Jose Henrique M. Oliveira

Blood meal-derived heme decreases ROS levels in the midgut of Aedes aegypti and allows proliferation of intestinal microbiota

The presence of bacteria in the midgut of mosquitoes antagonizes infectious agents, such as Dengue and Plasmodium, acting as a negative factor in the vectorial competence of the mosquito. Therefore, knowledge of the molecular mechanisms involved in the control of midgut microbiota could help in the development of new tools to reduce transmission. We hypothesized that toxic reactive oxygen species (ROS) generated by epithelial cells control bacterial growth in the midgut of Aedes aegypti, the vector of Yellow fever and Dengue viruses. We show that ROS are continuously present in the midgut of sugar-fed (SF) mosquitoes and a blood-meal immediately decreased ROS through a mechanism involving heme-mediated activation of PKC. This event occurred in parallel with an expansion of gut bacteria. Treatment of sugar-fed mosquitoes with increased concentrations of heme led to a dose dependent decrease in ROS levels and a consequent increase in midgut endogenous bacteria. In addition, gene silencing of dual oxidase (Duox) reduced ROS levels and also increased gut flora. Using a model of bacterial oral infection in the gut, we show that the absence of ROS resulted in decreased mosquito resistance to infection, increased midgut epithelial damage, transcriptional modulation of immune-related genes and mortality. As heme is a pro-oxidant molecule released in large amounts upon hemoglobin degradation, oxidative killing of bacteria in the gut would represent a burden to the insect, thereby creating an extra oxidative challenge to the mosquito. We propose that a controlled decrease in ROS levels in the midgut of Aedes aegypti is an adaptation to compensate for the ingestion of heme.

The Role of Reactive Oxygen Species in Anopheles aquasalis Response to Plasmodium vivax Infection

Malaria affects millions of people worldwide and hundreds of thousands of people each year in Brazil. The mosquito Anopheles aquasalis is an important vector of Plasmodium vivax, the main human malaria parasite in the Americas. Reactive oxygen species (ROS) have been shown to have a role in insect innate immune responses as a potent pathogen-killing agent. We investigated the mechanisms of free radicals modulation after A. aquasalis infection with P. vivax. ROS metabolism was evaluated in the vector by studying expression and activity of three key detoxification enzymes, one catalase and two superoxide dismutases (SOD3A and SOD3B). Also, the involvement of free radicals in the mosquito immunity was measured by silencing the catalase gene followed by infection of A. aquasalis with P. vivax. Catalase, SOD3A and SOD3B expression in whole A. aquasalis were at the same levels of controls at 24 h and upregulated 36 h after ingestion of blood containing P. vivax. However, in the insect isolated midgut, the mRNA for these enzymes was not regulated by P. vivax infection, while catalase activity was reduced 24 h after the infectious meal. RNAi-mediated silencing of catalase reduced enzyme activity in the midgut, resulted in increased P. vivax infection and prevalence, and decreased bacterial load in the mosquito midgut. Our findings suggest that the interactions between A. aquasalis and P. vivax do not follow the model of ROS-induced parasite killing. It appears that P. vivax manipulates the mosquito detoxification system in order to allow its own development. This can be an indirect effect of fewer competitive bacteria present in the mosquito midgut caused by the increase of ROS after catalase silencing. These findings provide novel information on unique aspects of the main malaria parasite in the Americas interaction with one of its natural vectors.

Tracking Resilience to Infections by Mapping Disease Space

Infected hosts differ in their responses to pathogens; some hosts are resilient and recover their original health, whereas others follow a divergent path and die. To quantitate these differences, we propose mapping the routes infected individuals take through “disease space.” We find that when plotting physiological parameters against each other, many pairs have hysteretic relationships that identify the current location of the host and predict the future route of the infection. These maps can readily be constructed from experimental longitudinal data, and we provide two methods to generate the maps from the cross-sectional data that is commonly gathered in field trials. We hypothesize that resilient hosts tend to take small loops through disease space, whereas nonresilient individuals take large loops. We support this hypothesis with experimental data in mice infected with Plasmodium chabaudi, finding that dying mice trace a large arc in red blood cells (RBCs) by reticulocyte space as compared to surviving mice. We find that human malaria patients who are heterozygous for sickle cell hemoglobin occupy a small area of RBCs by reticulocyte space, suggesting this approach can be used to distinguish resilience in human populations. This technique should be broadly useful in describing the in-host dynamics of infections in both model hosts and patients at both population and individual levels.

