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• W2009911844 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 H2O2. 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 (O2), that when dismutated would generate the local increase in H2O2 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. 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 H2O2. 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 (O2), that when dismutated would generate the local increase in H2O2 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) 1The abbreviations used are: NOS, nitric-oxide synthase; NO, nitric oxide; DAB, 3,3′-diaminobenzidine; AT, 3-amino-1,2,4-triazole; TMB, 3,3′,5,5′-tetramethylbenzidine, PBS, phosphate-buffered saline; BSA, bovine serum albumin; AgSRPN10, AgSerpin10; JNK, c-Jun-N-terminal kinase.1The abbreviations used are: NOS, nitric-oxide synthase; NO, nitric oxide; DAB, 3,3′-diaminobenzidine; AT, 3-amino-1,2,4-triazole; TMB, 3,3′,5,5′-tetramethylbenzidine, PBS, phosphate-buffered saline; BSA, bovine serum albumin; AgSRPN10, AgSerpin10; JNK, c-Jun-N-terminal kinase. as revealed by immunofluorescence (1Han Y.S. Thompson J. Kafatos F.C. Barillas-Mury C. EMBO J. 2000; 19: 6030-6040Crossref PubMed Scopus (293) Google Scholar) and increased NADPH-dependent nitroblue tetrazolium reduction activity (2Luckhart S. Vodovotz Y. Cui L. Rosenberg R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5700-5705Crossref PubMed Scopus (343) Google Scholar). NOS catalyzes the formation of nitric oxide (NO), a highly reactive and toxic molecule (3Gorren A.C. Mayer B. Biochemistry (Mosc). 1998; 63: 734-743PubMed Google Scholar, 4Brüne B. von Knethen A. Sandau K.B. Eur. J. Pharmacol. 1998; 351: 261-272Crossref PubMed Scopus (385) Google Scholar, 5Kim P.K.M. Zamora R. Petrosko P. Billiar T.R. Int. Immunopharmacol. 2001; 1: 1421-1441Crossref PubMed Scopus (322) Google Scholar). 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 (6Daiber A. Herold S. Schöneich C. Namgaladze D. Peterson J.A. Ullrich V. Eur. J. Biochem. 2000; 267: 6729-6739PubMed Google Scholar, 7Jiao K. Mandapati S. Skipper P.L. Tannenbaum S.R. Wishnok J.S. Anal. Biochem. 2001; 293: 43-52Crossref PubMed Scopus (68) Google Scholar). peroxynitrite has also been proposed to be the major mediator of protein nitration in vivo (8Liu D. Ling X. Wen J. Liu J. J. Neurochem. 2000; 75: 2144-2154Crossref PubMed Scopus (131) Google Scholar, 9Moulian N. Truffault F. Gaudry-Talarmain Y.M. Serraf A. Berrih-Aknin S. Blood. 2001; 97: 3521-3530Crossref PubMed Scopus (70) Google Scholar). NO production plays an important role limiting ookinete infection in the mosquito midgut, as the administration of Nω-nitro-l-arginine methyl ester (l-NAME), a NOS inhibitor, results in a 2-fold increase in the number of developing oocysts (2Luckhart S. Vodovotz Y. Cui L. Rosenberg R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5700-5705Crossref PubMed Scopus (343) Google Scholar). Previous studies indicate that in anophelines P. berghei invasion causes irreversible damage leading to cell death (1Han Y.S. Thompson J. Kafatos F.C. Barillas-Mury C. EMBO J. 2000; 19: 6030-6040Crossref PubMed Scopus (293) Google Scholar, 10Vlachou D. Zimmermann T. Cantera R. Janse C.J. Waters A.P. Kafatos F.C. Cell. Microbiol. 2004; 6: 671-685Crossref PubMed Scopus (139) Google Scholar). Some of the observed changes include loss of microvilli, genome fragmentation, nuclear picnosis (1Han Y.S. Thompson J. Kafatos F.C. Barillas-Mury C. EMBO J. 2000; 19: 6030-6040Crossref PubMed Scopus (293) Google Scholar), and activation of caspases (10Vlachou D. Zimmermann T. Cantera R. Janse C.J. Waters A.P. Kafatos F.C. Cell. Microbiol. 2004; 6: 671-685Crossref PubMed Scopus (139) Google Scholar). The damage inflicted by parasite invasion is repaired by “budding off” the damaged cells into the midgut lumen through an actin ring-mediated restitution mechanism (1Han Y.S. Thompson J. Kafatos F.C. Barillas-Mury C. EMBO J. 2000; 19: 6030-6040Crossref PubMed Scopus (293) Google Scholar). Invasion of Plasmodium gallinaceum ookinetes also damages Aedes aegypti midgut cells and results in activation of caspases and cell death (11Zieler H. Dvorak J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11516-11521Crossref PubMed Scopus (116) Google Scholar). Based on these studies using the A. stephensi-P. berghei model system we proposed the “time bomb model” of