Engineered single nucleotide polymorphisms in the mosquito MEK docking site alter Plasmodium berghei development in Anopheles gambiae.

BackgroundSusceptibility to Plasmodium infection in Anopheles gambiae has been proposed to result from naturally occurring polymorphisms that alter the strength of endogenous innate defenses. Despite the fact that some of these mutations are known to introduce non-synonymous substitutions in coding sequences, these mutations have largely been used to rationalize knockdown of associated target proteins to query the effects on parasite development in the mosquito host. Here, we assay the effects of engineered mutations on an immune signaling protein target that is known to control parasite sporogonic development. By this proof-of-principle work, we have established that naturally occurring mutations can be queried for their effects on mosquito protein function and on parasite development and that this important signaling pathway can be genetically manipulated to enhance mosquito resistance.MethodsWe introduced SNPs into the A. gambiae MAPK kinase MEK to alter key residues in the N-terminal docking site (D-site), thus interfering with its ability to interact with the downstream kinase target ERK. ERK phosphorylation levels in vitro and in vivo were evaluated to confirm the effects of MEK D-site mutations. In addition, overexpression of various MEK D-site alleles was used to assess P. berghei infection in A. gambiae.ResultsThe MEK D-site contains conserved lysine residues predicted to mediate protein-protein interaction with ERK. As anticipated, each of the D-site mutations (K3M, K6M) suppressed ERK phosphorylation and this inhibition was significant when both mutations were present. Tissue-targeted overexpression of alleles encoding MEK D-site polymorphisms resulted in reduced ERK phosphorylation in the midgut of A. gambiae. Furthermore, as expected, inhibition of MEK-ERK signaling due to D-site mutations resulted in reduction in P. berghei development relative to infection in the presence of overexpressed catalytically active MEK.ConclusionMEK-ERK signaling in A. gambiae, as in model organisms and humans, depends on the integrity of conserved key residues within the MEK D-site. Disruption of signal transmission via engineered SNPs provides a purposeful proof-of-principle model for the study of naturally occurring mutations that may be associated with mosquito resistance to parasite infection as well as an alternative genetic basis for manipulation of this important immune signaling pathway

Protein kinase C-dependent signaling controls the midgut epithelial barrier to malaria parasite infection in anopheline mosquitoes.

Anopheline mosquitoes are the primary vectors of parasites in the genus Plasmodium, the causative agents of malaria. Malaria parasites undergo a series of complex transformations upon ingestion by the mosquito host. During this process, the physical barrier of the midgut epithelium, along with innate immune defenses, functionally restrict parasite development. Although these defenses have been studied for some time, the regulatory factors that control them are poorly understood. The protein kinase C (PKC) gene family consists of serine/threonine kinases that serve as central signaling molecules and regulators of a broad spectrum of cellular processes including epithelial barrier function and immunity. Indeed, PKCs are highly conserved, ranging from 7 isoforms in Drosophila to 16 isoforms in mammals, yet none have been identified in mosquitoes. Despite conservation of the PKC gene family and their potential as targets for transmission-blocking strategies for malaria, no direct connections between PKCs, the mosquito immune response or epithelial barrier integrity are known. Here, we identify and characterize six PKC gene family members--PKCδ, PKCε, PKCζ, PKD, PKN, and an indeterminate conventional PKC--in Anopheles gambiae and Anopheles stephensi. Sequence and phylogenetic analyses of the anopheline PKCs support most subfamily assignments. All six PKCs are expressed in the midgut epithelia of A. gambiae and A. stephensi post-blood feeding, indicating availability for signaling in a tissue that is critical for malaria parasite development. Although inhibition of PKC enzymatic activity decreased NF-κB-regulated anti-microbial peptide expression in mosquito cells in vitro, PKC inhibition had no effect on expression of a panel of immune genes in the midgut epithelium in vivo. PKC inhibition did, however, significantly increase midgut barrier integrity and decrease development of P. falciparum oocysts in A. stephensi, suggesting that PKC-dependent signaling is a negative regulator of epithelial barrier function and a potential new target for transmission-blocking strategies

