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Sustained activation of Akt elicits mitochondrial dysfunction to block Plasmodium falciparum infection in the mosquito host.

Luckhart S, Giulivi C, Drexler AL, Antonova-Koch Y, Sakaguchi D, Napoli E, Wong S, Price MS, Eigenheer R, Phinney BS, Pakpour N, Pietri JE, Cheung K, Georgis M, Riehle M - PLoS Pathog. (2013)

Bottom Line: Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios.Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan.Further, quality control of mitochondrial function in the midgut is necessary for the maintenance of midgut health as reflected in energy homeostasis and tissue repair and renewal.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, Davis, California, United States of America. sluckhart@ucdavis.edu

ABSTRACT
The overexpression of activated, myristoylated Akt in the midgut of female transgenic Anopheles stephensi results in resistance to infection with the human malaria parasite Plasmodium falciparum but also decreased lifespan. In the present study, the understanding of mitochondria-dependent midgut homeostasis has been expanded to explain this apparent paradox in an insect of major medical importance. Given that Akt signaling is essential for cell growth and survival, we hypothesized that sustained Akt activation in the mosquito midgut would alter the balance of critical pathways that control mitochondrial dynamics to enhance parasite killing at some cost to survivorship. Toxic reactive oxygen and nitrogen species (RNOS) rise to high levels in the midgut after blood feeding, due to a combination of high NO production and a decline in FOXO-dependent antioxidants. Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios. In addition, mitochondrial mass was lower and accompanied by the presence of stalled autophagosomes in the posterior midgut, a critical site for blood digestion and stem cell-mediated epithelial maintenance and repair, and by functional degradation of the epithelial barrier. By 18 d, the age at which An. stephensi would transmit P. falciparum to human hosts, mitochondrial dysfunction coupled to Akt-mediated repression of autophagy/mitophagy was more evident and midgut epithelial structure was markedly compromised. Inhibition of RNOS by co-feeding of the nitric-oxide synthase inhibitor L-NAME at infection abrogated Akt-dependent killing of P. falciparum that begins within 18 h of infection in 3-5 d old mosquitoes. Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan. Further, quality control of mitochondrial function in the midgut is necessary for the maintenance of midgut health as reflected in energy homeostasis and tissue repair and renewal.

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EPR detection of NO indicated marked NOS catalytic activity in midguts from 3 d HM myrAkt An. stephensi relative to 3 d NTG mosquitoes.Whole midguts from 150 NTG and 150 HM An. stephensi at 3-d post-adult emergence were each hand-homogenized in 300 µl of 20 mM HEPES, pH 7.4 with protease inhibitors and phosphatase inhibitors), then incubated for 3 h at 20–22°C following addition of 100 µl reaction buffer (3 mM sodium N-methyl-D-glucamine dithiocarbamate [MGD] complexed with ferrous sulfate prepared fresh), 0.1 mM NADPH, 1 mM calcium chloride and 1 mM L-arginine in degassed 20 mM HEPES, pH 7.4; [104]. After the incubation, 50 to 100 µl of sample was loaded into an EPR tube and measured using a Bruker EPR and XEpr software. Instrument conditions were indicated under Methods. X-band EPR analysis at 190°K of midgut homogenates produced a strong, broad EPR signal with resonance positions (g tensor factors) at g = 2.04 (left arrow) with an associated triplet signal at g = 2.014 with hyperfine splitting of 17.5 Gauss (right arrow). The broad paramagnetic signal at g = 2.04 has been attributed to the trapping of nitric oxide by NMGD-Fe resulting in the formation of NMGD-Fe-NO adduct. The area and/or amplitude of the adduct signal (indicative of concentration of free radicals, in this case NO) was markedly increased in TG midguts.
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ppat-1003180-g009: EPR detection of NO indicated marked NOS catalytic activity in midguts from 3 d HM myrAkt An. stephensi relative to 3 d NTG mosquitoes.Whole midguts from 150 NTG and 150 HM An. stephensi at 3-d post-adult emergence were each hand-homogenized in 300 µl of 20 mM HEPES, pH 7.4 with protease inhibitors and phosphatase inhibitors), then incubated for 3 h at 20–22°C following addition of 100 µl reaction buffer (3 mM sodium N-methyl-D-glucamine dithiocarbamate [MGD] complexed with ferrous sulfate prepared fresh), 0.1 mM NADPH, 1 mM calcium chloride and 1 mM L-arginine in degassed 20 mM HEPES, pH 7.4; [104]. After the incubation, 50 to 100 µl of sample was loaded into an EPR tube and measured using a Bruker EPR and XEpr software. Instrument conditions were indicated under Methods. X-band EPR analysis at 190°K of midgut homogenates produced a strong, broad EPR signal with resonance positions (g tensor factors) at g = 2.04 (left arrow) with an associated triplet signal at g = 2.014 with hyperfine splitting of 17.5 Gauss (right arrow). The broad paramagnetic signal at g = 2.04 has been attributed to the trapping of nitric oxide by NMGD-Fe resulting in the formation of NMGD-Fe-NO adduct. The area and/or amplitude of the adduct signal (indicative of concentration of free radicals, in this case NO) was markedly increased in TG midguts.