ookinete midgut invasion, which states that cell invasion triggers a series of toxic reactions (a “bomb”) that leads to cell death and is also potentially toxic to the parasite (1Han Y.S. Thompson J. Kafatos F.C. Barillas-Mury C. EMBO J. 2000; 19: 6030-6040Crossref PubMed Scopus (293) Google Scholar, 12Han Y.S. Barillas-Mury C. Insect Biochem. Mol. Biol. 2002; 32: 1311-1316Crossref PubMed Scopus (61) Google Scholar). The model predicts that ookinete survival would depend on the parasite migrating out of the cell before the bomb detonates. In the present study we investigate the biochemistry of the reactions generating the toxic products mediating these defense responses. Our data indicate that in A. stephensi and A. gambiae, P. berghei ookinetes trigger tyrosine nitration as a two-step reaction in which NO generation by NOS is followed by local induction of peroxidase and probably also oxidase enzymes. Peroxidase induction appears to be the rate-limiting step to generate highly reactive nitrogen dioxide, which is predicted to mediate tyrosine nitration and to play a critical role in determining parasite survival and thus the vectorial capacity of the mosquito. Mosquitoes and Malaria Parasites—A. stephensi (NIH strain) and A. gambiae (G3 strain) were reared at 28 °C and 80% humidity on a 12-h light-dark cycle. Larvae were grown in tap water and fed on Whiskas™ cat food, whereas adults were fed ad libitum on 10% sucrose. P. berghei parasites were maintained by serial passage in 3–4-week-old female Balb/C mice and as frozen stocks. P. berghei Infections in Mosquitoes—Mosquito females were infected with P. berghei by feeding on anesthetized infected Balb/C mice 5 days postemergence. The infectivity of the mice was established by determining the parasitemia and by performing an exflagellation assay as described previously (13Billker O. Shaw M.K. Margos G. Sinden R.E. Parasitology. 1997; 115: 1-7Crossref PubMed Scopus (107) Google Scholar). In all the studies, mice having 3–4 exflagellations/field under ×40 objective were used to infect mosquitoes. Bloodfed infected and control mosquitoes were kept at 21 °C in a humidified environment, unless otherwise stated. Midgut Immunofluorescence Stainings—Mosquito midgut immunostainings were performed as described previously (1Han Y.S. Thompson J. Kafatos F.C. Barillas-Mury C. EMBO J. 2000; 19: 6030-6040Crossref PubMed Scopus (293) Google Scholar). Briefly, midguts from mosquitoes fed on healthy or P. berghei-infected mice were dissected, fixed for 1 min in 4% paraformaldehyde, and opened longitudinally in phosphate-buffered saline (PBS, pH 7.2) to remove the bolus contents. Clean, opened tissues were fixed for 1 h with 4% paraformaldehyde in PBS at room temperature and permeabilized with PBT solution (1% BSA, 0.1% Triton X-100 in PBS) for 2 h at room temperature. Midguts were incubated overnight with the primary antibodies (1:300 dilution in PBT) at 4 °C and 4 h at room temperature with Cy5-, Cy3- (Amersham Biosciences), or Alexa488-conjugated secondary antibodies (1:500 dilution in PBT). ToPro3 (Molecular Probes) was used to visualize DNA by confocal microscopy. The tissues were washed and mounted in Vectashield™ (Vector Laboratories, Inc.) containing 4′,6-diamidino-2-phenylindole to counter stain the nuclei. Immunostainings were analyzed by fluorescence microscopy. The final images were obtained and analyzed using confocal microscopy with a Fluoview system and software (Olympus) or regular light and fluorescence microscopy (Olympus) with a color digital camera. The following commercially available antibodies were used: Universal anti-NOS rabbit polyclonal antibody (Affinity Bioreagents, Inc., catalog no. PA1-039) and mouse anti-nitrotyrosine monoclonal antibody (Calbiochem, catalog no. 487923). Anti-Pbs21 monoclonal antibodies were kindly provided by Dr. Robert Sinden, and anti-AgSRPN10 rabbit antiserum was provided by Dr. Alberto Danielli. 