Activation of innate immunity selectively compromises mitochondrial complex I, proline oxidation, and flight activity in the major arbovirus vector Aedes aegypti.

Aedes aegypti females are natural vectors of important arboviruses such as dengue, zika, and yellow fever. Mosquitoes activate innate immune response signaling pathways upon infection, as a resistance mechanism to fight pathogens and limit their propagation. Despite the beneficial effects of immune activation for insect vectors, phenotypic costs ultimately affect their fitness. However, the underlying mechanisms that mediate these fitness costs remain poorly understood. Given the high energy required to mount a proper immune response, we hypothesized that systemic activation of innate immunity would impair flight muscle mitochondrial function, compromising tissue energy demand and flight activity. Here, we investigated the dynamic effects of activation of innate immunity by intra-thoracic zymosan injection on A. aegypti flight muscle mitochondrial metabolism. Zymosan injection significantly increased defensin A expression in fat bodies in a time-dependent manner that compromised flight activity. Although oxidant levels in flight muscle were hardly altered, ATP-linked respiratory rates driven by mitochondrial pyruvate+proline oxidation were significantly reduced at 24 h upon zymosan injection. Oxidative phosphorylation coupling was preserved regardless of innate immune response activation along 24 h. Importantly, rotenone-sensitive respiration and complex I-III activity were specifically reduced 24 h upon zymosan injection. Also, loss of complex I activity compromised ATP-linked and maximal respiratory rates mediated by mitochondrial proline oxidation. Finally, the magnitude of innate immune response activation negatively correlated with respiratory rates, regardless of the metabolic states. Collectively, we demonstrate that activation of innate immunity is strongly associated with reduced flight muscle complex I activity with direct consequences to mitochondrial proline oxidation and flight activity. Remarkably, our results indicate a trade-off between dispersal and immunity exists in an insect vector, underscoring the potential consequences of disrupted flight muscle mitochondrial energy metabolism to arbovirus transmission.

Export of heme by the feline leukemia virus C receptor regulates mitochondrial biogenesis and redox balance in the hematophagous insect Rhodnius prolixus.

Heme is a prosthetic group of proteins involved in vital physiological processes. It participates, for example, in redox reactions crucial for cell metabolism due to the variable oxidation state of its central iron atom. However, excessive heme can be cytotoxic due to its prooxidant properties. Therefore, the control of intracellular heme levels ensures the survival of organisms, especially those that deal with high concentrations of heme during their lives, such as hematophagous insects. The export of heme initially attributed to the feline leukemia virus C receptor (FLVCR) has recently been called into question, following the discovery of choline uptake by the same receptor in mammals. Here, we found that RpFLVCR is a heme exporter in the midgut of the hematophagous insect Rhodnius prolixus, a vector for Chagas disease. Silencing RpFLVCR decreased hemolymphatic heme levels and increased the levels of intracellular dicysteinyl-biliverdin, indicating heme retention inside midgut cells. FLVCR silencing led to increased expression of heme oxygenase (HO), ferritin, and mitoferrin mRNAs while downregulating the iron importers Malvolio 1 and 2. In contrast, HO gene silencing increased FLVCR and Malvolio expression and downregulated ferritin, revealing crosstalk between heme degradation/export and iron transport/storage pathways. Furthermore, RpFLVCR silencing strongly increased oxidant production and lipid peroxidation, reduced cytochrome c oxidase activity, and activated mitochondrial biogenesis, effects not observed in RpHO-silenced insects. These data support FLVCR function as a heme exporter, playing a pivotal role in heme/iron metabolism and maintenance of redox balance, especially in an organism adapted to face extremely high concentrations of heme.

Mechanical Permeabilization as a New Method for Assessment of Mitochondrial Function in Insect Tissues.

Respirometry analysis is an effective technique to assess mitochondrial physiology. Insects are valuable biochemical models to understand metabolism and human diseases. Insect flight muscle and brain have been extensively used to explore mitochondrial function due to dissection feasibility and the low sample effort to allow oxygen consumption measurements. However, adequate plasma membrane permeabilization is required for substrates/modulators to reach mitochondria. Here, we describe a new method for study of mitochondrial physiology in insect tissues based on mechanical permeabilization as a fast and reliable method that do not require the use of detergents for chemical permeabilization of plasma membrane, while preserves mitochondrial integrity.

