Toxoplasma gondii mitochondrial association factor 1b interactome reveals novel binding partners including Ral GTPase accelerating protein α1.

The intracellular parasite, Toxoplasma gondii, has developed sophisticated molecular strategies to subvert host processes and promote growth and survival. During infection, T. gondii replicates in a parasitophorous vacuole (PV) and modulates host functions through a network of secreted proteins. Of these, Mitochondrial Association Factor 1b (MAF1b) recruits host mitochondria to the PV, a process that confers an in vivo growth advantage, though the precise mechanisms remain enigmatic. To address this knowledge gap, we mapped the MAF1b interactome in human fibroblasts using a commercial Yeast-2-hybrid (Y2H) screen, which revealed several previously unidentified binding partners including the GAP domain of Ral GTPase Accelerating Protein α1 (RalGAPα1(GAP)). Recombinantly produced MAF1b and RalGAPα1(GAP) formed as a stable binary complex as shown by size exclusion chromatography with a Kd of 334 nM as measured by isothermal titration calorimetry (ITC). Notably, no binding was detected between RalGAPα1(GAP) and the structurally conserved MAF1b homolog, MAF1a, which does not recruit host mitochondria. Next, we used hydrogen deuterium exchange mass spectrometry (HDX-MS) to map the RalGAPα1(GAP)-MAF1b interface, which led to identification of the "GAP-binding loop" on MAF1b that was confirmed by mutagenesis and ITC to be necessary for complex formation. A high-confidence Alphafold model predicts the GAP-binding loop to lie at the RalGAPα1(GAP)-MAF1b interface further supporting the HDX-MS data. Mechanistic implications of a RalGAPα1(GAP)-MAF1b complex are discussed in the context of T. gondii infection and indicates that MAF1b may have evolved multiple independent functions to increase T. gondii fitness.

Differential contribution of two organelles of endosymbiotic origin to iron-sulfur cluster synthesis and overall fitness in Toxoplasma.

Iron-sulfur (Fe-S) clusters are one of the most ancient and ubiquitous prosthetic groups, and they are required by a variety of proteins involved in important metabolic processes. Apicomplexan parasites have inherited different plastidic and mitochondrial Fe-S clusters biosynthesis pathways through endosymbiosis. We have investigated the relative contributions of these pathways to the fitness of Toxoplasma gondii, an apicomplexan parasite causing disease in humans, by generating specific mutants. Phenotypic analysis and quantitative proteomics allowed us to highlight notable differences in these mutants. Both Fe-S cluster synthesis pathways are necessary for optimal parasite growth in vitro, but their disruption leads to markedly different fates: impairment of the plastidic pathway leads to a loss of the organelle and to parasite death, while disruption of the mitochondrial pathway trigger differentiation into a stress resistance stage. This highlights that otherwise similar biochemical pathways hosted by different sub-cellular compartments can have very different contributions to the biology of the parasites, which is something to consider when exploring novel strategies for therapeutic intervention.

Rhoptry kinase protein 39 (ROP39) is a novel factor that recruits host mitochondria to the parasitophorous vacuole of Toxoplasma gondii.

Most intracellular pathogens replicate in a vacuole to avoid the defense system of the host. A few pathogens recruit host mitochondria around those vacuoles, but the molecules responsible for mitochondrial recruitment remain unidentified. It is only in the apicomplexan parasite Toxoplasma gondii, that mitochondrial association factor 1b (MAF1b) has been identified as an association factor for host mitochondria. Here, we show that rhoptry kinase family protein 39 (ROP39) induces host mitochondrial recruitment in T. gondii. We found that the abundance of ROP39 was increased on host mitochondria extracted from human foreskin fibroblasts (HFFs) infected with T. gondii. ROP39 expressed exogenously in HFFs localized on host mitochondria, indicating that it has the potential to bind to host mitochondria without assistance from other parasite factors. Confocal microscopy revealed that ROP39 colocalized with host mitochondria on the membrane of parasitophorous vacuoles, in which the parasites reside. Moreover, we observed about a 10% reduction in the level of mitochondrial association in rop39-knockout parasites compared with a parental strain.

