Carolina Barillas-Mury

Evolutionary dynamics of immune-related genes and pathways in disease-vector mosquitoes

Mosquitoes are vectors of parasitic and viral diseases of immense importance for public health. The acquisition of the genome sequence of the yellow fever and Dengue vector, Aedes aegypti (Aa), has enabled a comparative phylogenomic analysis of the insect immune repertoire: in Aa, the malaria vector Anopheles gambiae (Ag), and the fruit fly Drosophila melanogaster (Dm). Analysis of immune signaling pathways and response modules reveals both conservative and rapidly evolving features associated with different functional gene categories and particular aspects of immune reactions. These dynamics reflect in part continuous readjustment between accommodation and rejection of pathogens and suggest how innate immunity may have evolved.

Molecular interactions between Anopheles stephensi midgut cells and Plasmodium berghei: the time bomb theory of ookinete invasion of mosquitoes

We present a detailed analysis of the interactions between Anopheles stephensi midgut epithelial cells and Plasmodium berghei ookinetes during invasion of the mosquito by the parasite. In this mosquito, P.berghei ookinetes invade polarized columnar epithelial cells with microvilli, which do not express high levels of vesicular ATPase. The invaded cells are damaged, protrude towards the midgut lumen and suffer other characteristic changes, including induction of nitric oxide synthase (NOS) expression, a substantial loss of microvilli and genomic DNA fragmentation. Our results indicate that the parasite inflicts extensive damage leading to subsequent death of the invaded cell. Ookinetes were found to be remarkably plastic, to secrete a subtilisin‐like serine protease and the GPI‐anchored surface protein Pbs21 into the cytoplasm of invaded cells, and to be capable of extensive lateral movement between cells. The epithelial damage inflicted is repaired efficiently by an actin purse‐string‐mediated restitution mechanism, which allows the epithelium to ‘bud off’ the damaged cells without losing its integrity. A new model, the time bomb theory of ookinete invasion, is proposed and its implications are discussed.

Genome expression analysis of Anopheles gambiae: Responses to injury, bacterial challenge, and malaria infection

The complex gene expression responses of Anopheles gambiae to microbial and malaria challenges, injury, and oxidative stress (in the mosquito and/or a cultured cell line) were surveyed by using cDNA microarrays constructed from an EST-clone collection. The expression profiles were broadly subdivided into induced and down-regulated gene clusters. Gram+ and Gram− bacteria and microbial elicitors up-regulated a diverse set of genes, many belonging to the immunity class, and the response to malaria partially overlapped with this response. Oxidative stress activated a distinctive set of genes, mainly implicated in oxidoreductive processes. Injury up- and down-regulated gene clusters also were distinctive, prominently implicating glycolysis-related genes and citric acid cycle/oxidative phosphorylation/redox-mitochondrial functions, respectively. Cross-comparison of in vivo and in vitro responses indicated the existence of tightly coregulated gene groups that may correspond to gene pathways.

Hemocyte Differentiation Mediates Innate Immune Memory in Anopheles gambiae Mosquitoes

Mosquito Malarial Memory During their life cycle malaria parasites produce vast numbers of successive proliferative stages in their vertebrate hosts, and yet in the field most mosquitoes are free of parasites. Rodrigues et al. (p. 1353) report that the immune system of mosquitoes is primed early-on when the malaria parasite (Plasmodium spp.) first crosses the mosquito gut epithelial barrier. A substantial (2- to 3.2-fold) increase in a single type of hemocyte (macrophage-like insect immune cells) is implicated in long-lived antiplasmodial immunity. This work may prove important for malaria control and for understanding immune memory in invertebrates. Early immune priming limits malaria parasite infection of mosquitoes. Mosquito midgut invasion by ookinetes of the malaria parasite Plasmodium disrupts the barriers that normally prevent the gut microbiota from coming in direct contact with epithelial cells. This triggers a long-lived response characterized by increased abundance of granulocytes, a subpopulation of hemocytes that circulates in the insect’s hemocoel, and enhanced immunity to bacteria that indirectly reduces survival of Plasmodium parasites upon reinfection. In mosquitoes, differentiation of hemocytes was necessary and sufficient to confer innate immune memory.

The role of reactive oxygen species on Plasmodium melanotic encapsulation in Anopheles gambiae.

Malaria transmission depends on the competence of some Anopheles mosquitoes to sustain Plasmodium development (susceptibility). A genetically selected refractory strain of Anopheles gambiae blocks Plasmodium development, melanizing, and encapsulating the parasite in a reaction that begins with tyrosine oxidation, and involves three quantitative trait loci. Morphological and microarray mRNA expression analysis suggest that the refractory and susceptible strains have broad physiological differences, which are related to the production and detoxification of reactive oxygen species. Physiological studies corroborate that the refractory strain is in a chronic state of oxidative stress, which is exacerbated by blood feeding, resulting in increased steady-state levels of reactive oxygen species, which favor melanization of parasites as well as Sephadex beads.

