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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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Over-expression of myrAKT was associated with changes suggestive of stalled mitochondrial fission.No differences were observed for percentages of round midgut mitochondria among NTG, HT, or HM myrAkt transgenic An. stephensi at 3 d and 18 d post-emergence (ANOVA following arcsin transformation; alpha = 0.05). In all groups except for 18 d HM, midgut mitochondria were associated with the brush border. In 18 d HM, midgut mitochondria were distributed throughout the cell. Further, size distributions of round mitochondria in NTGs were significantly different from those in HT and HM midguts at 3 d (NTG vs HT: χ2 = 20.5, df = 2, P<0.0001; NTG vs HM: χ2 = 19.6, df = 2, P<0.0001) and at 18 d (NTG vs HT: χ2 = 54.8, df = 2, P<0.0001; NTG vs HM: χ2 = 244, df = 2, P<0.0001). In addition, within each TG genotype (HT and HM), there were significant differences in size distributions of round mitochondria between samples analyzed from 3 d and 18 d An. stephensi (3 d HT vs 18 d HT: χ2 = 182, df = 2, P<0.0001; 3 d HM vs 18 d HM: χ2 = 457, df = 2, P<0.0001).
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ppat-1003180-g006: Over-expression of myrAKT was associated with changes suggestive of stalled mitochondrial fission.No differences were observed for percentages of round midgut mitochondria among NTG, HT, or HM myrAkt transgenic An. stephensi at 3 d and 18 d post-emergence (ANOVA following arcsin transformation; alpha = 0.05). In all groups except for 18 d HM, midgut mitochondria were associated with the brush border. In 18 d HM, midgut mitochondria were distributed throughout the cell. Further, size distributions of round mitochondria in NTGs were significantly different from those in HT and HM midguts at 3 d (NTG vs HT: χ2 = 20.5, df = 2, P<0.0001; NTG vs HM: χ2 = 19.6, df = 2, P<0.0001) and at 18 d (NTG vs HT: χ2 = 54.8, df = 2, P<0.0001; NTG vs HM: χ2 = 244, df = 2, P<0.0001). In addition, within each TG genotype (HT and HM), there were significant differences in size distributions of round mitochondria between samples analyzed from 3 d and 18 d An. stephensi (3 d HT vs 18 d HT: χ2 = 182, df = 2, P<0.0001; 3 d HM vs 18 d HM: χ2 = 457, df = 2, P<0.0001).

Mentions: The overrepresentation of mitochondrial proteins (3–5 d; Table 1), the accumulation of autophagosomes (3 d HM and HT; Table 2), apparent altered autophagy (18 d HM; Fig. 4), and changes in size, number, and distribution of mitochondria in the midguts of 3 d HM (Fig. 5D,Table 2) and 18 d HT (Fig. 5D) An. stephensi were indicative of defective organelle maintenance in TG mosquitoes. Accordingly, we quantified the percentages of round versus non-round mitochondria to assess the balance of fission-fusion with the assumption that functional midgut mitochondria exhibit a tubular, elongated shape while round mitochondria form in cells undergoing a response to cellular oxidative damage. We examined the shape of 2524 mitochondria in 3 d NTG, 2979 in 3 d HT, 1638 in 3 d HM, 2080 in 18 d NTG, 2154 in 18 d HT, and 2147 mitochondria in 18 d HM An. stephensi. The number of midgut epithelial cells from which we analyzed mitochondrial shape could not be determined for 18 d HT and 18 d HM mosquitoes due to extensive tissue damage induced by transgene expression in older mosquitoes. Thus, mitochondria were counted over an identical midgut area for all treatments. As expected, a high percentage of elongated, tubular mitochondria were observed in NTG midguts (Fig. 6). Contrary to our expectations, however, no significant differences in the percentages of round mitochondria among midguts of NTG, HM, and HT females at 3 d or 18 d or between matched genotypes at 3 d or 18 d were observed (P = 0.088). However, analysis of the distributions of small (<50,000 nm2), medium (50,000–100,000 nm2), and large (>100,000 nm2) round mitochondria showed significant differences between and among NTG, HT, and HM females. The distributions of small, medium, and large round mitochondria were comparable in midguts from 3 d and 18 d NTGs (Fig. 6). However, the distributions of round mitochondria in NTGs were significantly different from those in HT and HM midguts at both 3 d and 18 d (P<0.0001). In addition, within each transgenic genotype (HT or HM), there were significant differences in mitochondrial size distributions between samples analyzed from 3 d and 18 d An. stephensi (P<0.0001). At 18 d, the occurrence of small, round mitochondria showed a gene-dose dependence from NTG to HT to HM females (30% to 48% to 65%; Fig. 6). The increased percentages of small mitochondria were accompanied by losses in both medium and large sized mitochondria, with percentages of both sizes trending downward from NTG to HT to HM females at 18 d. These changes in morphology appeared to be consistent with persistence of oxidative stress-induced fission, resulting in the formation of small, round mitochondria that can persist when fusion is inhibited [60]–[63].