Analysis of mitochondrial enzymatic activity in blood lymphomonocyte fractions during infection with different Trypanosoma cruzi strains.

Proinflammatory and inflammatory mediators induced by Trypanosoma cruzi infection increase the oxidative stress, generating toxicity for cells targeting mitochondria of different tissues. We studied the activity of citrate synthase and complexes I-IV of respiratory chain in mitochondria of blood lymphomonocyte fraction, from albino Swiss mice infected with different isolates of T. cruzi , during Chagas disease evolution. Complexes I-IV were modified in infected groups (p<0.05) in all the stages, and an inflammatory process of different magnitudes was detected in the heart and skeletal muscle according to the isolate. The citrate synthase activity presented modifications in the SGO Z12 and the Tulahuen group (p<0.05). Hearts showed fiber fragmentation and fibrosis; skeletal muscle presented inflammatory infiltrates and in the Tulahuen infected group, there were also amastigote nests. The inflammatory processes produced an oxidative stress that induced different alterations of mitochondrial enzymes activities in the lymphomonocyte fraction that can be detected by a simple blood extraction, suggesting that they could be used as disease markers, especially in the indeterminate phase of Chagas disease.

Measuring mitochondrial respiration in adherent cells infected with Trypanosoma cruzi Chagas, 1909 using Seahorse extracellular flux analyser.

Infection with Trypanosoma cruzi Chagas, 1909 is reported to increase the production of reactive oxygen species in patients with Chagas disease. Mitochondria dysfunction, host inflammatory response and inadequate antioxidant response are described as the main factors leading to oxidative stress during acute and chronic stages of the disease. The Seahorse XFe24 extracellular flux platform allows energy metabolism determination through mitochondrial respiration and glycolysis measurements. XFe24 platform can be used in in vitro models of T. cruzi-infected cells, which allow the assessment and even modulation of endogenous conditions of infected cells, generating readouts of real-time cellular bioenergetics changes. In this protocol, we standardised the use of XFe24 technology in T. cruzi infected AC16 cardiomyocytes and SGHPL-5 trophoblasts. In addition, we provide a list of optimised assay specifications, advantages and critical steps to be considered during the process. Cardiomyocytes and trophoblasts are attractive target cells to evaluate the metabolic environment in acute, chronic and congenital Chagas transmission scenarios.

Leishmania amazonensis ferric iron reductase (LFR1) is a bifunctional enzyme: Unveiling a NADPH oxidase activity.

Leishmania amazonensis is one of leishmaniasis' causative agents, a disease that has no cure and leads to the appearance of cutaneous lesions. Recently, our group showed that heme activates a Na+/K+ ATPase in these parasites through a signaling cascade involving hydrogen peroxide (H2O2) generation. Heme has a pro-oxidant activity and signaling capacity, but the mechanism by which this molecule increases H2O2 levels in L. amazonensis has not been elucidated. Here we investigated the source of H2O2 stimulated by heme, ruling out the participation of mitochondria and raising the possibility of a role for a NADPH oxidase (Nox) activity. Despite the absence of a classical Nox sequence in trypanosomatid genomes, L. amazonensis expresses a surface ferric iron reductase (LFR1). Interestingly, Nox enzymes are thought to have evolved from ferric iron reductases because they share same core domain and are very similar in structure. The main difference is that Nox catalyses electron flow from NADPH to oxygen, generating reactive oxygen species (ROS), while ferric iron reductase promotes electron flow to ferric iron, generating ferrous iron. Using L. amazonensis overexpressing or knockout for LFR1 and heterologous expression of LFR1 in mammalian embryonic kidney (HEK 293) cells, we show that this enzyme is bifunctional, being able to generate both ferrous iron and H2O2. It was previously described that protozoans knockout for LFR1 have their differentiation to virulent forms (amastigote and metacyclic promastigote) impaired. In this work, we observed that LFR1 overexpression stimulates protozoan differentiation to amastigote forms, reinforcing the importance of this enzyme in L. amazonensis life cycle regulation. Thus, we not only identified a new source of ROS production in Leishmania, but also described, for the first time, an enzyme with both ferric iron reductase and Nox activities.

First Report of Echinococcus ortleppi in Human Cases of Cystic Echinococcosis in Poland.