3,3′-Diaminobenzidine (DAB) Activity of the Midgut Tissue—Control or infected blood-fed midguts were dissected 24 h postfeeding, fixed for 1 min in paraformaldehyde, and opened longitudinally to remove the blood meal. For direct DAB stainings, midguts were fixed in 0.5% glutaraldehyde for 10 min at room temperature, washed, and developed for DAB activity. Samples were incubated at room temperature with 2.5 mm DAB (Sigma) and 1 mm H2O2 (Sigma) in PBS (pH 6.5) and continuously observed under the microscope. For experiments involving dual DAB and immunofluorescence staining, midguts were fixed in 4% paraformaldehyde in PBS for 1 h at room temperature after removal of the blood meal and stained with anti-Pbs21 antibody as described above under “Midgut Immunofluorescence Staining.” The DAB reaction was performed as the last step before mounting the sample. In some experiments, midguts were pre-incubated for 10 min with 10 mg/ml sodium azide (NaN3) or 10 mg/ml 3-amino-1,2,4-triazole (AT) in PBS (pH 6.5) before measuring DAB activity. 3,3′,5,5′-Tetramethylbenzidine (TMB) Peroxidase Assay—Peroxidase assays using TMB as a substrate were performed following the manufacturer's instructions (Kirkegaard & Perry Laboratories, Inc.). Briefly, five uninfected or infected blood-fed midguts were fixed for 1 h at room temperature in PBS containing 4% paraformaldehyde and 1% glutaraldehyde, washed, transferred to 100 μl of a TMB/H2O2 solution, and triturated. After a 10-min incubation at 37 °C in the dark, the midgut tissue was removed by centrifugation and the reaction stopped by adding 100 μlof3 n HCl. The relative concentration of the end products was determined based on the absorbance at 450 nm in an Emax microplate reader (Molecular Devices). To evaluate the effect of specific inhibitors, triturated midguts were pre-incubated for 5 min at room temperature with different concentrations (1, 2, 5, and 10 mg/ml) of NaN3 or AT before adding the TMB solution. Effect of Catalase on Peroxidase Activity—The activity of horseradish peroxidase (0.3 units/ml) (Invitrogen) after the addition of increasing amounts (10, 20, and 40 units/ml) of bovine liver catalase (EC 1.11.1.6, Sigma, catalog no. C3155) was determined using the TMB assay as described above, incubating the reactions for 5 min at 37 °C in the dark. To assess the effect of AT on the inhibitory effect of catalase, 0.3 units/ml peroxidase and 10 units/ml catalase were pre-incubated for 5 min at room temperature with increasing amounts (1, 2, and 5 mg/ml) of AT before performing the TMB assay. In Vitro Protein Nitration Assay—The ability of midgut homogenates to mediate protein nitration in vitro was evaluated by incubating blood-fed control or infected triturated midguts 24 h postfeeding (after removal of the blood bolus and fixation with 4% paraformaldehyde and 1% glutaraldehyde solution for 1 h at room temperature) with 100 μg of BSA in the presence of 1 mm H2O2 and1mm sodium nitrite (NaNO2) for 30 min at 37 °C. Midgut tissue was removed by centrifugation, and 3 μg of BSA was subjected to 10% SDS-PAGE and electroblotted to a polyvinylidene difluoride membrane. The membrane was treated with 1 mm levamisole solution to inhibit any internal phosphatase activity and blocked with 5% milk protein in Tris-buffered saline buffer (150 mm NaCl, 10 mm Tris-HCl, pH 7.6). The presence of nitrotyrosine in BSA was established by Western blot analysis, using an anti-nitrotyrosine mouse primary antibody (Calbiochem, catalog no. 487923) at a 1:3,000 dilution and a secondary alkaline phosphatase-conjugated antibody (Calbiochem, catalog no. 401212) at a 1:10,000 dilution. The membrane was developed for the enzymatic reaction following the manufacturer's instructions. In parallel, 0.3 unit/ml commercial horseradish peroxidase (Invitrogen) was used as a positive control. The participation of peroxidase activity from midgut homogenates or commercial sources in the catalysis of tyrosine nitration was tested by pre-incubating the sample with 5 mg/ml sodium azide (peroxidase inhibitor) before the reaction was carried out. Induction of Cell Death by Actimomycin D—Female mosquitoes were fed on 10% BSA with 5 mg/ml sodium bicarbonate in the presence or absence of 10 μg/ml actimomycin D, using a Hemotek artificial feeder (Discovery Workshop). Midguts were dissected 8 h postfeeding, opened longitudinally to remove the bolus content, and fixed in 0.5% glutaraldehyde solution at room temperature for 10 min. DAB staining to detect peroxidase activity was carried out in the presence of 10 mg/ml AT (catalase inhibitor) as described above. Reverse Transcription-PCR Analysis—Poly(A) mRNA was isolated from a group of 20 A. gambiae mosquito midguts 28 h after feeding using Oligotex-dT beads (Qiagen), following the manufacturer's instructions. First strand cDNA was synthesized by using random hexamers and Superscript