Mitochondrial Reactive Oxygen Species Modulate Mosquito Susceptibility to Plasmodium Infection

Background Mitochondria perform multiple roles in cell biology, acting as the site of aerobic energy-transducing pathways and as an important source of reactive oxygen species (ROS) that modulate redox metabolism. Methodology/Principal Findings We demonstrate that a novel member of the mitochondrial transporter protein family, Anopheles gambiae mitochondrial carrier 1 (AgMC1), is required to maintain mitochondrial membrane potential in mosquito midgut cells and modulates epithelial responses to Plasmodium infection. AgMC1 silencing reduces mitochondrial membrane potential, resulting in increased proton-leak and uncoupling of oxidative phosphorylation. These metabolic changes reduce midgut ROS generation and increase A. gambiae susceptibility to Plasmodium infection. Conclusion We provide direct experimental evidence indicating that ROS derived from mitochondria can modulate mosquito epithelial responses to Plasmodium infection.

Energy metabolism affects susceptibility of Anopheles gambiae mosquitoes to Plasmodium infection

Previous studies showed that Anopheles gambiae L3-5 females, which are refractory (R) to Plasmodium infection, express higher levels of genes involved in redox-metabolism and mitochondrial respiration than susceptible (S) G3 females. Our studies revealed that R females have reduced longevity, faster utilization of lipid reserves, impaired mitochondrial state-3 respiration, increased rate of mitochondrial electron leak and higher expression levels of several glycolytic enzyme genes. Furthermore, when state-3 respiration was reduced in S females by silencing expression of the adenine nucleotide translocator (ANT), hydrogen peroxide generation was higher and the mRNA levels of lactate dehydrogenase increased in the midgut, while the prevalence and intensity of Plasmodium berghei infection were significantly reduced. We conclude that there are broad metabolic differences between R and S An. gambiae mosquitoes that influence their susceptibility to Plasmodium infection.

Antioxidant pathways are up-regulated during biological nitrogen fixation to prevent ROS-induced nitrogenase inhibition in Gluconacetobacter diazotrophicus

Gluconacetobacter diazotrophicus, an endophyte isolated from sugarcane, is a strict aerobe that fixates N2. This process is catalyzed by nitrogenase and requires copious amounts of ATP. Nitrogenase activity is extremely sensitive to inhibition by oxygen and reactive oxygen species (ROS). However, the elevated oxidative metabolic rates required to sustain biological nitrogen fixation (BNF) may favor an increased production of ROS. Here, we explored this paradox and observed that ROS levels are, in fact, decreased in nitrogen-fixing cells due to the up-regulation of transcript levels of six ROS-detoxifying genes. A cluster analyses based on common expression patterns revealed the existence of a stable cluster with 99.8% similarity made up of the genes encoding the α-subunit of nitrogenase Mo–Fe protein (nifD), superoxide dismutase (sodA) and catalase type E (katE). Finally, nitrogenase activity was inhibited in a dose-dependent manner by paraquat, a redox cycler that increases cellular ROS levels. Our data revealed that ROS can strongly inhibit nitrogenase activity, and G. diazotrophicus alters its redox metabolism during BNF by increasing antioxidant transcript levels resulting in a lower ROS generation. We suggest that careful controlled ROS production during this critical phase is an adaptive mechanism to allow nitrogen fixation.

Blood-Feeding Induces Reversible Functional Changes in Flight Muscle Mitochondria of Aedes aegypti Mosquito

Background Hematophagy poses a challenge to blood-feeding organisms since products of blood digestion can exert cellular deleterious effects. Mitochondria perform multiple roles in cell biology acting as the site of aerobic energy-transducing pathways, and also an important source of reactive oxygen species (ROS), modulating redox metabolism. Therefore, regulation of mitochondrial function should be relevant for hematophagous arthropods. Here, we investigated the effects of blood-feeding on flight muscle (FM) mitochondria from the mosquito Aedes aegypti, a vector of dengue and yellow fever. Methodology/Principal Findings Blood-feeding caused a reversible reduction in mitochondrial oxygen consumption, an event that was parallel to blood digestion. These changes were most intense at 24 h after blood meal (ABM), the peak of blood digestion, when oxygen consumption was inhibited by 68%. Cytochromes c and a+a 3 levels and cytochrome c oxidase activity of the electron transport chain were all reduced at 24 h ABM. Ultrastructural and molecular analyses of FM revealed that mitochondria fuse upon blood meal, a condition related to reduced ROS generation. Consistently, BF induced a reversible decrease in mitochondrial H2O2 formation during blood digestion, reaching their lowest values at 24 h ABM where a reduction of 51% was observed. Conclusion Blood-feeding triggers functional and structural changes in hematophagous insect mitochondria, which may represent an important adaptation to blood feeding.