Cystic echinococcosis is considered as an emerging zoonosis that can develop asymptomatically for years, clinically nonpathognomic. The disease is of public health importance due to often late, difficult diagnostics, uncertain results of treatment, the need to remove hydatid cysts surgically in advanced cases, and poor prognosis in untreated patients. Six Polish female patients with diagnosed cystic echinococcosis (CE) were examined. DNA extracted from the liver and lung samples served for amplification of mitochondrial nad1 gene fragment. Sequence alignments of 5 isolates showed identity with the pig strain, Echinococcus canadensis G7. One case was in 100% identical with Echinococcus ortleppi G5, the cattle strain. These data demonstrate first report of E. ortleppi, regarded as extinct species, causing human cystic echinococcosis in Poland, where the most frequent causative agent of human CE is E. canadensis.

Leishmania donovani Parasites Are Inhibited by the Benzoxaborole AN2690 Targeting Leucyl-tRNA Synthetase.

Visceral leishmaniasis is an important public health threat in parts of India. It is caused by a protozoan parasite, Leishmania donovani Currently available drugs manifest severe side effects. Hence, there is a need to identify new drug targets and drugs. Aminoacyl-tRNA synthetases, required for protein synthesis, are known drug targets for bacterial and fungal pathogens. The aim of the present study was to obtain essentiality data for Leishmania donovani leucyl-tRNA synthetase (LdLRS) by gene replacement. Gene replacement studies indicate that this enzyme plays an essential role in the viability of this pathogenic organism and appears to be indispensable for its survival in vitro The heterozygous mutant parasites demonstrated a growth deficit and reduced infectivity in mouse macrophages compared to the wild-type cells. We also report that Leishmania donovani recombinant LRS displayed aminoacylation activity and that the protein localized to both the cytosol and the mitochondrion. A broad-spectrum antifungal, 5-fluoro-1,3-dihydro-1-hydroxy-2,1-benzoxaborole (AN2690), was found to inhibit parasite growth in both the promastigote and amastigote stages in vitro as well as in vivo in BALB/c mice. This compound exhibited low toxicity to mammalian cells. AN2690 was effective in inhibiting the aminoacylation activity of the recombinant LdLRS. We provide preliminary chemical validation of LdLRS as a drug target by showing that AN2690 is an inhibitor both of L. donovani LRS and of L. donovani cell growth.

Atovaquone enhances doxorubicin's efficacy via inhibiting mitochondrial respiration and STAT3 in aggressive thyroid cancer.

The clinical management of anaplastic thyroid carcinoma and follicular thyroid carcinoma is challenging and requires an alternative therapeutic strategy. Although atovaquone is an FDA-approved anti-malarial drug, studies has recently demonstrated its anti-cancer activities. In line with these efforts, our study shows that atovaquone is an attractive candidate for thyroid cancer treatment. We show that atovaquone significantly inhibits growth, migration and survival in a concentration-dependent manner in 8505C and FTC113 cells. Mechanistically, atovaquone inhibits mitochondrial complex III activity, leading to mitochondrial respiration inhibition and reduction of ATP production in thyroid cancer cells. The inhibitory effects of atovaquone is reversed in mitochondrial respiration-deficient 8505C ρ0 cells, confirming mitochondrial respiration as the mechanism of atovaquone's action in thyroid cancer. In addition, atovaquone suppresses phosphorylation of STAT3 in thyroid cancer wildype but not ρ0 cells, demonstrating that STAT3 phosphorylation inhibition by atovaquone is a consequence of mitochondrial respiration inhibition. Notably, we further demonstrate that atovaquone significantly augments doxorubicin's inhibitory effects via suppressing mitochondrial respiration and STAT3. Our findings suggest that atovaquone can be repurposed for thyroid cancer treatment. Our work also highlights that targeting mitochondrial respiration may represent potential therapeutic strategy in thyroid cancer.

Antileishmanial Activity, Cytotoxicity and Mechanism of Action of Clioquinol Against Leishmania infantum and Leishmania amazonensis Species.

