Reactive species and pathogen antioxidant networks during phagocytosis

Abstract

The generation of phagosomal cytotoxic reactive species (i.e., free radicals and oxidants) by activated macrophages and neutrophils is a crucial process for the control of intracellular pathogens. The chemical nature of these species, the reactions they are involved in, and the subsequent effects are multifaceted and depend on several host- and pathogen-derived factors that influence their production rates and catabolism inside the phagosome. Pathogens rely on an intricate and synergistic antioxidant armamentarium that ensures their own survival by detoxifying reactive species. In this review, we discuss the generation, kinetics, and toxicity of reactive species generated in phagocytes, with a focus on the response of macrophages to internalized pathogens and concentrating on Mycobacterium tuberculosis and Trypanosoma cruzi as examples of bacterial and parasitic infection, respectively. The ability of pathogens to deal with host-derived reactive species largely depends on the competence of their antioxidant networks at the onset of invasion, which in turn can tilt the balance toward pathogen survival, proliferation, and virulence over redox-dependent control of infection.

Figure 1.

Reactive species at the phagosome. (A) In macrophages (phagosome pH ≤ 5), NADPH oxidase activation (blue sphere) leads to O₂^•− generation toward the phagocytic vacuole (1). O₂^•− dismutates to H₂O₂ (2) and/or protonates to generate HO^•₂ radical (3). O₂^•− may reach the pathogen by means of anion channels, whereas both H₂O₂ and HO₂^• can diffuse across membranes. Immune-stimulated macrophages expressing iNOS (red sphere) produce ^•NO (4), which diffuses to the phagosome while reacting fast with O₂^•− (5) to yield peroxynitrite anion (ONOO⁻). ONOO⁻ can protonate to peroxynitrous acid (ONOOH); it also permeates the parasite and reacts with CO₂ (6) to yield ^•NO₂ and CO₃^•− radicals (inset). Both ONOO⁻ and ONOOH (7) promote the oxidation and nitration of membrane lipids and proteins (8). (B) In neutrophils (phagosome pH ≥ 7.5), MPO-derived HOCl is the dominant oxidant generated in this phagocyte (3) and promotes oxidation and chlorination reactions (9). Although the de novo production of ^•NO (4) by human neutrophils has rarely been documented, ^•NO may arise from exogenous sources existing in inflammatory foci and permeate the neutrophil plasma membrane. The other numbers in B denote the same processes described above for A.

Figure 2.

Pathogen antioxidant networks: The examples of Mtb and T. cruzi. (A) Mtb. NADPH oxidase-derived (O₂^•−) and iNOS-derived (^•NO) radicals can react with specific enzymes (Fe- or Cu-containing SODs to form H₂O₂; trHbN to form nitrate) or recombine to form peroxynitrite. H₂O₂, peroxynitritous acid (ONOOH), and organic hydroperoxides (ROOH) are reduced particularly by KatG and peroxiredoxins (Prx). Most Mtb Prx (AhpC, TPx, PrxQB) can use Trx as reducing substrate. AhpC can also be reduced by AhpD, an adaptor protein that links antioxidant with metabolic enzymes and NADH. NADPH—derived from the pentose phosphate pathway (PPP), isocitrate dehydrogenase (IDH), malic enzyme (ME), and H⁺-transhydrogenases (TH)—is the final electron donor for TrxR and MshR. TrxR reduces Trx B and C (collectively indicated herein as Trx’s). The one-Cys Prx AhpE is reduced by Mrx-1 either directly (data not shown) or through the formation of a mixed disulfide with MSH, which is reduced by Mrx-1 in a monothiolic mechanism that leads to mycothiolated Mrx-1 (Mrx-1-SM) and is resolved by MSH/MshR/NADPH. MsrA and B reduce S– and R–Met-SO, respectively, and accept electrons from the Trx/TrxR/NADPH system. (B) T. cruzi. Enzymatic and nonenzymatic redox-active molecules use reducing equivalents from NADPH—derived from the PPP, IDH, ME, and TH—are funneled through the T(SH)₂, GSH, ascorbate (ASC), and/or TXN-I/II redox systems. H₂O₂ is metabolized by APxCcP at the endoplasmic reticulum, mitochondria, and plasma membrane using ASC/Cyt c^II as the electron donors. Dehidroascorbate (DHA) is reduced by a direct reaction with T(SH)₂. Organic hydroperoxides (ROOH) are substrates for GPX-II that uses GSH. T(SH)₂ reduces oxidized GSH (GSSG), while TR reduces TS₂. Mitochondrial O₂^•− formation by the electron transport chain (mainly at complex III) is detoxified by Fe-SODA with H₂O₂ generation. ^•NO can reach the mitochondria and inhibit respiration at complex IV (CIV; with an enhanced O₂^•− generation). Fe-SODA outcompetes for O₂^•− inhibiting ONOO⁻ formation. Mitochondrial peroxiredoxin (MPX) decomposes H₂O₂ and/or ONOO⁻, probably using reduced TXN-II and T(SH)₂ as the reducing substrate (dashed arrows). Met-SO is repaired by the action of MsrB at the expense of TXN. In the cytosol, cytosolic peroxiredoxin (CPX) detoxifies ROOH. T(SH)₂ is synthesized from two molecules of GSH and one spermidine in a reaction catalyzed by the enzyme trypanothione synthetase (TS). Met-SO is repaired by the presence of MsrA and B in the different compartments.