Mitochondrial NAD+-dependent malic enzyme from Anopheles stephensi: a possible novel target for malaria mosquito control

<p>Abstract</p> <p>Background</p> <p><it>Anopheles stephensi </it>mitochondrial malic enzyme (ME) emerged as having a relevant role in the provision of pyruvate for the Krebs' cycle because inhibition of this enzyme results in the complete abrogation of oxygen uptake by mitochondria. Therefore, the identification of ME in mitochondria from immortalized <it>A. stephensi </it>(ASE) cells and the investigation of the stereoselectivity of malate analogues are relevant in understanding the physiological role of ME in cells of this important malaria parasite vector and its potential as a possible novel target for insecticide development.</p> <p>Methods</p> <p>To characterize the mitochondrial ME from immortalized ASE cells (Mos. 43; ASE), mass spectrometry analyses of trypsin fragments of ME, genomic sequence analysis and biochemical assays were performed to identify the enzyme and evaluate its activity in terms of cofactor dependency and inhibitor preference.</p> <p>Results</p> <p>The encoding gene sequence and primary sequences of several peptides from mitochondrial ME were found to be highly homologous to the mitochondrial ME from <it>Anopheles gambiae </it>(98%) and 59% homologous to the mitochondrial NADP⁺-dependent ME isoform from <it>Homo sapiens</it>. Measurements of ME activity in mosquito mitochondria isolated from ASE cells showed that (<it>i</it>) <it>V_max</it>with NAD⁺was 3-fold higher than that with NADP⁺, (<it>ii</it>) addition of Mg²⁺or Mn²⁺increased the <it>V_max</it>by 9- to 21-fold, with Mn²⁺2.3-fold more effective than Mg²⁺, (<it>iii</it>) succinate and fumarate increased the activity by 2- and 5-fold, respectively, at sub-saturating concentrations of malate, (<it>iv</it>) among the analogs of L-malate tested as inhibitors of the NAD⁺-dependent ME catalyzed reaction, small (2- to 3-carbons) organic diacids carrying a 2-hydroxyl/keto group behaved as the most potent inhibitors of ME activity (e.g., oxaloacetate, tartronic acid and oxalate).</p> <p>Conclusions</p> <p>The biochemical characterization of <it>Anopheles stephensi </it>ME is of critical relevance given its important role in bioenergetics, suggesting that it is a suitable target for insecticide development.</p

MAPK ERK Signaling Regulates the TGF-β1-Dependent Mosquito Response to Plasmodium falciparum

Malaria is caused by infection with intraerythrocytic protozoa of the genus Plasmodium that are transmitted by Anopheles mosquitoes. Although a variety of anti-parasite effector genes have been identified in anopheline mosquitoes, little is known about the signaling pathways that regulate these responses during parasite development. Here we demonstrate that the MEK-ERK signaling pathway in Anopheles is controlled by ingested human TGF-β1 and finely tunes mosquito innate immunity to parasite infection. Specifically, MEK-ERK signaling was dose-dependently induced in response to TGF-β1 in immortalized cells in vitro and in the A. stephensi midgut epithelium in vivo. At the highest treatment dose of TGF-β1, inhibition of ERK phosphorylation increased TGF-β1-induced expression of the anti-parasite effector gene nitric oxide synthase (NOS), suggesting that increasing levels of ERK activation negatively feed back on induced NOS expression. At infection levels similar to those found in nature, inhibition of ERK activation reduced P. falciparum oocyst loads and infection prevalence in A. stephensi and enhanced TGF-β1-mediated control of P. falciparum development. Taken together, our data demonstrate that malaria parasite development in the mosquito is regulated by a conserved MAPK signaling pathway that mediates the effects of an ingested cytokine

Anopheles stephensi p38 MAPK signaling regulates innate immunity and bioenergetics during Plasmodium falciparum infection.