In this study, a quinoline derivate, clioquinol (5-chloro-7-iodoquinolin-8-ol), was evaluated against Leishmania amazonensis and Leishmania infantum promastigotes and amastigotes. The cytotoxicity in murine macrophages and human red blood cells, as well as the efficacy in treating infected macrophages and the inhibition of infection using pre-treated parasites were also evaluated. Results showed that clioquinol inhibited L. amazonensis and L. infantum promastigotes with effective concentration 50% (EC50 ) values of 2.55 ± 0.25 and 1.44 ± 0.35 µg/mL, respectively, and of 1.88 ± 0.13 and 0.98 ± 0.17 µg/mL against axenic amastigotes, respectively. The cytotoxic EC50 concentrations of clioquinol in murine macrophages and human red blood cells were, respectively, 255 ± 23 and 489 ± 20 µg/mL. With these results, the selectivity index was calculated, showing values of 99.9 and 177.1 against promastigotes, respectively, and of 135.6 and 260.1 against axenic amastigotes, respectively. Significant reductions in the percentage of infected macrophages after treatment using clioquinol were also observed, as well as when parasites were pre-treated with clioquinol and used to infect murine macrophages. The mechanism of action of clioquinol was investigated in L. amazonensis, and results revealed morphological and biochemical alterations in the clioquinol-treated parasites, including reduction in cell volume, loss of mitochondrial membrane potential, increase in the ROS production and rupture of the plasma membrane. The externalization of phosphatidylserine (PS) at the cell surface was evaluated in treated parasites that had been doubly labelled with annexin and propidium iodide (PI). The results showed no significant difference for PS exposure when compared to the untreated control, although a significant increase in the PI/annexin V-labelled cell population was found in the treated parasites. Results suggest that clioquinol induces a discontinuity of the parasite membrane, possibly related to a characteristic event of cell death caused by necrosis. This study demonstrates, for the first time, the antileishmanial activity of clioquinol against two relevant Leishmania species and suggests that the mitochondria of the parasites may be a possible biological target leading to parasite necrosis. Our findings suggest that clioquinol may have a potential application in treatment of leishmaniasis and further studies should be performed in infected mammalian hosts.

Cardiomyocyte diffusible redox mediators control Trypanosoma cruzi infection: role of parasite mitochondrial iron superoxide dismutase.

Chagas disease (CD), caused by the protozoa Trypanosoma cruzi, is a chronic illness in which parasites persist in the host-infected tissues for years. T. cruzi invasion in cardiomyocytes elicits the production of pro-inflammatory mediators [TNF-α, IL-1ß, IFN-γ; nitric oxide (·NO)], leading to mitochondrial dysfunction with increased superoxide radical (O2·-), hydrogen peroxide (H2O2) and peroxynitrite generation. We hypothesize that these redox mediators may control parasite proliferation through the induction of intracellular amastigote programmed cell death (PCD). In this work, we show that T. cruzi (CL-Brener strain) infection in primary cardiomyocytes produced an early (24 h post infection) mitochondrial dysfunction with H2O2 generation and the establishment of an oxidative stress evidenced by FoxO3 activation and target host mitochondrial protein expression (MnSOD and peroxiredoxin 3). TNF-α/IL-1ß-stimulated cardiomyocytes were able to control intracellular amastigote proliferation compared with unstimulated cardiomyocytes. In this condition leading to oxidant formation, an enhanced number of intracellular apoptotic amastigotes were detected. The ability of H2O2 to induce T. cruzi PCD was further confirmed in the epimastigote stage of the parasite. H2O2 treatment induced parasite mitochondrial dysfunction together with intra-mitochondrial O2·- generation. Importantly, parasites genetically engineered to overexpress mitochondrial Fe-superoxide dismutase (Fe-SODA) were more infective to TNF-α/IL-1ß-stimulated cardiomyocytes with less apoptotic amastigotes; this result underscores the role of this enzyme in parasite survival. Our results indicate that cardiomyocyte-derived diffusible mediators are able to control intracellular amastigote proliferation by triggering T. cruzi PCD and that parasite Fe-SODA tilts the process toward survival as part of an antioxidant-based immune evasion mechanism.

A glutaredoxin in the mitochondrial intermembrane space has stage-specific functions in the thermo-tolerance and proliferation of African trypanosomes.