Mentions: To test these possibilities, oxidative/nitrative damage was assessed by evaluating Tyr nitration of the beta subunit of ATPase (ATPB; Fig. S2), a sensitive marker for mitochondrial protein nitration [30], [31], given that Complex V activity was significantly lower than controls (50 to 70% of NTG). NOS activity was evaluated by detecting NO using electron paramagnetic resonance in conjunction with spin trapping technique. Nitration of midgut proteins in HM females was significantly increased at 3 d (8-fold of controls; P = 0.02) and at 18 d (2-fold of controls; P = 0.05) while nitration of mitochondrial ATPB was 3-fold (P = 0.003) and 2-fold (P = 0.048) of controls at each time point (Table 3,Fig. 8). The production of NO was 2-fold of controls at 3 d (Table 3,Fig. 9). Thus, nitrative/oxidative stress was enhanced in midguts from HM An. stephensi – even at 3 d post-emergence – consistent with the increased ATPB nitration and activity loss [65]. Further, NO is an inhibitor of Complex IV [66], [67] through competitive and noncompetitive pathways [66], suggesting increased NO production could block electron transport at the terminal oxidase, even when no changes in activity are detected, enhancing the RNOS-mediated damage of individual Complexes and/or mitochondrial targets and negating compensatory biogenesis.


Sustained activation of Akt elicits mitochondrial dysfunction to block Plasmodium falciparum infection in the mosquito host.

Luckhart S, Giulivi C, Drexler AL, Antonova-Koch Y, Sakaguchi D, Napoli E, Wong S, Price MS, Eigenheer R, Phinney BS, Pakpour N, Pietri JE, Cheung K, Georgis M, Riehle M - PLoS Pathog. (2013)

EPR detection of NO indicated marked NOS catalytic activity in midguts from 3 d HM myrAkt An. stephensi relative to 3 d NTG mosquitoes.Whole midguts from 150 NTG and 150 HM An. stephensi at 3-d post-adult emergence were each hand-homogenized in 300 µl of 20 mM HEPES, pH 7.4 with protease inhibitors and phosphatase inhibitors), then incubated for 3 h at 20–22°C following addition of 100 µl reaction buffer (3 mM sodium N-methyl-D-glucamine dithiocarbamate [MGD] complexed with ferrous sulfate prepared fresh), 0.1 mM NADPH, 1 mM calcium chloride and 1 mM L-arginine in degassed 20 mM HEPES, pH 7.4; [104]. After the incubation, 50 to 100 µl of sample was loaded into an EPR tube and measured using a Bruker EPR and XEpr software. Instrument conditions were indicated under Methods. X-band EPR analysis at 190°K of midgut homogenates produced a strong, broad EPR signal with resonance positions (g tensor factors) at g = 2.04 (left arrow) with an associated triplet signal at g = 2.014 with hyperfine splitting of 17.5 Gauss (right arrow). The broad paramagnetic signal at g = 2.04 has been attributed to the trapping of nitric oxide by NMGD-Fe resulting in the formation of NMGD-Fe-NO adduct. The area and/or amplitude of the adduct signal (indicative of concentration of free radicals, in this case NO) was markedly increased in TG midguts.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585164&req=5