Anti-Virulence Strategy against the Multidrug-Resistant Bacterial Pathogen Pseudomonas aeruginosa: Pseudolysin (Elastase B) as a Potential Druggable Target.

Pseudomonas aeruginosa is a non-fermentative, gram-negative bacterium that is one of the most common pathogens responsible for hospital-acquired infections worldwide. The management of the infections caused by P. aeruginosa represents a huge challenge in the healthcare settings due to the increased emergence of resistant isolates, some of them resistant to all the currently available antimicrobials, which results in elevated morbimortality rates. Consequently, the development of new therapeutic strategies against multidrug-resistant P. aeruginosa is urgent and needful. P. aeruginosa is wellrecognized for its extreme genetic versatility and its ability to produce a lush variety of virulence factors. In this context, pseudolysin (or elastase B) outstands as a pivotal virulence attribute during the infectious process, playing multifunctional roles in different aspects of the pathogen-host interaction. This protein is a 33-kDa neutral zinc-dependent metallopeptidase that is the most abundant peptidase found in pseudomonal secretions, which contributes to the invasiveness of P. aeruginosa due to its ability to cleave several extracellular matrix proteins and to disrupt the basolateral intercellular junctions present in the host tissues. Moreover, pseudolysin makes P. aeruginosa able to overcome host defenses by the hydrolysis of many immunologically relevant molecules, including antibodies and complement components. The attenuation of this striking peptidase therefore emerges as an alternative and promising antivirulence strategy to combat antibiotic-refractory infections caused by P. aeruginosa. The anti-virulence approach aims to disarm the P. aeruginosa infective arsenal by inhibiting the expression/activity of bacterial virulence factors in order to reduce the invasiveness of P. aeruginosa, avoiding the emergence of resistance since the proliferation is not affected. This review summarizes the most relevant features of pseudolysin and highlights this enzyme as a promising target for the development of new anti-virulence compounds.

Modulation of mitochondrial metabolism as a biochemical trait in blood feeding organisms: the redox vampire hypothesis redux.

Hematophagous organisms undergo remarkable metabolic changes during the blood digestion process, increasing fermentative glucose metabolism, and reducing respiratory rates, both consequence of functional mitochondrial remodeling. Here, we review the pathways involved in energy metabolism and mitochondrial functionality in a comparative framework across different hematophagous species, and consider how these processes regulate redox homeostasis during blood digestion. The trend across distinct species indicate that a switch in energy metabolism might represent an important defensive mechanism to avoid the potential harmful interaction of oxidants generated from aerobic energy metabolism with products derived from blood digestion. Indeed, in insect vectors, blood feeding transiently reduces respiratory rates and oxidant production, irrespective of tissue and insect model. On the other hand, a different scenario is observed in several unrelated parasite species when exposed to blood digestion products, as respiratory rates reduce and mitochondrial oxidant production increase. The emerging picture indicates that re-wiring of energy metabolism, through reduced mitochondrial function, culminates in improved tolerance to redox insults and seems to represent a key step for hematophagous organisms to cope with the overwhelming and potentially toxic blood meal.

Promoting Drp1-mediated mitochondrial fission in midlife prolongs healthy lifespan of Drosophila melanogaster.

The accumulation of dysfunctional mitochondria has been implicated in aging, but a deeper understanding of mitochondrial dynamics and mitophagy during aging is missing. Here, we show that upregulating Drp1-a Dynamin-related protein that promotes mitochondrial fission-in midlife, prolongs Drosophila lifespan and healthspan. We find that short-term induction of Drp1, in midlife, is sufficient to improve organismal health and prolong lifespan, and observe a midlife shift toward a more elongated mitochondrial morphology, which is linked to the accumulation of dysfunctional mitochondria in aged flight muscle. Promoting Drp1-mediated mitochondrial fission, in midlife, facilitates mitophagy and improves both mitochondrial respiratory function and proteostasis in aged flies. Finally, we show that autophagy is required for the anti-aging effects of midlife Drp1-mediated mitochondrial fission. Our findings indicate that interventions that promote mitochondrial fission could delay the onset of pathology and mortality in mammals when applied in midlife.Mitochondrial fission and fusion are important mechanisms to maintain mitochondrial function. Here, the authors report that middle-aged flies have more elongated, or 'hyper-fused' mitochondria, and show that induction of mitochondrial fission in midlife, but not in early life, extends the health and life of flies.