Trypanosoma brucei glutaredoxin 2 (Grx2) is a dithiol glutaredoxin that is specifically located in the mitochondrial intermembrane space. Bloodstream form parasites lacking Grx2 or both, Grx2 and the cytosolic Grx1, are viable in vitro and infectious to mice suggesting that neither oxidoreductase is needed for survival or infectivity to mammals. A 37 °C to 39 °C shift changes the cellular redox milieu of bloodstream cells to more oxidizing conditions and induces a significantly stronger growth arrest in wildtype parasites compared to the mutant cells. Grx2-deficient cells ectopically expressing the wildtype form of Grx2 with its C31QFC34 active site, but not the C34S mutant, regain the sensitivity of the parental strain, indicating that the physiological role of Grx2 requires both active site cysteines. In the procyclic insect stage of the parasite, Grx2 is essential. Both alleles can be replaced if procyclic cells ectopically express authentic or C34S, but not C31S/C34S Grx2, pointing to a redox role that relies on a monothiol mechanism. RNA-interference against Grx2 causes a virtually irreversible proliferation defect. The cells adopt an elongated morphology but do not show any significant alteration in the cell cycle. The growth retardation is attenuated by high glucose concentrations. Under these conditions, procyclic cells obtain ATP by substrate level phosphorylation suggesting that Grx2 might regulate a respiratory chain component.

Targeting host mitochondria: A role for the Trypanosoma cruzi amastigote flagellum.

Trypanosoma cruzi is the kinetoplastid protozoan parasite that causes human Chagas disease, a chronic disease with complex outcomes including severe cardiomyopathy and sudden death. In mammalian hosts, T. cruzi colonises a wide range of tissues and cell types where it replicates within the host cell cytoplasm. Like all intracellular pathogens, T. cruzi amastigotes must interact with its immediate host cell environment in a manner that facilitates access to nutrients and promotes a suitable niche for replication and survival. Although potentially exploitable to devise strategies for pathogen control, fundamental knowledge of the host pathways co-opted by T. cruzi during infection is currently lacking. Here, we report that intracellular T. cruzi amastigotes establish close contact with host mitochondria via their single flagellum. Given the key bioenergetic and homeostatic roles of mitochondria, this striking finding suggests a functional role for host mitochondria in the infection process and points to the T. cruzi amastigote flagellum as an active participant in pathogenesis. Our study establishes the basis for future investigation of the molecular and functional consequences of this intriguing host-parasite interaction.

Copromyxa laresi n. sp. (Amoebozoa: Tubulinea) and Transfer of Cashia limacoides (Page, 1967) to Copromyxa Zopf, 1885.

Five amoeboid organisms of different origin (isolated from fish organs, soil and digestive tract of earthworm) that shared light microscopical and ultrastructural features including type and arrangement of mitochondrial cristae were subjected to phylogenetic analyses based on sequences of SSU rDNA and protein coding genes (actin, cytochrome oxidase I, and eukaryotic elongation factor 2). The reconstruction of multigene phylogeny of the strains studied (i) revealed that they belong to the same single-genus Copromyxa clade; (ii) strongly supported position of Copromyxa cantabrigiensis (syn. Hartmannella cantabrigiensis) within the genus; (iii) together with comparisons of light and electron microscopy data justified reclassification of Cashia limacoides (syn. Vexillifera expectata) to Copromyxa limacoides n. comb., and (iv) justified description of a new species, Copromyxa laresi n. sp.

Proteomic Profiling of Mouse Liver following Acute Toxoplasma gondii Infection.

