data are significantly greater than the other sampled time points at P < 0.05 (Student's t test).

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