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)

Over-expression of myrAKT was associated with changes suggestive of stalled mitochondrial fission.No differences were observed for percentages of round midgut mitochondria among NTG, HT, or HM myrAkt transgenic An. stephensi at 3 d and 18 d post-emergence (ANOVA following arcsin transformation; alpha = 0.05). In all groups except for 18 d HM, midgut mitochondria were associated with the brush border. In 18 d HM, midgut mitochondria were distributed throughout the cell. Further, size distributions of round mitochondria in NTGs were significantly different from those in HT and HM midguts at 3 d (NTG vs HT: χ2 = 20.5, df = 2, P<0.0001; NTG vs HM: χ2 = 19.6, df = 2, P<0.0001) and at 18 d (NTG vs HT: χ2 = 54.8, df = 2, P<0.0001; NTG vs HM: χ2 = 244, df = 2, P<0.0001). In addition, within each TG genotype (HT and HM), there were significant differences in size distributions of round mitochondria between samples analyzed from 3 d and 18 d An. stephensi (3 d HT vs 18 d HT: χ2 = 182, df = 2, P<0.0001; 3 d HM vs 18 d HM: χ2 = 457, df = 2, P<0.0001).
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Related In: Results  -  Collection

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ppat-1003180-g006: Over-expression of myrAKT was associated with changes suggestive of stalled mitochondrial fission.No differences were observed for percentages of round midgut mitochondria among NTG, HT, or HM myrAkt transgenic An. stephensi at 3 d and 18 d post-emergence (ANOVA following arcsin transformation; alpha = 0.05). In all groups except for 18 d HM, midgut mitochondria were associated with the brush border. In 18 d HM, midgut mitochondria were distributed throughout the cell. Further, size distributions of round mitochondria in NTGs were significantly different from those in HT and HM midguts at 3 d (NTG vs HT: χ2 = 20.5, df = 2, P<0.0001; NTG vs HM: χ2 = 19.6, df = 2, P<0.0001) and at 18 d (NTG vs HT: χ2 = 54.8, df = 2, P<0.0001; NTG vs HM: χ2 = 244, df = 2, P<0.0001). In addition, within each TG genotype (HT and HM), there were significant differences in size distributions of round mitochondria between samples analyzed from 3 d and 18 d An. stephensi (3 d HT vs 18 d HT: χ2 = 182, df = 2, P<0.0001; 3 d HM vs 18 d HM: χ2 = 457, df = 2, P<0.0001).
Mentions: The overrepresentation of mitochondrial proteins (3–5 d; Table 1), the accumulation of autophagosomes (3 d HM and HT; Table 2), apparent altered autophagy (18 d HM; Fig. 4), and changes in size, number, and distribution of mitochondria in the midguts of 3 d HM (Fig. 5D,Table 2) and 18 d HT (Fig. 5D) An. stephensi were indicative of defective organelle maintenance in TG mosquitoes. Accordingly, we quantified the percentages of round versus non-round mitochondria to assess the balance of fission-fusion with the assumption that functional midgut mitochondria exhibit a tubular, elongated shape while round mitochondria form in cells undergoing a response to cellular oxidative damage. We examined the shape of 2524 mitochondria in 3 d NTG, 2979 in 3 d HT, 1638 in 3 d HM, 2080 in 18 d NTG, 2154 in 18 d HT, and 2147 mitochondria in 18 d HM An. stephensi. The number of midgut epithelial cells from which we analyzed mitochondrial shape could not be determined for 18 d HT and 18 d HM mosquitoes due to extensive tissue damage induced by transgene expression in older mosquitoes. Thus, mitochondria were counted over an identical midgut area for all treatments. As expected, a high percentage of elongated, tubular mitochondria were observed in NTG midguts (Fig. 6). Contrary to our expectations, however, no significant differences in the percentages of round mitochondria among midguts of NTG, HM, and HT females at 3 d or 18 d or between matched genotypes at 3 d or 18 d were observed (P = 0.088). However, analysis of the distributions of small (<50,000 nm2), medium (50,000–100,000 nm2), and large (>100,000 nm2) round mitochondria showed significant differences between and among NTG, HT, and HM females. The distributions of small, medium, and large round mitochondria were comparable in midguts from 3 d and 18 d NTGs (Fig. 6). However, the distributions of round mitochondria in NTGs were significantly different from those in HT and HM midguts at both 3 d and 18 d (P<0.0001). In addition, within each transgenic genotype (HT or HM), there were significant differences in mitochondrial size distributions between samples analyzed from 3 d and 18 d An. stephensi (P<0.0001). At 18 d, the occurrence of small, round mitochondria showed a gene-dose dependence from NTG to HT to HM females (30% to 48% to 65%; Fig. 6). The increased percentages of small mitochondria were accompanied by losses in both medium and large sized mitochondria, with percentages of both sizes trending downward from NTG to HT to HM females at 18 d. These changes in morphology appeared to be consistent with persistence of oxidative stress-induced fission, resulting in the formation of small, round mitochondria that can persist when fusion is inhibited [60]–[63].

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