Sustained Activation of Akt Elicits Mitochondrial Dysfunction to Block <em>Plasmodium falciparum</em> Infection in the Mosquito Host

<div><p>The overexpression of activated, myristoylated Akt in the midgut of female transgenic <i>Anopheles stephensi</i> results in resistance to infection with the human malaria parasite <i>Plasmodium falciparum</i> 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 <i>An. stephensi</i> would transmit <i>P. falciparum</i> 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 <i>L</i>-NAME at infection abrogated Akt-dependent killing of <i>P. falciparum</i> 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.</p> </div

Tyrosine nitration of midgut ATPase beta subunit was increased in HM myrAkt An. stephensi relative to NTG females at 3 d and 18 d post-emergence.

<p>Top panel, representative western blots probed for nitrotyrosine (nY) and total ATPase beta subunit (Beta) of midgut proteins from HM and NTG <i>An. stephensi</i> at 3 d and 18 d post-adult emergence. Bottom panel, quantified ECL signals for nY were normalized to total ATPase beta subunit and represented as fold change relative to NTG <i>An. stephensi</i> (indicated as dotted line at 1.0). Data were analyzed using Student's t-test (alpha = 0.05); calculated <i>P</i> values indicate significant differences in levels of nitration of midgut ATPase beta subunit between age-matched midguts of HM and NTG <i>An. stephensi</i>.</p

Over-expression of myrAKT was associated with changes in midgut mitochondria number and size.

<p>The number and size of mitochondria in the posterior midgut of myrAkt TG and NTG mosquitoes were determined for young (3 d) and old (18 d) mosquitoes within 85.90 µm of the brush border. (A) Average mitochondrial size was significantly decreased in midguts of 18 d old HM females compared to 18 d NTG and to 3 d HM. (B, C) The total number of mitochondria and mitochondrial density was significantly lower in 3 d HM mosquitoes relative to 3 d HT. In addition, the number and density of mitochondria significantly increased as the HM mosquitoes aged. (D) Total mitochondria content (percent area occupied by mitochondria) was significantly decreased in 18 d HT and HM mosquitoes and in 3 d HM mosquitoes relative to NTG controls and 3 d HT mosquitoes. Measurements from 5 midguts for each 3 d genotype and 4 midguts for each 18 d genotype were analyzed using a two-way ANOVA followed by Tukey-Kramer HSD test. Different letters indicate significant differences (alpha = 0.05).</p

<i>Atg6</i> and <i>Atg8</i> expression levels were reduced in 18 d HM myrAkt relative to NTG <i>An. stephensi</i>.

<p>Midguts were dissected from 18 d HM myrAkt and NTG <i>An. stephensi</i> for RNA isolation and quantitative RT-PCR as described in the <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003180#s4" target="_blank">Methods</a>. The analyses were performed on midgut RNAs from three independent cohorts of <i>An. stephensi</i>. Each data point represents <i>Atg6</i> or <i>Atg8</i> expression from one of three biological replicate samples; values were normalized to NTG levels (indicated as 1.0). Means are indicated as bars for each treatment. Data were analyzed by paired Student's t-test (alpha = 0.05) and <i>P</i> values are noted on the graph.</p

EPR detection of NO indicated marked NOS catalytic activity in midguts from 3 d HM myrAkt <i>An. stephensi</i> relative to 3 d NTG mosquitoes.

<p>Whole midguts from 150 NTG and 150 HM <i>An. stephensi</i> 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 <i>N</i>-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; <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003180#ppat.1003180-Steffen1" target="_blank">[104]</a>. 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003180#s4" target="_blank">Methods</a>. 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.</p

Over-expression of myrAKT led to morphological changes in the epithelium and mitochondria of the <i>An. stephensi</i> midgut.

<p>Posterior midgut morphology of NTG <i>An. stephensi</i> (3 d (A) and 18 d (B) after adult emergence), HT myrAkt <i>An. stephensi</i> (3 d (C) and 18 d (D) after adult emergence), and HM myrAkt <i>An. stephensi</i> (3 d (E) and 18 d (F) after adult emergence). Midguts from 3 d NTG, HT, and HM <i>An. stephensi</i> had an intact brush border (BB) and mitochondria (M) localized near the brush border (A, C, E). Stalled autophagosomes (SA) were observed in the midguts of 3 d HM (E) and 18 d HM and HT mosquitoes (D, F). Giant stalled autophagosomes (GSA) were found in the midguts of 18 d HM and HT mosquitoes (D, F). Many SAs contained electron dense material consistent with engulfed mitochondria (E, F). An example of a clear, double membrane, autophagic vacuole fusing with a small, apparently damaged, mitochondrion in the midgut of 3 d HM (white arrowhead, E inset) indicated mitophagy in process. In addition, the mitochondria in 18 d HM mosquitoes were not localized to the brush border, but distributed through the cytoplasm (F). An inset shows example of a stalled autophagosome containing three partially degraded mitochondria in an 18 d HT midgut (F inset). Representative TEM images taken at a magnification of 2,650× are shown. Inset in (E) is at magnification of 15,000×, and inset in (F) is at 8,800×. Midgut epithelium microvilli or brush border, BB; lysosomes, L; basal lamina mitochondria, M; cell nucleus, N; nucleolus, n; stalled autophagosomes, SA; giant autophagosomes with brush border inside, GSA; secretory granules, SG; white arrows point at representative structures.</p