II (Invitrogen). For the expression studies, PCRs were performed by using 20 pmol of each primer in 50-μl reactions and AmpliTaq (PerkinElmer Life Sciences) with standard buffer conditions (1.5 mm MgCl2). DNA was denatured initially for 3 min at 94 °C followed by 24 cycles of amplification (1 min denaturation at 94 °C, 1 min at the annealing temperature of the specific primer pair, 1 min extension at 72 °C) and a final 10-min extension at 72 °C. For information regarding the protein identification number, primer pair sequence, annealing temperature, and product size for each peroxidase reverse transcription-PCR product see the online supplemental material. Amplification of the ribosomal protein gene S7 (14Salazar C.E. Mills-Hamm D. Kumar V. Collins F.H. Nucleic Acids Res. 1993; 21: 4147Crossref PubMed Scopus (77) Google Scholar) using primers 5′-GGCGATCATCATCTACGTGC-3′ and 5′-GTAGCTGCTGCAAACTTCGG-3′ (461 bp) provided the internal control for the amount of cDNA template used in the PCR reactions. The PCR products were analyzed by agarose gel electrophoresis and photographed. Ookinete-invaded Cells Undergo Protein Nitration—To investigate the mechanism of protein nitration in ookinete-invaded cells, control and infected A. stephensi midguts were double stained for NOS and nitrotyrosine 24 h postfeeding. In non-infected midguts, a low level of NOS expression was observed in the cytoplasm of healthy epithelial cells, but neither cells protruding into the lumen nor nitrotyrosine staining could be detected (data not shown). In contrast, cells invaded by P. berghei ookinetes protruded into the lumen and exhibited high levels of NOS protein expression (Fig. 1, A–D) as described previously (1Han Y.S. Thompson J. Kafatos F.C. Barillas-Mury C. EMBO J. 2000; 19: 6030-6040Crossref PubMed Scopus (293) Google Scholar). These two parameters were used as markers of parasite invasion. To our surprise, only some of the invaded cells protruding and expressing high NOS levels had also undergone extensive tyrosine nitration (Fig. 1, A and B). One possible explanation is a time delay in the activation of other components critical for nitration. To begin to address a temporal difference we took advantage of the fact that single ookinetes often migrate laterally, invading two or more adjacent epithelial cells, as revealed by Pbs21 trails left behind during migration (Pbs21 is a glycosylphosphatidylinositol-anchored surface protein continuously released by migrating P. berghei ookinetes), cell protrusion, loss of microvilli, and NOS induction (1Han Y.S. Thompson J. Kafatos F.C. Barillas-Mury C. EMBO J. 2000; 19: 6030-6040Crossref PubMed Scopus (293) Google Scholar). Sequential lateral invasions of P. gallinaceum ookinetes in A. aegypti, and of P. berghei in A. gambiae and A. stephensi midgut cells, have been observed directly in vivo by video microscopy (11Zieler H. Dvorak J.A. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 11516-11521Crossref PubMed Scopus (116) Google Scholar) and real-time confocal microscopy (10Vlachou D. Zimmermann T. Cantera R. Janse C.J. Waters A.P. Kafatos F.C. Cell. Microbiol. 2004; 6: 671-685Crossref PubMed Scopus (139) Google Scholar), respectively. Cells invaded sequentially provide information regarding the relative timing of the cellular responses to the parasite, as more time has elapsed since the invasion of the first than the second, the second than the third cell, and so forth. The location of the ookinete was determined by light microscopy; it is shown in the Fig. 1 as an asterisk and indicates the last (and thus the most recently) invaded cell (Fig. 1, B and C and F and G). We consistently found that, at the time when all cells invaded by the same parasite have already protruded to the lumen and increased NOS expression, tyrosine nitration appeared with a time lag and was often confined to the first cell invaded by the parasite (Fig. 1, B and C). Cells in advanced stages of degeneration, in the process of “budding off” from the midgut epithelium, are heavily nitrated (Fig. 1D). These results indicate that induction of NOS expression is necessary but not sufficient to mediate tyrosine nitration. Occasionally, highly nitrated degenerate cells are negative for NOS staining (Fig. 1C). This could be due to either a decrease in NOS expression or to chemical damage of the epitopes recognized by the anti-NOS antibody. AgSerpin10 (AgSRPN10), a serine protease inhibitor, is highly induced in A. gambiae midguts in response to parasite invasion (15Danielli A. Kafatos F.C. Loukeris T.G. J. Biol. Chem. 2003; 278: 4184-4193Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). AgSRPN10 undergoes dramatic changes in subcellular localization and protein expression in a sequential manner (16Danielli A. Barillas-Mury A. Kumar S. Kafatos F.C. Loukeris T.G. Cell. Microbiol. 