Heme modulates Trypanosoma cruzi bioenergetics inducing mitochondrial ROS production.

Trypanosoma cruzi is the causative agent of Chagas disease and has a single mitochondrion, an organelle responsible for ATP production and the main site for the formation of reactive oxygen species (ROS). T. cruzi is an obligate intracellular parasite with a complex life cycle that alternates between vertebrate and invertebrate hosts, therefore the development of survival strategies and morphogenetic adaptations to deal with the various environments is mandatory. Over the years our group has been studying the vector-parasite interactions using heme as a physiological oxidant molecule that triggered epimastigote proliferation however, the source of ROS induced by heme remained unknown. In the present study we demonstrate the involvement of heme in the parasite mitochondrial metabolism, decreasing oxygen consumption leading to increased mitochondrial ROS and membrane potential. First, we incubated epimastigotes with carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP), an uncoupler of oxidative phosphorylation, which led to decreased ROS formation and parasite proliferation, even in the presence of heme, correlating mitochondrial ROS and T. cruzi survival. This hypothesis was confirmed after the mitochondria-targeted antioxidant ((2-(2,2,6,6 Tetramethylpiperidin-1-oxyl-4-ylamino)-2-oxoethyl) triphenylphosphonium chloride (MitoTEMPO) decreased both heme-induced ROS and epimastigote proliferation. Furthermore, heme increased the percentage of tetramethylrhodamine methyl ester (TMRM) positive parasites tremendously-indicating the hyperpolarization and increase of potential of the mitochondrial membrane (ΔΨm). Assessing the mitochondrial functional metabolism, we observed that in comparison to untreated parasites, heme-treated epimastigotes decreased their oxygen consumption, and increased the complex II-III activity. These changes allowed the electron flow into the electron transport system, even though the complex IV (cytochrome c oxidase) activity decreased significantly, showing that heme-induced mitochondrial ROS appears to be a consequence of the enhanced mitochondrial physiological modulation. Finally, the parasites that were submitted to high concentrations of heme presented no alterations in the ultrastructure. Consequently, our results suggest that heme released by the insect vector after the blood meal, modify epimastigote mitochondrial physiology to increase ROS as a metabolic mechanism to maintain epimastigote survival and proliferation.

Sexual Preferences in Nutrient Utilization Regulate Oxygen Consumption and Reactive Oxygen Species Generation in Schistosoma mansoni: Potential Implications for Parasite Redox Biology.

Schistosoma mansoni, one of the causative agents of human schistosomiasis, has a unique antioxidant network that is key to parasite survival and a valuable chemotherapeutic target. The ability to detoxify and tolerate reactive oxygen species increases along S. mansoni development in the vertebrate host, suggesting that adult parasites are more exposed to redox challenges than young stages. Indeed, adult parasites are exposed to multiple redox insults generated from blood digestion, activated immune cells, and, potentially, from their own parasitic aerobic metabolism. However, it remains unknown how reactive oxygen species are produced by S. mansoni metabolism, as well as their biological effects on adult worms. Here, we assessed the contribution of nutrients and parasite gender to oxygen utilization pathways, and reactive oxygen species generation in whole unpaired adult S. mansoni worms. We also determined the susceptibilities of both parasite sexes to a pro-oxidant challenge. We observed that glutamine and serum importantly contribute to both respiratory and non-respiratory oxygen utilization in adult worms, but with different proportions among parasite sexes. Analyses of oxygen utilization pathways revealed that respiratory rates were high in male worms, which contrast with high non-respiratory rates in females, regardless nutritional sources. Interestingly, mitochondrial complex I-III activity was higher than complex IV specifically in females. We also observed sexual preferences in substrate utilization to sustain hydrogen peroxide production towards glucose in females, and glutamine in male worms. Despite strikingly high oxidant levels and hydrogen peroxide production rates, female worms were more resistant to a pro-oxidant challenge than male parasites. The data presented here indicate that sexual preferences in nutrient metabolism in adult S. mansoni worms regulate oxygen utilization and reactive oxygen species production, which may differently contribute to redox biology among parasite sexes.