Mitochondrial outcomes in midguts of non-transgenic (NTG) and myrAkt homozygous (HM) <i>An. stephensi</i> females at 3 d and 18 d post emergence.

<p>Identical superscripts indicate values significant differences with the following <i>P</i> values: (a) 0.001, (b) 1×10⁻⁵, (c) 5×10⁻⁸, (d) 2×10⁻³, (e) 1×10⁻⁴, (f) 8×10⁻⁴, (g) 5×10⁻⁴, (h) 0.03, (i) 0.01, (j) 4×10⁻⁵, (k) 4×10⁻⁵, (l) 2×10⁻⁶, (m) 8×10⁻⁵, (n) 1×10⁻⁴, (o) 0.043, (p) 2×10⁻⁵, (q) 5×10⁻⁴; CS = citrate synthase; nd = not determined;</p>*<p>defined as EPR area of spin adduct MGD-NO obtained under optimal conditions for NOS activity (meaning maximum NO produced by NOS, but not the NO produced by each type of mosquito after 3 d).</p

Midgut metabolite profiles in HM myrAkt <i>An. stephensi</i> suggested marked energy deficiencies relative to NTG mosquitoes at 3 d and 18 d post-emergence.

<p>All metabolites were evaluated by HPLC as described under <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003180#s4" target="_blank">Methods</a>. ATP, ADP, AMP, AN (ATP+ADP+AMP), NAD, NADH and NAD+NADH were calculated as nmol/midgut and data are represented as fold of NTG values. The NTG values were the average of 3 d and 18 d given that no statistical differences were observed between these two days. Data were analyzed relative to NTG values with Student's t-test (alpha = 0.05).*<i>P</i><0.05 relative to NTG values.</p

HM myrAkt <i>An. stephensi</i> exhibited enhanced killing of early stage GFP-<i>Plasmodium yoelii yoelii</i> 17XNL (Pyy 17XNL) and <i>P. falciparum</i> relative to NTG females.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003180#ppat.1003180-Vaughan1" target="_blank">[76]</a>. A secondary drop in infection levels was evident at 48 h, which coincides with early oocyst development for Pyy 17XNL <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003180#ppat.1003180-Vaughan1" target="_blank">[76]</a>. (B) <i>Plasmodium falciparum</i> parasites were quantified using real-time reverse-transcriptase PCR of <i>A18S</i> rRNA (for total parasites) as well as <i>Pfs16</i> and <i>Pfs25</i>, markers for sexual stage development <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003180#ppat.1003180-Dechering1" target="_blank">[73]</a>, <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003180#ppat.1003180-Rodriguez1" target="_blank">[74]</a>. Changes in <i>Pfs16</i> expression were not significant but trended downward relative to expression in NTG <i>An. stephensi</i> at 18 h and 48 h post-infection. A reduction in <i>Pfs25</i> expression at 6 h suggested that early ookinetes were reduced in HM <i>An. stephensi</i>, with significant killing at 18 h post-infection, a time at which all ookinetes are still present in the midgut lumen <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003180#ppat.1003180-Meis1" target="_blank">[77]</a>. 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003180#ppat.1003180-Meis1" target="_blank">[77]</a>. The data are represented as the average fold change ± SEM in the number of parasites for HM <i>An. stephensi</i> (black bars) compared to NTG <i>An. stephensi</i> at the same timepoints (indicated as the transition at 1.0). Data from independent experiments with three (A) or four (B) separate cohorts of <i>An. stephensi</i> females were analyzed by Student's t-test (alpha = 0.05) and <i>P</i> values are noted on the graph.</p

Nucleotide contents in whole bodies of non-transgenic (NTG) and myrAkt homozygous (HM) female <i>An. stephensi</i> at 3 d post-emergence.

*<p>Different from controls with <i>P</i>≤0.05. ECP = energy charge potential.</p