2004; (doi:10.1111/j. 1462-5822.2004.00445.x)Google Scholar), and thus, it also provides some information regarding the timing of tyrosine nitration. Healthy uninfected cells express low levels of AgSRPN10 predominantly in the nucleus and are always negative for tyrosine nitration (16Danielli A. Barillas-Mury A. Kumar S. Kafatos F.C. Loukeris T.G. Cell. Microbiol. 2004; (doi:10.1111/j. 1462-5822.2004.00445.x)Google Scholar) (Fig. 1E). Parasite invasion immediately triggers translocation of the protein from the nucleus to the cytoplasm, which is followed by the induction of high expression levels (16Danielli A. Barillas-Mury A. Kumar S. Kafatos F.C. Loukeris T.G. Cell. Microbiol. 2004; (doi:10.1111/j. 1462-5822.2004.00445.x)Google Scholar). Tyrosine nitration is first detected when the cytoplasmic levels of AgSRPN10 begin to increase (Fig. 1E) and becomes stronger as AgSRPN10 expression further increases (Fig. 1, F and G). All cells positive for nitrotyrosine staining express high AgSRPN10 levels; however, cells with high AgSRPN10 expression are not always nitrated (Fig. 1, F and G). These data indicate that protein nitration takes place during the later stages of the cell death process triggered by parasite invasion. Ookinete Invasion Induces a Glutaraldehyde-resistant Peroxidase Activity—The classic model of peroxynitrite-mediated tyrosine nitration in vertebrates has been challenged based on kinetic studies in murine RAW 264.7 macrophage cells (17Pfeiffer S. Lass A. Schmidt K. Mayer B. J. Biol. Chem. 2001; 276: 34051-34058Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Both NO and superoxide anion levels were found to increase following immune stimulation with interferon-γ/lipopolysaccharide or interferon-γ/zymosan A, but they rapidly decreased to base-line levels several hours before tyrosine nitration could be detected. NO formation resulted in nitrite accumulation, which was proposed to serve as a substrate for a myeloperoxidase (MPO)-mediated tyrosine nitration reaction (17Pfeiffer S. Lass A. Schmidt K. Mayer B. J. Biol. Chem. 2001; 276: 34051-34058Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Experiments using multiple distinct models of acute inflammation with eosinophil peroxidase (EPO)- and MPO knock-out mice indicate that leukocyte peroxidases participate in nitrotyrosine formation in vivo (18Brennan M.L. Wu W. Fu X. Shen Z. Song W. Frost H. Vadseth C. Narine L. Lenkiewicz E. Borchers M.T. Lusis A.J. Lee J.J. Lee N.A. Abu-Soud H.M. Ischiropoulos H. Hazen S.L. J. Biol. Chem. 2002; 277: 17415-17427Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). In some models, MPO and EPO played a dominant role, accounting for the majority of nitrotyrosine formed. However, in other leukocyte-rich acute inflammatory models, neither MPO nor EPO contributed to nitrotyrosine formation, implying the existence of alternative nitration pathways (18Brennan M.L. Wu W. Fu X. Shen Z. Song W. Frost H. Vadseth C. Narine L. Lenkiewicz E. Borchers M.T. Lusis A.J. Lee J.J. Lee N.A. Abu-Soud H.M. Ischiropoulos H. Hazen S.L. J. Biol. Chem. 2002; 277: 17415-17427Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). Based on the vertebrate data we decided to test the hypothesis that protein nitration of invaded midgut cells was mediated by ookinete-induced peroxidase(s). Midguts of females fed on healthy (Ctl) or malaria-infected (Inf) mice were fixed briefly with glutaraldehyde and assayed for peroxidase activity using DAB and hydrogen peroxide as substrates (Fig. 2A). Within a few minutes of incubation some of the malaria-infected cells protruding into the lumen stained very strongly with DAB (Fig. 2A, right panel), whereas no staining was detected in control samples incubated for the same amount of time (Fig. 2A, left panel). The cells positive for DAB staining are in close association with invading ookinetes. Furthermore, when two adjacent cells are invaded by the same parasite, the peroxidase activity is usually much higher in the cell that was invaded first (Fig. 2, B and C), in a pattern very similar to that described above for tyrosine nitration (Fig. 1, B and C). When P. berghei-infected females were kept at 28 °C, a non-permissive temperature for ookinete development, neither cells protruding into the midgut lumen nor DAB staining was observed (data not shown), implying that these two events are triggered by ookinete invasion. As both catalase and peroxidase activities can give rise to a positive DAB reaction (19Angermuller S. Fahimi H.D. Histochemistry. 