Flavivirus susceptibility in Aedes aegypti

Aedes aegypti is the primary vector of yellow fever (YF) and dengue fever (DF) flaviviruses worldwide. In this review we focus on past and present research on genetic components and environmental factors in Aedes aegypti that appear to control flavivirus transmission. We review genetic relationships among Ae. aegypti populations throughout the world and discuss how variation in vector competence is correlated with overall genetic differences among populations. We describe current research into how genetic and environmental factors jointly affect distribution of vector competence in natural populations. Based on this information, we propose a population genetic model for vector competence and discuss our recent progress in testing this model. We end with a discussion of approaches being taken to identify the genes that may control flavivirus susceptibility in Ae. aegypti.

A Role for Insect Galectins in Parasite Survival

Insect galectins are associated with embryonic development or immunity against pathogens. Here, we show that they can be exploited by parasites for survival in their insect hosts. PpGalec, a tandem repeat galectin expressed in the midgut of the sandfly Phlebotomus papatasi, is used by Leishmania major as a receptor for mediating specific binding to the insect midgut, an event crucial for parasite survival, and accounts for species-specific vector competence for the most widely distributed form of cutaneous leishmaniasis in the Old World. In addition, these studies demonstrate the feasibility of using midgut receptors for parasite ligands as target antigens for transmission-blocking vaccines.

Anopheles gambiae Ag‐STAT, a new insect member of the STAT family, is activated in response to bacterial infection

A new insect member of the STAT family of transcription factors (Ag‐STAT) has been cloned from the human malaria vector Anopheles gambiae. The domain involved in DNA interaction and the SH2 domain are well conserved. Ag‐STAT is most similar to Drosophila D‐STAT and to vertebrate STATs 5 and 6, constituting a proposed ancient class A of the STAT family. The mRNA is expressed at all developmental stages, and the protein is present in hemocytes, pericardial cells, midgut, skeletal muscle and fat body cells. There is no evidence of transcriptional activation following bacterial challenge. However, bacterial challenge results in nuclear translocation of Ag‐STAT protein in fat body cells and induction of DNA‐binding activity that recognizes a STAT target site. In vitro treatment with pervanadate (vanadate and H2O2) translocates Ag‐STAT to the nucleus in midgut epithelial cells. This is the first evidence of direct participation of the STAT pathway in immune responses in insects.

Reactive Oxygen Species Modulate Anopheles gambiae Immunity against Bacteria and Plasmodium

The involvement of reactive oxygen species (ROS) in mosquito immunity against bacteria and Plasmodium was investigated in the malaria vector Anopheles gambiae. Strains of An. gambiae with higher systemic levels of ROS survive a bacterial challenge better, whereas reduction of ROS by dietary administration of antioxidants significantly decreases survival, indicating that ROS are required to mount effective antibacterial responses. Expression of several ROS detoxification enzymes increases in the midgut and fat body after a blood meal. Furthermore, expression of several of these enzymes increases to even higher levels when mosquitoes are fed a Plasmodium berghei-infected meal, indicating that the oxidative stress after a blood meal is exacerbated by Plasmodium infection. Paradoxically, a complete lack of induction of catalase mRNA and lower catalase activity were observed in P. berghei-infected midguts. This suppression of midgut catalase expression is a specific response to ookinete midgut invasion and is expected to lead to higher local levels of hydrogen peroxide. Further reduction of catalase expression by double-stranded RNA-mediated gene silencing promoted parasite clearance by a lytic mechanism and reduced infection significantly. High mosquito mortality is often observed after P. berghei infection. Death appears to result in part from excess production of ROS, as mortality can be decreased by oral administration of uric acid, a strong antioxidant. We conclude that ROS modulate An. gambiae immunity and that the mosquito response to P. berghei involves a local reduction of detoxification of hydrogen peroxide in the midgut that contributes to limit Plasmodium infection through a lytic mechanism.

A peroxidase/dual oxidase system modulates midgut epithelial immunity in Anopheles gambiae.

Mosquito Double Act Peroxidase/dual oxidase (duox) systems act in concert to catalyze the nonspecific formation of dityrosine bonds, which cross-link a variety of proteins. Knowing that these reactions are involved in fine-tuning insect immune responses, Kumar et al. (p. 1644, published online 11 March) investigated how the peroxidase/duox system in malaria-vector mosquitoes protects the gut flora by modulating midgut antibacterial responses. Generating immune reactions resulted in a loss of mosquito egg viability, but modulating host responses allowed malaria parasites to persist among the surviving commensal flora. The peroxidase/duox system appears to promote dityrosine bond formation between proteins across the surface of midgut epithelial cells to form a layer that inhibits immune recognition and mediator release. Interference with the formation of this layer might provide a target for mosquito and malaria control. Bonding between cell-surface proteins forms a physical barrier in mosquito guts to prevent microbe invasion. Extracellular matrices in diverse biological systems are cross-linked by dityrosine covalent bonds catalyzed by the peroxidase/oxidase system. We show that a peroxidase, secreted by the Anopheles gambiae midgut, and dual oxidase form a dityrosine network that decreases gut permeability to immune elicitors. This network protects the microbiota by preventing activation of epithelial immunity. It also provides a suitable environment for malaria parasites to develop within the midgut lumen without inducing nitric oxide synthase expression. Disruption of this barrier results in strong and effective pathogen-specific immune responses.