Staphylococcus aureus sortase A contributes to the Trojan horse mechanism of immune defense evasion with its intrinsic resistance to Cys184 oxidation

Abstract

Staphylococcus aureus is a Gram-positive bacterial pathogen that causes serious infections which have become increasingly difficult to treat due to antimicrobial resistance and natural virulence strategies. Bacterial sortase enzymes are important virulence factors and good targets for future antibiotic development. It has recently been shown that sortase enzymes are integral to bacterial survival of phagocytosis, an underappreciated, but vital, step in S. aureus pathogenesis. Of note, the reaction mechanism of sortases relies on a solvent-accessible cysteine for transpeptidation. Because of the common strategy of oxidative damage employed by professional phagocytes to kill pathogens, it is possible that this cysteine may be oxidized inside the phagosome, thereby inhibiting the enzyme. This study addresses this apparent paradox by assessing the ability of physiological reactive oxygen species, hydrogen peroxide and hypochlorite, to inhibit sortase A (SrtA) from S. aureus. Surprisingly, we found that SrtA is highly resistant to oxidative inhibition, both in vitro and in vivo. The mechanism of resistance to oxidative damage is likely mediated by maintaining a high reduction potential of the catalytic cysteine residue, Cys184. This is due to the unusual active site utilized by S. aureus SrtA, which employs a reverse protonation mechanism for transpeptidation, resulting in a high pK(a) as well as reduction potential for Cys184. The results of this study suggest that S. aureus SrtA is able to withstand the extreme conditions encountered in the phagosome and maintain function, contributing to survival of phagocytotic killing.

Figure 1

Time-dependent inhibition kinetics of S. aureus SrtA. ν_i/ν₀ vs time graphs were fit to obtain k_obs values and k_obs vs [ROS] graphs were fit to obtain K₁ and k_inact. H₂O₂ concentrations were 1 mM, 10 mM, 25 mM, 50 mM, 75 mM, and 100 mM. NaOCl concentrations were 500 μM, 1 mM, 2.5 mM, 5 mM, 7.5 mM, and 10 mM. (A) H₂O₂ kinetic data, (B) H₂O₂ k_obs fit, (C) NaOCl kinetic data, (D) NaOCl k_obs fit.

Figure 2

Active site residues (His142, Cys208, and Arg216) of Streptococcus pyogenes SrtA crystal structure with Cys208 oxidized to a sulfenic acid (PDB ID 3FN6). 82 × 43mm (450 × 450 DPI)

Figure 3

LC-MS/MS analysis of S. aureus SrtA_ΔN24 after oxidation by H₂O₂ and NaOCl. The percent of Cys184 oxidized to the sulfonic acid form was calculated based on the sum of all peak intensities containing free Cys 184 or triply-oxidized Cys184 for each of three treatment conditions (water, 1 mM H₂O₂, or 1 mM NaOCl for 2 h). Water showed no oxidation, H₂O₂ only 6 %, and NaOCl 63 % oxidation.

Figure 4

Analysis of Protein A anchoring in S. aureus strain Newman grown in TSB media at pH 7 under different conditions. Rows: (A) No ROS, (B) 1 mM H₂O₂, (C) 1 mM NaOCl, (D) S. aureus ΔSrtA.

Figure 5

Cysteine reduction potential. (A) Representative cyclic voltammogram of L-cysteine (blue) and SrtA_ΔN24 (red) at pH 5 used to determine reduction potential. (B) Pourbaix diagram showing pH-dependence of the measured reduction potential of L-cysteine (blue ▲) and SrtA_ΔN24 (red ●) in 50 mM phosphate buffered solution at 4 °C over pH 3-11.

Figure 6

Model of SrtA oxidation during phagocytosis of S. aureus. Oxidation of SrtA (to sulfenic, sulfinic, or sulfonic acid) inside phagocytes results in inhibition of SrtA and death of S. aureus. To combat this, SrtA is resistant to oxidation, allowing S. aureus to survive within the phagocyte. 82×34mm (600 × 600 DPI)

Scheme 1. Oxidation States of a Cysteine Residue

Double arrows indicate physiogically reversible steps. Sulfinic acid formation has only been found to be reversible in a few specific cases when catalyzed by the enzyme sulfiredoxin(49). Disulfide formation is employed by enzymes like AhpC and sulfenyl amide formation by enzymes like PTP1B to protect the enzymes from irreversible oxidation. 82×77mm (600 × 600 DPI)