Detergent-Mediated Formation of ß-Hematin: Heme Crystallization Promoted by Detergents Implicates Nanostructure Formation for Use as a Biological Mimic.

Hemozoin is a unique biomineral that results from the sequestration of toxic free heme liberated as a consequence of hemoglobin degradation in the malaria parasite. Synthetic neutral lipid droplets (SNLDs) and phospholipids were previously shown to support the rapid formation of ß-hematin, abiological hemozoin, under physiologically relevant pH and temperature, though the mechanism by which heme crystallization occurs remains unclear. Detergents are particularly interesting as a template because they are amphiphilic molecules that spontaneously organize into nanostructures and have been previously shown to mediate ß-hematin formation. Here, 11 detergents were investigated to elucidate the physicochemical properties that best recapitulate crystal formation in the parasite. A strong correlation between the detergent's molecular structure and the corresponding kinetics of ß-hematin formation was observed, where higher molecular weight polar chains promoted faster reactions. The larger hydrophilic chains correlated to the detergent's ability to rapidly sequester heme into the lipophilic core, allowing for crystal nucleation to occur. The data presented here suggest that detergent nanostructures promote ß-hematin formation in a similar manner to SNLDs and phospholipids. Through understanding mediator properties that promote optimal crystal formation, we are able to establish an in vitro assay to probe this drug target pathway.

Classical ROS-dependent and early/rapid ROS-independent release of Neutrophil Extracellular Traps triggered by Leishmania parasites.

Neutrophil extracellular traps (NETs) extruded from neutrophils upon activation are composed of chromatin associated with cytosolic and granular proteins, which ensnare and kill microorganisms. This microbicidal mechanism named classical netosis has been shown to dependent on reactive oxygen species (ROS) generation by NADPH oxidase and also chromatin decondensation dependent upon the enzymes (PAD4), neutrophil elastase (NE) and myeloperoxidase (MPO). NET release also occurs through an early/rapid ROS-independent mechanism, named early/rapid vital netosis. Here we analyze the role of ROS, NE, MPO and PAD4 in the netosis stimulated by Leishmania amazonensis promastigotes in human neutrophils. We demonstrate that promastigotes induce a classical netosis, dependent on the cellular redox imbalance, as well as by a chloroamidine sensitive and elastase activity mechanism. Additionally, Leishmania also induces the early/rapid NET release occurring only 10 minutes after neutrophil-parasite interaction. We demonstrate here, that this early/rapid mechanism is dependent on elastase activity, but independent of ROS generation and chloroamidine. A better understanding of both mechanisms of NET release, and the NETs effects on the host immune system modulation, could support the development of new potential therapeutic strategies for leishmaniasis.

Silencing of maternal heme-binding protein causes embryonic mitochondrial dysfunction and impairs embryogenesis in the blood sucking insect Rhodnius prolixus.

The heme molecule is the prosthetic group of many hemeproteins involved in essential physiological processes, such as electron transfer, transport of gases, signal transduction, and gene expression modulation. However, heme is a pro-oxidant molecule capable of propagating reactions leading to the generation of reactive oxygen species. The blood-feeding insect Rhodnius prolixus releases enormous amounts of heme during host blood digestion in the midgut lumen when it is exposed to a physiological oxidative challenge. Additionally, this organism produces a hemolymphatic heme-binding protein (RHBP) that transports heme to pericardial cells for detoxification and to growing oocytes for yolk granules and as a source of heme for embryo development. Here, we show that silencing of RHBP expression in female fat bodies reduced total RHBP circulating in the hemolymph, promoting oxidative damage to hemolymphatic proteins. Moreover, RHBP knockdown did not cause reduction in oviposition but led to the production of heme-depleted eggs (white eggs). A lack of RHBP did not alter oocyte fecundation. However, produced white eggs were nonviable. Embryo development cellularization and vitellin yolk protein degradation, processes that normally occur in early stages of embryogenesis, were compromised in white eggs. Total cytochrome c content, cytochrome c oxidase activity, citrate synthase activity, and oxygen consumption, parameters that indicate mitochondrial function, were significantly reduced in white eggs compared with normal dark red eggs. Our results showed that reduction of heme transport from females to growing oocytes by RHBP leads to embryonic mitochondrial dysfunction and impaired embryogenesis.