Inducible Peroxidases Mediate Nitration of Anopheles Midgut Cells Undergoing Apoptosis in Response to Plasmodium Invasion

Abstract

Plasmodium berghei invasion of Anopheles stephensi midgut cells causes severe damage, induces expression of nitric-oxide synthase, and leads to apoptosis. The present study indicates that invasion results in tyrosine nitration, catalyzed as a two-step reaction in which nitric-oxide synthase induction is followed by increased peroxidase activity. Ookinete invasion induced localized expression of peroxidase enzymes, which catalyzed protein nitration in vitro in the presence of nitrite and H 2 O 2 . Histochemical stainings revealed that when a parasite migrates laterally and invades more than one cell, the pattern of induced peroxidase activity is similar to that observed for tyrosine nitration. In Anopheles gambiae, ookinete invasion elicited similar responses; it induced expression of 5 of the 16 peroxidase genes predicted by the genome sequence and decreased mRNA levels of one of them. One of these inducible peroxidases has a C-terminal oxidase domain homologous to the catalytic moiety of phagocyte NADPH oxidase and could provide high local levels of superoxide anion (O 2 . ), that when dismutated would generate the local increase in H 2 O 2 required for nitration. Chemically induced apoptosis of midgut cells also activated expression of four ookinete-induced peroxidase genes, suggesting their involvement in general apoptotic responses. The two-step nitration reaction provides a mechanism to precisely localize and circumscribe the toxic products generated by defense reactions involving nitration. The present study furthers our understanding of the biochemistry of midgut defense reactions to parasite invasion and how these may influence the efficiency of malaria transmission by anopheline mosquitoes.Anopheline mosquitoes are the natural vectors of human malaria worldwide. When a female mosquito takes a blood meal from a malaria-infected host, the ingested Plasmodium gametocytes complete their differentiation into mature gametes in the midgut lumen. Following fertilization, zygotes mature into motile ookinetes, which traverse the midgut epithelium and form oocysts in the space between the epithelial cells and the basal lamina. Oocysts grow, mature, and eventually rupture, releasing a large number of sporozoites into the hemolymph. The sporozoites invade the salivary glands and are injected into a new vertebrate host when an infected female takes a second blood meal.In Anopheles stephensi, midgut invasion of Plasmodium berghei ookinetes takes place around 24 h after blood feeding and induces the expression of nitric-oxide synthase (NOS) 1 as revealed by immunofluorescence (1) and increased NADPHdependent nitroblue tetrazolium reduction activity (2). NOS catalyzes the formation of nitric oxide (NO), a highly reactive and toxic molecule (3-5). NO is unstable and reacts readily with other molecules, generating multiple reactive nitrogen intermediates. Peroxynitrite is formed by a rapid reaction between NO and a superoxide anion and readily nitrates proteins in vitro (6, 7). peroxynitrite has also been proposed to be t...

A mosquito 2-Cys peroxiredoxin protects against nitrosative and oxidative stresses associated with malaria parasite infection

“…Values in excess of 75 μM are consistent with inflammatory levels of nitrite/nitrate reported in human serum under conditions of sepsis [66,67] and therefore indicate that the NO-mediated response of A. stephensi to Plasmodium infection is an inflammatory response. In addition to this extracellular accumulation of toxic RNOS, strong, localized tyrosine nitration is correlated with advanced stages of apoptosis in ookinete-invaded A. stephensi midgut cells [45], confirming that the NOmediated response to parasite infection is deleterious to the host. However, AsNOS induction is often uncoupled from tyrosine nitration in parasite-invaded cells [45].…”

“…In addition to this extracellular accumulation of toxic RNOS, strong, localized tyrosine nitration is correlated with advanced stages of apoptosis in ookinete-invaded A. stephensi midgut cells [45], confirming that the NOmediated response to parasite infection is deleterious to the host. However, AsNOS induction is often uncoupled from tyrosine nitration in parasite-invaded cells [45]. Given the protection afforded by AsPrx-4783 against nitrating species and other damaging RNOS, we propose that induction of AsPrx-4783 in the midgut epithelium of A. stephensi to levels as high as sevenfold relative to control insects is perhaps one of several mechanisms to limit self-induced damage (e.g., nitration of tyrosine) that is a by-product of the NO-mediated defense against parasite development.…”

“…PN can also oxidize sulfhydryls, leading to thiyl radical formation [43]. Major nitration targets of PN include phenols such as tyrosine residues in proteins; indeed, nitrotyrosine formation has been associated with a variety of inflammatory conditions [44] and has been observed in ookinete-invaded cells of the A. gambiae midgut [45].…”

Nitric oxide metabolites induced in Anopheles stephensi control malaria parasite infection

“…Ag-Duox encodes an N-terminal peroxidase domain and a C-terminal NADPH oxidase domain [39]. As such, the local induction of Ag-Duox could lead to high local levels of O 2 ·− as has been shown in An.…”

“…In addition to NOS, a putative dual function oxidase, Ag-Duox, is highly expressed in the A. gambiae midgut in response to ookinete invasion [39]. Ag-Duox encodes an N-terminal peroxidase domain and a C-terminal NADPH oxidase domain [39].…”