Methionine Sulfoxide Reductase in Helicobacter pylori: Interaction with Methionine-Rich Proteins and Stress-Induced Expression

(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.

(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).

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.

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).

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 (P_msr-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.