1981; 71: 33-44Crossref PubMed Scopus (120) Google Scholar), the relative contribution of these two enzymes was evaluated by performing stainings in the presence of specific inhibitors. The addition of AT, a catalase inhibitor, had no effect, whereas sodium azide, a peroxidase inhibitor, completely abolished DAB staining (Fig. 2C), indicating that the DAB reaction was due to an inducible peroxidase. To confirm the induction of peroxidase activity in response to malaria infection, the activity was also determined in midgut homogenates by performing a colorimetric assay using TMB, a peroxidase-specific chromophore, and H2O2 as substrates. Parasite infection resulted in a marked increase in peroxidase activity when homogenates from midguts that had been fixed with a mixture of glutaraldehyde and paraformaldehyde were used in the assay (Fig. 2D). However, no difference in total peroxidase activity could be observed between control and infected samples when unfixed tissues or tissues fixed only with paraformaldehyde were used (data not shown). This observation suggests that parasite-induced peroxidase(s) is more resistant to fixation than other peroxidases constitutively expressed in control samples. For this reason, all activity assays presented here were performed in midgut tissues fixed previously with a mixture of glutaraldehyde and paraformaldehyde (see “Experimental Procedures” for details). As expected, sodium azide had a strong inhibitory effect on this inducible peroxidase activity in contrast to the catalase inhibitor AT, which had the opposite effect, slightly increasing peroxidase activity (Fig. 2E). This activity enhancement is probably because of a competition between catalase and peroxidases present in the midgut homogenate for hydrogen peroxide, a common substrate. We confirmed that the addition of commercial catalase to a commercial peroxidase did have an inhibitory effect (Fig. 2F) that could be alleviated by inhibiting catalase activity with AT (Fig. 2G). The Ookinete-induced Peroxidase Activity Can Mediate Protein Nitration—An in vitro nitration assay was performed to determine whether the ookinete-induced peroxidase activity could mediate protein nitration. Midgut homogenates from infected or non-infected mosquitoes were incubated with BSA in the presence of H2O2 and sodium nitrite (NaNO2). Following incubation, BSA was subjected to SDS-PAGE and electroblotted, and nitrotyrosine was detected by Western blot analysis using the same anti-nitrotyrosine antibody used for immunofluorescence staining. The BSA sample incubated with infected midgut homogenate had a higher level of nitrotyrosine staining relative to the control uninfected samples (Fig. 3). Tyrosine nitration required all components to be present, a source of peroxidase activity, sodium nitrite, and hydrogen peroxide; the removal of any of them completely inhibited the reaction. As expected, the addition of sodium azide inhibited nitration mediated either by midgut homogenates or by a commercial peroxidase used as a positive control for the reaction (Fig. 3). Ookinete-induced Peroxidase Activity in A. gambiae Midguts Correlates with Transcriptional Activation of Several Peroxidase Genes—The invasion of A. gambiae midgut cells by P. berghei also induced cell protrusion, localized peroxidase activity (Fig. 4A), and" @default.
• W2009911844 created "2016-06-24" @default.
• W2009911844 creator A5024718203 @default.
• W2009911844 creator A5033723381 @default.
• W2009911844 creator A5050790088 @default.
• W2009911844 creator A5081752921 @default.
• W2009911844 date "2004-12-01" @default.
• W2009911844 modified "2023-09-26" @default.
• W2009911844 title "Inducible Peroxidases Mediate Nitration of Anopheles Midgut Cells Undergoing Apoptosis in Response to Plasmodium Invasion" @default.
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