Toxoplasma gondii remains a global public health problem. However, its pathophysiology is still not-completely understood particularly the impact of infection on host liver metabolism. We performed iTRAQ-based proteomic analysis to evaluate early liver protein responses in BALB/c mice following infection with T. gondii PYS strain (genotype ToxoDB#9) infection. Our data revealed modification of protein expression in key metabolic pathways, as indicated by the upregulation of immune response and downregulation of mitochondrial respiratory chain, and the metabolism of fatty acids, lipids and xenobiotics. T. gondii seems to hijack host PPAR signaling pathway to downregulate the metabolism of fatty acids, lipids and energy in the liver. The metabolism of over 400 substances was affected by the downregulation of genes involved in xenobiotic metabolism. The top 10 transcription factors used by upregulated genes were Stat2, Stat1, Irf2, Irf1, Sp2, Egr1, Stat3, Klf4, Elf1 and Gabpa, while the top 10 transcription factors of downregulated genes were Hnf4A, Ewsr1, Fli1, Hnf4g, Nr2f1, Pparg, Rxra, Hnf1A, Foxa1 and Foxo1. These findings indicate global reprogramming of the metabolism of the mouse liver after acute T. gondii infection. Functional characterization of the altered proteins may enhance understanding of the host responses to T. gondii infection and lead to the identification of new therapeutic targets.

Leishmania donovani activates SREBP2 to modulate macrophage membrane cholesterol and mitochondrial oxidants for establishment of infection.

Establishment of infection by an intracellular pathogen depends on successful internalization with a concomitant neutralization of host defense machinery. Leishmania donovani, an intramacrophage pathogen, targets host SREBP2, a critical transcription factor, to regulate macrophage plasma membrane cholesterol and mitochondrial reactive oxygen species generation, favoring parasite invasion and persistence. Leishmania infection triggered membrane-raft reorientation-dependent Lyn-PI3K/Akt pathway activation which in turn deactivated GSK3ß to stabilize nuclear SREBP2. Moreover, cells perceiving less available intracellular cholesterol due to its sequestration at the plasma membrane resulted in the deregulation of the ER-residing SCAP-SREBP2-Insig circuit thereby assisting increased nuclear translocation of SREBP2. Both increased nuclear transport and stabilization of SREBP2 caused HMGCR-catalyzed cholesterol biosynthesis-mediated plasma membrane cholesterol enrichment leading to decreased membrane-fluidity and plausibly assisting delay in phagosomal acidification. Parasite survival ensuing entry was further ensured by SREBP2-dependent transcriptional up-regulation of UCP2, which suppressed mitochondrial ROS generation, one of the primary microbicidal molecules in macrophages recognized for its efficacy against Leishmania. Functional knock-down of SREBP2 both in vitro and in vivo was associated with reduction in macrophage plasma membrane cholesterol, increased ROS production and lower parasite survival. To our knowledge, this study, for the first time, reveals that Leishmania exploits macrophage cholesterol-dependent SREBP2 circuit to facilitate its entry and survival within the host.

Toxoplasma effector MAF1 mediates recruitment of host mitochondria and impacts the host response.

Recent information has revealed the functional diversity and importance of mitochondria in many cellular processes including orchestrating the innate immune response. Intriguingly, several infectious agents, such as Toxoplasma, Legionella, and Chlamydia, have been reported to grow within vacuoles surrounded by host mitochondria. Although many hypotheses have been proposed for the existence of host mitochondrial association (HMA), the causes and biological consequences of HMA have remained unanswered. Here we show that HMA is present in type I and III strains of Toxoplasma but missing in type II strains, both in vitro and in vivo. Analysis of F1 progeny from a type II×III cross revealed that HMA is a Mendelian trait that we could map. We use bioinformatics to select potential candidates and experimentally identify the polymorphic parasite protein involved, mitochondrial association factor 1 (MAF1). We show that introducing the type I (HMA+) MAF1 allele into type II (HMA-) parasites results in conversion to HMA+ and deletion of MAF1 in type I parasites results in a loss of HMA. We observe that the loss and gain of HMA are associated with alterations in the transcription of host cell immune genes and the in vivo cytokine response during murine infection. Lastly, we use exogenous expression of MAF1 to show that it binds host mitochondria and thus MAF1 is the parasite protein directly responsible for HMA. Our findings suggest that association with host mitochondria may represent a novel means by which Toxoplasma tachyzoites manipulate the host. The existence of naturally occurring HMA+ and HMA- strains of Toxoplasma, Legionella, and Chlamydia indicates the existence of evolutionary niches where HMA is either advantageous or disadvantageous, likely reflecting tradeoffs in metabolism, immune regulation, and other functions of mitochondria.

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

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.