Protein disulfide isomerase (PDI) associates with NADPH oxidase and is required for phagocytosis of Leishmania chagasi promastigotes by macrophages

Abstract

PDI, a redox chaperone, is involved in host cell uptake of bacteria/viruses, phagosome formation, and vascular NADPH oxidase regulation. PDI involvement in phagocyte infection by parasites has been poorly explored. Here, we investigated the role of PDI in in vitro infection of J774 macrophages by amastigote and promastigote forms of the protozoan Leishmania chagasi and assessed whether PDI associates with the macrophage NADPH oxidase complex. Promastigote but not amastigote phagocytosis was inhibited significantly by macrophage incubation with thiol/PDI inhibitors DTNB, bacitracin, phenylarsine oxide, and neutralizing PDI antibody in a parasite redox-dependent way. Binding assays indicate that PDI preferentially mediates parasite internalization. Bref-A, an ER-Golgi-disrupting agent, prevented PDI concentration in an enriched macrophage membrane fraction and promoted a significant decrease in infection. Promastigote phagocytosis was increased further by macrophage overexpression of wild-type PDI and decreased upon transfection with an antisense PDI plasmid or PDI siRNA. At later stages of infection, PDI physically interacted with L. chagasi, as revealed by immunoprecipitation data. Promastigote uptake was inhibited consistently by macrophage preincubation with catalase. Additionally, loss- or gain-of-function experiments indicated that PMA-driven NADPH oxidase activation correlated directly with PDI expression levels. Close association between PDI and the p22phox NADPH oxidase subunit was shown by confocal colocalization and coimmunoprecipitation. These results provide evidence that PDI not only associates with phagocyte NADPH oxidase but also that PDI is crucial for efficient macrophage infection by L. chagasi.

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

“…However, pathways that adjust Nox activation to specific physiologic programs in cells are unclear. Our group has provided evidence that the endoplasmic reticulum (ER) redox chaperone protein disulfide isomerase (PDI) functionally regulates NADPH oxidase and associates with its subunits in distinct cell types (7)(8)(9)(10). PDI is the founding member of a large protein family belonging to the thioredoxin superfamily of dithiol-disulfide oxidoreductases, displaying thiol isomerase, oxidase, and reductase activities (11)(12)(13)(14).…”

“…Contrarily, induced PDI overexpression promotes preemptive spontaneous ROS generation, NADPH oxidase activity, and Nox1, but not Nox4 mRNA expression (8). In J-774 macrophages, PDI overexpression increases, whereas PDI silencing decreases Leishmania chagasi phagocytosis and NADPH oxidase activation (9). Moreover, we recently showed PDI requirement for activation of neutrophil NADPH oxidase, in both cell-free and whole cell systems (7).…”

Roles of Cellular Redox Factors in Pathogen and Toxin Entry in the Endocytic Pathways

“…This suggests that the oxidative stress induced by NADPH oxidase activation may have a favorable effect, instead of the expected anti-microbial effect, with regard to parasite infectivity. Recently, PDI has been shown to be involved in phagocytosis of Leishmania chagasi through regulation of NADPH oxidase, in which PDI was found to closely associate with the NADPH oxidase, and inhibitors of PDI (bacitracin, phenylarsine oxide, anti-PDI antibody) significantly blocked promastigote phagocytosis (40). These results correlate well with, and are supported by, the previous findings that proteomic study of macrophages showed that PDI is involved in the formation of the phagosomes during phagocytosis of some parasites, including Leishmania (66,81).…”

“…diphtheria toxin [29][30][31][32][33], cholera toxin [34], botulinum neurotoxins [35,36], anthrax toxin [37]) are apparently dependent on the redox states (either reduced or oxidized) of the specific disulfides of either the toxin molecules, or the host receptors. At the same time, protein disulfide isomerase (PDI) [38] and other redox factors, such as gamma-interferoninducible lysosomal thiol reductase (GILT) [39]) and NADPH oxidase [40][41][42], have been implicated in regulating the redox states of the disulfides. Similarly, PDI and others are also involved in the entry of numerous pathogenic bacteria (e.g.…”

“…Similarly, PDI and others are also involved in the entry of numerous pathogenic bacteria (e.g. Chlamydia [43,44], Listeria [39]), viruses (e. g. HIV [45] and SV40 (46,47]) and parasites (e. g. Leishmania [40]) through endocytosis. This review will present the current major findings on the roles of cellular redox factors in pathogen and toxin entry with an attempt to outline the strategies and mechanisms that microbial pathogens and toxins utilize to hijack the cellular redox factors within the endocytic pathways.…”