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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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Evaluation of midgut permeability in HM myrAkt relative to NTG An. stephensi.(A) 3–5 d old, age-matched non-blood-fed NTG (nbf NTG), (B) blood-fed (bf) NTG and (C) HM female mosquitoes, were fed fluorescent beads (3–3.5 µM, Spherotech) in a reconstituted human blood meal through a Hemotek Insect Feeding System (Discovery Workshops). Prior to feeding (nbf NTG) and at 48 h post-blood feeding (bf NTG, bf HM), five whole mosquitoes from each group were cold-anesthetized and placed in 24-well plates. Mosquitoes were photographed under normal light (left panels) and also imaged on a Nikon TE 200 inverted fluorescent microscope at 4× with identical settings for all fluorescent images (right panels). Autofluorescence is visible on the mosquito thorax in all images. Arrowheads in nbf NTG mark location of mosquito leg and in bf NTG and bf HM images mark fluorescent beads in mosquito legs, an indication of bead passage through the midgut epithelium into the hemocoel. Note larger number of beads in bf HM compared to bf NTG An. stephensi. (D) Bead numbers per three whole mosquitoes minus bead numbers in three paired midguts from the same groups at 48 h post-feeding are represented as individual dots (means indicated as bars). Midgut beads averaged 236 for bf NTG and 225 for bf HM at 48 h post-feeding, so midgut beads accounted for less than 10% and less than 5%, respectively, of NTG and HM whole body bead counts. Data were analyzed by Student's t-test (alpha = 0.05).
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ppat-1003180-g003: Evaluation of midgut permeability in HM myrAkt relative to NTG An. stephensi.(A) 3–5 d old, age-matched non-blood-fed NTG (nbf NTG), (B) blood-fed (bf) NTG and (C) HM female mosquitoes, were fed fluorescent beads (3–3.5 µM, Spherotech) in a reconstituted human blood meal through a Hemotek Insect Feeding System (Discovery Workshops). Prior to feeding (nbf NTG) and at 48 h post-blood feeding (bf NTG, bf HM), five whole mosquitoes from each group were cold-anesthetized and placed in 24-well plates. Mosquitoes were photographed under normal light (left panels) and also imaged on a Nikon TE 200 inverted fluorescent microscope at 4× with identical settings for all fluorescent images (right panels). Autofluorescence is visible on the mosquito thorax in all images. Arrowheads in nbf NTG mark location of mosquito leg and in bf NTG and bf HM images mark fluorescent beads in mosquito legs, an indication of bead passage through the midgut epithelium into the hemocoel. Note larger number of beads in bf HM compared to bf NTG An. stephensi. (D) Bead numbers per three whole mosquitoes minus bead numbers in three paired midguts from the same groups at 48 h post-feeding are represented as individual dots (means indicated as bars). Midgut beads averaged 236 for bf NTG and 225 for bf HM at 48 h post-feeding, so midgut beads accounted for less than 10% and less than 5%, respectively, of NTG and HM whole body bead counts. Data were analyzed by Student's t-test (alpha = 0.05).

Mentions: Midgut epithelium is a highly aerobic tissue and deficits in energy from mitochondria, perhaps associated with altered mitophagy, would likely result in electrolyte imbalance, defective secondary active transport of cations and solutes, and increased permeability [57]. To determine whether histological changes observed in HM midguts were accompanied by altered epithelial integrity, 3–5 d old NTG and HM mosquitoes were fed with fluorescent beads in reconstituted human blood meals. After 48 h – to allow complete digestion of the blood – bead numbers were quantified by flow cytometry in washed, lysed midguts and in whole mosquitoes to estimate transport of beads across the epithelium. Body bead counts (minus midgut beads) from NTG females were 2,805±593 (mean ± SEM), whereas body bead counts from HM females were nearly 2-fold higher (5,073±534; P = 0.011; Fig. 3). Therefore, the permeability of the midgut epithelium was significantly increased in 3–5 d old HM females compared to age-matched NTG females, confirming that observed histological changes in the midgut epithelium of 3–5 d old HM myrAkt An. stephensi were functionally significant.


