Synergistic roles of Helicobacter pylori methionine sulfoxide reductase and GroEL in repairing oxidant-damaged catalase

Abstract

Hypochlorous acid (HOCl) produced via the enzyme myeloperoxidase is a major antibacterial oxidant produced by neutrophils, and Met residues are considered primary amino acid targets of HOCl damage via conversion to Met sulfoxide. Met sulfoxide can be repaired back to Met by methionine sulfoxide reductase (Msr). Catalase is an important antioxidant enzyme; we show it constitutes 4-5% of the total Helicobacter pylori protein levels. msr and katA strains were about 14- and 4-fold, respectively, more susceptible than the parent to killing by the neutrophil cell line HL-60 cells. Catalase activity of an msr strain was much more reduced by HOCl exposure than for the parental strain. Treatment of pure catalase with HOCl caused oxidation of specific MS-identified Met residues, as well as structural changes and activity loss depending on the oxidant dose. Treatment of catalase with HOCl at a level to limit structural perturbation (at a catalase/HOCl molar ratio of 1:60) resulted in oxidation of six identified Met residues. Msr repaired these residues in an in vitro reconstituted system, but no enzyme activity could be recovered. However, addition of GroEL to the Msr repair mixture significantly enhanced catalase activity recovery. Neutrophils produce large amounts of HOCl at inflammation sites, and bacterial catalase may be a prime target of the host inflammatory response; at high concentrations of HOCl (1:100), we observed loss of catalase secondary structure, oligomerization, and carbonylation. The same HOCl-sensitive Met residue oxidation targets in catalase were detected using chloramine-T as a milder oxidant.

FIGURE 1.

Survivability of SS1, msr, and katA derivative strains from SS1 to differentiated HL-60 cells. Cells were infected with bacteria at a multiplicity of infection of 1:50. At the times indicated, the HL-60 cells were lysed, and the suspensions were serially diluted and plated onto blood agar plates. Colonies were enumerated after incubation of the plates at 2% O₂ for 5–7 days. Data are presented as mean ± S.D. (n = 6). The two mutant strains were significantly less viable than SS1 at both 15 and 60 min post-exposure (p < 0.001), although the mutant strains differed from each other at p < 0.04.

FIGURE 2.

Catalase activity and expression in SS1/msr strains following HOCl treatment. A, parent strain SS1 or the msr derivative from SS1 was incubated with HOCl for 4 h at 2% O₂, and catalase activities were determined in crude lysates. Catalase activity of SS1 + PBS was considered as 100%. Data are presented as mean ± S.D. (n = 4). B, expression of catalase following 4 h of HOCl treatment. Lysates (10 μg of protein each) were subjected to SDS-PAGE followed by Western blotting using anti-catalase antibodies. The immunoreactive band is indicated by the arrow.

FIGURE 3.

A, oxidant destruction of H. pylori catalase activity in a dose-dependent fashion. 10 μm catalase was incubated with the indicated fold excess of oxidant for 15 (HOCl) or 30 min (ONOO⁻, ChT, H₂O₂, and nitrite), and excess oxidants were quenched by adding excess l-Met. Catalase activities were measured spectrophotometrically by dissipation of H₂O₂ and presented as % of nonoxidized samples. Data are presented as mean ± S.D. from four independent assays. B, role of Msr and GroEL in repairing HOCl-damaged catalase. HOCl-treated samples were repaired with Msr (1-fold molar excess relative to catalase) and GroEL/ES (2-fold molar excess to catalase) or with buffer only but in the presence of 400 μm NADPH, 5 μm trx, 100 nm trxR, 5 mm ATP, and 100 μm DTT at 37 °C for 1 h. Addition of GroEL/ES components or Msr components alone resulted in not more than 5% activity recovery. Data are presented as mean ± S.D. (n = 4).

FIGURE 4.

A, MS/MS spectrum of peptide 369–392 following trypsin digestion of previously 60× HOCl-treated catalase. Fragmentation pattern of peptide 369–392 is displayed. The designation +O on b- and y-ions represents addition of 16 Da to the predicted product ion of the unoxidized peptide, consistent with the addition of one oxygen atom to Met³⁷². B, identification of Met residues after oxidation and repair of catalase. 10 μm catalase was incubated with 60× HOCl for 15 min, and excess oxidant was quenched by adding excess l-Met. Following dialysis-based removal, oxidized samples were incubated with Msr in the presence of DTT. Samples were digested separately with trypsin or Asp-N, and Met residues were identified and quantified by LC-MS/MS.

FIGURE 5.

Far-UV CD spectra of catalase treated with various fold excesses of HOCl. A, catalase was incubated with the indicated molar fold excess of HOCl for 15 min, and oxidant was quenched by addition of excess l-Met. Following dialysis, samples were adjusted to 200 μg of protein/ml (see text) and scanned from 195 to 250 nm. B, correlation between HOCl concentrations and CD ellipticity values (millidegree) at 220 nm.

FIGURE 6.

HOCl treatment causes oligomerization of catalase. Gel filtration chromatography of catalase following HOCl (A) or ChT (B) treatments. After oxidation and quenching, catalase samples were subjected to gel filtration chromatography. Chromatograms were integrated and analyzed using Unicorn 5.01 software.

FIGURE 7.

Proposed model indicating roles of Msr and GroEL in repairing HOCl-damaged catalase. Host-generated oxidants such as HOCl oxidize Met residues in catalase that leads to unfolding. However, Msr converts Met-SO to Met residues, but GroEL is required for proper folding to aid activity recovery.