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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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HM myrAkt An. stephensi exhibited enhanced killing of early stage GFP-Plasmodium yoelii yoelii 17XNL (Pyy 17XNL) and P. falciparum relative to NTG females.(A) Pyy 17XNL parasites were quantified in dissected midguts at 6, 20 and 48 h post-infection by fluorescence detection (485 nm excitation/535 nm emission wavelengths). Significant killing was noted by 20 h post infection, when Pyy 17XNL are present as fully matured ookinetes in the midgut lumen and in transit across the midgut epithelium [76]. A secondary drop in infection levels was evident at 48 h, which coincides with early oocyst development for Pyy 17XNL [76]. (B) Plasmodium falciparum parasites were quantified using real-time reverse-transcriptase PCR of A18S rRNA (for total parasites) as well as Pfs16 and Pfs25, markers for sexual stage development [73], [74]. Changes in Pfs16 expression were not significant but trended downward relative to expression in NTG An. stephensi at 18 h and 48 h post-infection. A reduction in Pfs25 expression at 6 h suggested that early ookinetes were reduced in HM An. stephensi, with significant killing at 18 h post-infection, a time at which all ookinetes are still present in the midgut lumen [77]. A secondary significant drop was evident at 48 h, at which time ookinetes have traversed the midgut epithelium and are starting to transition to oocysts [77]. The data are represented as the average fold change ± SEM in the number of parasites for HM An. stephensi (black bars) compared to NTG An. stephensi at the same timepoints (indicated as the transition at 1.0). Data from independent experiments with three (A) or four (B) separate cohorts of An. stephensi females were analyzed by Student's t-test (alpha = 0.05) and P values are noted on the graph.
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ppat-1003180-g011: HM myrAkt An. stephensi exhibited enhanced killing of early stage GFP-Plasmodium yoelii yoelii 17XNL (Pyy 17XNL) and P. falciparum relative to NTG females.(A) Pyy 17XNL parasites were quantified in dissected midguts at 6, 20 and 48 h post-infection by fluorescence detection (485 nm excitation/535 nm emission wavelengths). Significant killing was noted by 20 h post infection, when Pyy 17XNL are present as fully matured ookinetes in the midgut lumen and in transit across the midgut epithelium [76]. A secondary drop in infection levels was evident at 48 h, which coincides with early oocyst development for Pyy 17XNL [76]. (B) Plasmodium falciparum parasites were quantified using real-time reverse-transcriptase PCR of A18S rRNA (for total parasites) as well as Pfs16 and Pfs25, markers for sexual stage development [73], [74]. Changes in Pfs16 expression were not significant but trended downward relative to expression in NTG An. stephensi at 18 h and 48 h post-infection. A reduction in Pfs25 expression at 6 h suggested that early ookinetes were reduced in HM An. stephensi, with significant killing at 18 h post-infection, a time at which all ookinetes are still present in the midgut lumen [77]. A secondary significant drop was evident at 48 h, at which time ookinetes have traversed the midgut epithelium and are starting to transition to oocysts [77]. The data are represented as the average fold change ± SEM in the number of parasites for HM An. stephensi (black bars) compared to NTG An. stephensi at the same timepoints (indicated as the transition at 1.0). Data from independent experiments with three (A) or four (B) separate cohorts of An. stephensi females were analyzed by Student's t-test (alpha = 0.05) and P values are noted on the graph.

Mentions: To determine whether the effects of NO were attributable specifically to direct, toxic effects on developing parasites, we examined more closely the timing of parasite death in HM females relative to parasite development in NTG An. stephensi. For these studies, we used a mouse malaria parasite infection model (GFP-expressing Plasmodium yoelii yoelii 17XNL; kindly provided by A. Rodriguez [72]) in addition to mosquito infection with P. falciparum. This design allowed us to examine mosquito infection using independent, quantitative measures and to determine whether NO-dependent parasite killing in myrAkt An. stephensi was unique to P. falciparum or more broadly effective against unrelated parasite species. Infection with P. y. yoelii was monitored using fluorescence quantitation, while P. falciparum infection levels were assessed with quantitative, reverse-transcriptase PCR for two markers of sexual stage parasite development, Pfs16 and Pfs25[73]–[75]. In infections with both species of Plasmodium, significant parasite death was first observed by 18–20 h after infection (Figs. 11A, B). At 20 h post-infection, P. y. yoelii 17XNL parasites are present as mature ookinetes, with some in the midgut lumen and a large percentage in transit across the midgut epithelium [76]. At 18 h post-infection, all P. falciparum parasites are present only as ookinetes in the midgut lumen of An. stephensi[77]. A secondary significant drop in infection levels of HM An. stephensi relative to NTG females was observed by 48 h after infection for both parasite species – a time that coincides with ookinete to oocyst transition on the outside of the midgut epithelium for P. falciparum[77] and early oocyst development for P. y. yoelii[76] – suggesting that RNOS-mediated anti-parasite killing occurs over a broad period of parasite development. These data confirmed that anti-P. falciparum resistance in myrAkt An. stephensi is initiated as direct, early toxic effects of mosquito NO/RNOS on parasites prior to invasion of the midgut epithelium.