ppat-1003180-g009: EPR detection of NO indicated marked NOS catalytic activity in midguts from 3 d HM myrAkt An. stephensi relative to 3 d NTG mosquitoes.Whole midguts from 150 NTG and 150 HM An. stephensi at 3-d post-adult emergence were each hand-homogenized in 300 µl of 20 mM HEPES, pH 7.4 with protease inhibitors and phosphatase inhibitors), then incubated for 3 h at 20–22°C following addition of 100 µl reaction buffer (3 mM sodium N-methyl-D-glucamine dithiocarbamate [MGD] complexed with ferrous sulfate prepared fresh), 0.1 mM NADPH, 1 mM calcium chloride and 1 mM L-arginine in degassed 20 mM HEPES, pH 7.4; [104]. After the incubation, 50 to 100 µl of sample was loaded into an EPR tube and measured using a Bruker EPR and XEpr software. Instrument conditions were indicated under Methods. X-band EPR analysis at 190°K of midgut homogenates produced a strong, broad EPR signal with resonance positions (g tensor factors) at g = 2.04 (left arrow) with an associated triplet signal at g = 2.014 with hyperfine splitting of 17.5 Gauss (right arrow). The broad paramagnetic signal at g = 2.04 has been attributed to the trapping of nitric oxide by NMGD-Fe resulting in the formation of NMGD-Fe-NO adduct. The area and/or amplitude of the adduct signal (indicative of concentration of free radicals, in this case NO) was markedly increased in TG midguts.
Mentions: To test these possibilities, oxidative/nitrative damage was assessed by evaluating Tyr nitration of the beta subunit of ATPase (ATPB; Fig. S2), a sensitive marker for mitochondrial protein nitration [30], [31], given that Complex V activity was significantly lower than controls (50 to 70% of NTG). NOS activity was evaluated by detecting NO using electron paramagnetic resonance in conjunction with spin trapping technique. Nitration of midgut proteins in HM females was significantly increased at 3 d (8-fold of controls; P = 0.02) and at 18 d (2-fold of controls; P = 0.05) while nitration of mitochondrial ATPB was 3-fold (P = 0.003) and 2-fold (P = 0.048) of controls at each time point (Table 3,Fig. 8). The production of NO was 2-fold of controls at 3 d (Table 3,Fig. 9). Thus, nitrative/oxidative stress was enhanced in midguts from HM An. stephensi – even at 3 d post-emergence – consistent with the increased ATPB nitration and activity loss [65]. Further, NO is an inhibitor of Complex IV [66], [67] through competitive and noncompetitive pathways [66], suggesting increased NO production could block electron transport at the terminal oxidase, even when no changes in activity are detected, enhancing the RNOS-mediated damage of individual Complexes and/or mitochondrial targets and negating compensatory biogenesis.

Bottom Line: Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios.Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan.Further, quality control of mitochondrial function in the midgut is necessary for the maintenance of midgut health as reflected in energy homeostasis and tissue repair and renewal.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical Microbiology and Immunology, School of Medicine, University of California, Davis, Davis, California, United States of America. sluckhart@ucdavis.edu

ABSTRACT
The overexpression of activated, myristoylated Akt in the midgut of female transgenic Anopheles stephensi results in resistance to infection with the human malaria parasite Plasmodium falciparum but also decreased lifespan. In the present study, the understanding of mitochondria-dependent midgut homeostasis has been expanded to explain this apparent paradox in an insect of major medical importance. Given that Akt signaling is essential for cell growth and survival, we hypothesized that sustained Akt activation in the mosquito midgut would alter the balance of critical pathways that control mitochondrial dynamics to enhance parasite killing at some cost to survivorship. Toxic reactive oxygen and nitrogen species (RNOS) rise to high levels in the midgut after blood feeding, due to a combination of high NO production and a decline in FOXO-dependent antioxidants. Despite an apparent increase in mitochondrial biogenesis in young females (3 d), energy deficiencies were apparent as decreased oxidative phosphorylation and increased [AMP]/[ATP] ratios. In addition, mitochondrial mass was lower and accompanied by the presence of stalled autophagosomes in the posterior midgut, a critical site for blood digestion and stem cell-mediated epithelial maintenance and repair, and by functional degradation of the epithelial barrier. By 18 d, the age at which An. stephensi would transmit P. falciparum to human hosts, mitochondrial dysfunction coupled to Akt-mediated repression of autophagy/mitophagy was more evident and midgut epithelial structure was markedly compromised. Inhibition of RNOS by co-feeding of the nitric-oxide synthase inhibitor L-NAME at infection abrogated Akt-dependent killing of P. falciparum that begins within 18 h of infection in 3-5 d old mosquitoes. Hence, Akt-induced changes in mitochondrial dynamics perturb midgut homeostasis to enhance parasite resistance and decrease mosquito infective lifespan. Further, quality control of mitochondrial function in the midgut is necessary for the maintenance of midgut health as reflected in energy homeostasis and tissue repair and renewal.

Show MeSH
Related in: MedlinePlus