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)

Evaluation of midgut permeability in HM myrAkt relative to NTG An. stephensi.(A) 3–5 d old, age-matched non-blood-fed NTG (nbf NTG), (B) blood-fed (bf) NTG and (C) HM female mosquitoes, were fed fluorescent beads (3–3.5 µM, Spherotech) in a reconstituted human blood meal through a Hemotek Insect Feeding System (Discovery Workshops). Prior to feeding (nbf NTG) and at 48 h post-blood feeding (bf NTG, bf HM), five whole mosquitoes from each group were cold-anesthetized and placed in 24-well plates. Mosquitoes were photographed under normal light (left panels) and also imaged on a Nikon TE 200 inverted fluorescent microscope at 4× with identical settings for all fluorescent images (right panels). Autofluorescence is visible on the mosquito thorax in all images. Arrowheads in nbf NTG mark location of mosquito leg and in bf NTG and bf HM images mark fluorescent beads in mosquito legs, an indication of bead passage through the midgut epithelium into the hemocoel. Note larger number of beads in bf HM compared to bf NTG An. stephensi. (D) Bead numbers per three whole mosquitoes minus bead numbers in three paired midguts from the same groups at 48 h post-feeding are represented as individual dots (means indicated as bars). Midgut beads averaged 236 for bf NTG and 225 for bf HM at 48 h post-feeding, so midgut beads accounted for less than 10% and less than 5%, respectively, of NTG and HM whole body bead counts. Data were analyzed by Student's t-test (alpha = 0.05).
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003180-g003: Evaluation of midgut permeability in HM myrAkt relative to NTG An. stephensi.(A) 3–5 d old, age-matched non-blood-fed NTG (nbf NTG), (B) blood-fed (bf) NTG and (C) HM female mosquitoes, were fed fluorescent beads (3–3.5 µM, Spherotech) in a reconstituted human blood meal through a Hemotek Insect Feeding System (Discovery Workshops). Prior to feeding (nbf NTG) and at 48 h post-blood feeding (bf NTG, bf HM), five whole mosquitoes from each group were cold-anesthetized and placed in 24-well plates. Mosquitoes were photographed under normal light (left panels) and also imaged on a Nikon TE 200 inverted fluorescent microscope at 4× with identical settings for all fluorescent images (right panels). Autofluorescence is visible on the mosquito thorax in all images. Arrowheads in nbf NTG mark location of mosquito leg and in bf NTG and bf HM images mark fluorescent beads in mosquito legs, an indication of bead passage through the midgut epithelium into the hemocoel. Note larger number of beads in bf HM compared to bf NTG An. stephensi. (D) Bead numbers per three whole mosquitoes minus bead numbers in three paired midguts from the same groups at 48 h post-feeding are represented as individual dots (means indicated as bars). Midgut beads averaged 236 for bf NTG and 225 for bf HM at 48 h post-feeding, so midgut beads accounted for less than 10% and less than 5%, respectively, of NTG and HM whole body bead counts. Data were analyzed by Student's t-test (alpha = 0.05).
Mentions: Midgut epithelium is a highly aerobic tissue and deficits in energy from mitochondria, perhaps associated with altered mitophagy, would likely result in electrolyte imbalance, defective secondary active transport of cations and solutes, and increased permeability [57]. To determine whether histological changes observed in HM midguts were accompanied by altered epithelial integrity, 3–5 d old NTG and HM mosquitoes were fed with fluorescent beads in reconstituted human blood meals. After 48 h – to allow complete digestion of the blood – bead numbers were quantified by flow cytometry in washed, lysed midguts and in whole mosquitoes to estimate transport of beads across the epithelium. Body bead counts (minus midgut beads) from NTG females were 2,805±593 (mean ± SEM), whereas body bead counts from HM females were nearly 2-fold higher (5,073±534; P = 0.011; Fig. 3). Therefore, the permeability of the midgut epithelium was significantly increased in 3–5 d old HM females compared to age-matched NTG females, confirming that observed histological changes in the midgut epithelium of 3–5 d old HM myrAkt An. stephensi were functionally significant.

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