Catalase protects Aedes aegypti from oxidative stress and increases midgut infection prevalence of Dengue but not Zika

Background Digestion of blood in the midgut of Aedes aegypti results in the release of pro-oxidant molecules that can be toxic to the mosquito. We hypothesized that after a blood meal, the antioxidant capacity of the midgut is increased to protect cells against oxidative stress. Concomitantly, pathogens present in the blood ingested by mosquitoes, such as the arboviruses Dengue and Zika, also have to overcome the same oxidative challenge, and the antioxidant program induced by the insect is likely to influence infection status of the mosquito and its vectorial competence. Methodology/Principal findings We found that blood-induced catalase mRNA and activity in the midgut peaked 24 h after feeding and returned to basal levels after the completion of digestion. RNAi-mediated silencing of catalase (AAEL013407-RB) reduced enzyme activity in the midgut epithelia, increased H2O2 leakage and decreased fecundity and lifespan when mosquitoes were fed H2O2. When infected with Dengue 4 and Zika virus, catalase-silenced mosquitoes showed no alteration in infection intensity (number of plaque forming units/midgut) 7 days after the infectious meal. However, catalase knockdown reduced Dengue 4, but not Zika, infection prevalence (percent of infected midguts). Conclusion/Significance Here, we showed that blood ingestion triggers an antioxidant response in the midgut through the induction of catalase. This protection facilitates the establishment of Dengue virus in the midgut. Importantly, this mechanism appears to be specific for Dengue because catalase silencing did not change Zika virus prevalence. In summary, our data suggest that redox balance in the midgut modulates mosquito vectorial competence to arboviral infections.

Microbiota activates IMD pathway and limits Sindbis infection in Aedes aegypti

BackgroundAedes aegypti is the main vector of important arboviruses such as dengue, Zika and chikungunya. During infections mosquitoes can activate the immune pathways Toll, IMD and JAK/STAT to limit pathogen replication.ResultsHere, we evaluate the immune response profile of Ae. aegypti against Sindbis virus (SINV). We analyzed gene expression of components of Toll, IMD and JAK/STAT pathways and showed that a blood meal and virus infection upregulated aaREL2 in a microbiota-dependent fashion, since this induction was prevented by antibiotic. The presence of the microbiota activates IMD and impaired the replication of SINV in the midgut. Constitutive activation of the IMD pathway, by Caspar depletion, leads to a decrease in microbiota levels and an increase in SINV loads.ConclusionTogether, these results suggest that a blood meal is able to activate innate immune pathways, through a nutrient induced growth of microbiota, leading to upregulation of aaREL2 and IMD activation. Microbiota levels seemed to have a reciprocal interaction, where the proliferation of the microbiota activates IMD pathway that in turn controls bacterial levels, allowing SINV replication in Ae. aegypti mosquitoes. The activation of the IMD pathway seems to have an indirect effect in SINV levels that is induced by the microbiota.

The Dose Makes the Poison: Nutritional Overload Determines the Life Traits of Blood-Feeding Arthropods

Vertebrate blood composition is heavily biased towards proteins, and hemoglobin, which is a hemeprotein, is by far the most abundant protein. Typically, hematophagous insects ingest blood volumes several times their weight before the blood meal. This barbarian feast offers an abundance of nutrients, but the degradation of blood proteins generates toxic concentrations of amino acids and heme, along with unparalleled microbiota growth. Despite this challenge, hematophagous arthropods have successfully developed mechanisms that bypass the toxicity of these molecules. While these adaptations allow hematophagous arthropods to tolerate their diet, they also constitute a unique mode of operation for cell signaling, immunity, and metabolism, the study of which may offer insights into the biology of disease vectors and may lead to novel vector-specific control methods.