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)

HM myrAkt An. stephensi exhibited enhanced killing of early stage GFP-Plasmodium yoelii yoelii 17XNL (Pyy 17XNL) and P. falciparum relative to NTG females.(A) Pyy 17XNL parasites were quantified in dissected midguts at 6, 20 and 48 h post-infection by fluorescence detection (485 nm excitation/535 nm emission wavelengths). Significant killing was noted by 20 h post infection, when Pyy 17XNL are present as fully matured ookinetes in the midgut lumen and in transit across the midgut epithelium [76]. A secondary drop in infection levels was evident at 48 h, which coincides with early oocyst development for Pyy 17XNL [76]. (B) Plasmodium falciparum parasites were quantified using real-time reverse-transcriptase PCR of A18S rRNA (for total parasites) as well as Pfs16 and Pfs25, markers for sexual stage development [73], [74]. Changes in Pfs16 expression were not significant but trended downward relative to expression in NTG An. stephensi at 18 h and 48 h post-infection. A reduction in Pfs25 expression at 6 h suggested that early ookinetes were reduced in HM An. stephensi, with significant killing at 18 h post-infection, a time at which all ookinetes are still present in the midgut lumen [77]. A secondary significant drop was evident at 48 h, at which time ookinetes have traversed the midgut epithelium and are starting to transition to oocysts [77]. The data are represented as the average fold change ± SEM in the number of parasites for HM An. stephensi (black bars) compared to NTG An. stephensi at the same timepoints (indicated as the transition at 1.0). Data from independent experiments with three (A) or four (B) separate cohorts of An. stephensi females were analyzed by Student's t-test (alpha = 0.05) and P values are noted on the graph.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1003180-g011: HM myrAkt An. stephensi exhibited enhanced killing of early stage GFP-Plasmodium yoelii yoelii 17XNL (Pyy 17XNL) and P. falciparum relative to NTG females.(A) Pyy 17XNL parasites were quantified in dissected midguts at 6, 20 and 48 h post-infection by fluorescence detection (485 nm excitation/535 nm emission wavelengths). Significant killing was noted by 20 h post infection, when Pyy 17XNL are present as fully matured ookinetes in the midgut lumen and in transit across the midgut epithelium [76]. A secondary drop in infection levels was evident at 48 h, which coincides with early oocyst development for Pyy 17XNL [76]. (B) Plasmodium falciparum parasites were quantified using real-time reverse-transcriptase PCR of A18S rRNA (for total parasites) as well as Pfs16 and Pfs25, markers for sexual stage development [73], [74]. Changes in Pfs16 expression were not significant but trended downward relative to expression in NTG An. stephensi at 18 h and 48 h post-infection. A reduction in Pfs25 expression at 6 h suggested that early ookinetes were reduced in HM An. stephensi, with significant killing at 18 h post-infection, a time at which all ookinetes are still present in the midgut lumen [77]. A secondary significant drop was evident at 48 h, at which time ookinetes have traversed the midgut epithelium and are starting to transition to oocysts [77]. The data are represented as the average fold change ± SEM in the number of parasites for HM An. stephensi (black bars) compared to NTG An. stephensi at the same timepoints (indicated as the transition at 1.0). Data from independent experiments with three (A) or four (B) separate cohorts of An. stephensi females were analyzed by Student's t-test (alpha = 0.05) and P values are noted on the graph.
Mentions: To determine whether the effects of NO were attributable specifically to direct, toxic effects on developing parasites, we examined more closely the timing of parasite death in HM females relative to parasite development in NTG An. stephensi. For these studies, we used a mouse malaria parasite infection model (GFP-expressing Plasmodium yoelii yoelii 17XNL; kindly provided by A. Rodriguez [72]) in addition to mosquito infection with P. falciparum. This design allowed us to examine mosquito infection using independent, quantitative measures and to determine whether NO-dependent parasite killing in myrAkt An. stephensi was unique to P. falciparum or more broadly effective against unrelated parasite species. Infection with P. y. yoelii was monitored using fluorescence quantitation, while P. falciparum infection levels were assessed with quantitative, reverse-transcriptase PCR for two markers of sexual stage parasite development, Pfs16 and Pfs25[73]–[75]. In infections with both species of Plasmodium, significant parasite death was first observed by 18–20 h after infection (Figs. 11A, B). At 20 h post-infection, P. y. yoelii 17XNL parasites are present as mature ookinetes, with some in the midgut lumen and a large percentage in transit across the midgut epithelium [76]. At 18 h post-infection, all P. falciparum parasites are present only as ookinetes in the midgut lumen of An. stephensi[77]. A secondary significant drop in infection levels of HM An. stephensi relative to NTG females was observed by 48 h after infection for both parasite species – a time that coincides with ookinete to oocyst transition on the outside of the midgut epithelium for P. falciparum[77] and early oocyst development for P. y. yoelii[76] – suggesting that RNOS-mediated anti-parasite killing occurs over a broad period of parasite development. These data confirmed that anti-P. falciparum resistance in myrAkt An. stephensi is initiated as direct, early toxic effects of mosquito NO/RNOS on parasites prior to invasion of the midgut epithelium.

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