BackgroundFruit flies and mammals protect themselves against infection by mounting immune and metabolic responses that must be balanced against the metabolic needs of the pathogens. In this context, p38 mitogen-activated protein kinase (MAPK)-dependent signaling is critical to regulating both innate immunity and metabolism during infection. Accordingly, we asked to what extent the Asian malaria mosquito Anopheles stephensi utilizes p38 MAPK signaling during infection with the human malaria parasite Plasmodium falciparum.MethodsA. stephensi p38 MAPK (AsP38 MAPK) was identified and patterns of signaling in vitro and in vivo (midgut) were analyzed using phospho-specific antibodies and small molecule inhibitors. Functional effects of AsP38 MAPK inhibition were assessed using P. falciparum infection, quantitative real-time PCR, assays for reactive oxygen species and survivorship under oxidative stress, proteomics, and biochemical analyses.ResultsThe genome of A. stephensi encodes a single p38 MAPK that is activated in the midgut in response to parasite infection. Inhibition of AsP38 MAPK signaling significantly reduced P. falciparum sporogonic development. This phenotype was associated with AsP38 MAPK regulation of mitochondrial physiology and stress responses in the midgut epithelium, a tissue critical for parasite development. Specifically, inhibition of AsP38 MAPK resulted in reduction in mosquito protein synthesis machinery, a shift in glucose metabolism, reduced mitochondrial metabolism, enhanced production of mitochondrial reactive oxygen species, induction of an array of anti-parasite effector genes, and decreased resistance to oxidative stress-mediated damage. Hence, P. falciparum-induced activation of AsP38 MAPK in the midgut facilitates parasite infection through a combination of reduced anti-parasite immune defenses and enhanced host protein synthesis and bioenergetics to minimize the impact of infection on the host and to maximize parasite survival, and ultimately, transmission.ConclusionsThese observations suggest that, as in mammals, innate immunity and mitochondrial responses are integrated in mosquitoes and that AsP38 MAPK-dependent signaling facilitates mosquito survival during parasite infection, a fact that may attest to the relatively longer evolutionary relationship of these parasites with their invertebrate compared to their vertebrate hosts. On a practical level, improved understanding of the balances and trade-offs between resistance and metabolism could be leveraged to generate fit, resistant mosquitoes for malaria control

Abundance, behavior and entomological inoculation rates of anthropophilic anophelines from a primary Colombian malaria endemic area

ABSTARCT: In Colombia for several years, the Urabá-Bajo Cauca and Alto Sinú region has registered the highest numbers of malaria cases in the country. Malaria vector incrimination and the characterization of entomological parameters will allow for a better understanding of malaria transmission dynamics and the design of effective vector control strategies for this region. Methods. We conducted a longitudinal survey between November 2008 and June 2010 to quantify entomological (abundance and biting activity) and transmission parameters, including infection rate (IR) and entomological inoculation rate (EIR), to incriminate potential anopheline vectors in three localities of a major Colombian malaria endemic region, the Urabá-Bajo Cauca and Alto Sinú: La Capilla, Juan Jose and El Loro. Results: A total of 5,316 anopheline mosquitoes corresponding to seven species were collected. Anopheles nuneztovari (69.5%) and Anopheles darlingi (22.2%) were the most abundant species, followed by Anopheles pseudopunctipennis (4.5%), Anopheles albitarsis s.l. (2%), Anopheles triannulatus lineage Northwest (1.8%), Anopheles punctimacula and Anopheles argyritarsis (at < 1%, each). Three species were naturally infected with Plasmodium vivax, An. nuneztovari, An. darlingi (IRs < 1%) and An. triannulatus (IR = 1.5%). Annual EIRs for these species ranged from 3.5 to 4.8 infective bites per year. Conclusions: These results indicate that An. nuneztovari and An. darlingi continue to be the most important malaria vectors in this region. Anopheles triannulatus, a species of local importance in other South American countries was found naturally infected with Plasmodium vivax VK247; therefore, further work should be directed to understand if this species has a role in malaria transmission in this regio

Identification of three single nucleotide polymorphisms in Anopheles gambiae immune signaling genes that are associated with natural Plasmodium falciparum infection

<p>Abstract</p> <p>Background</p> <p>Laboratory studies have demonstrated that a variety of immune signaling pathways regulate malaria parasite infection in <it>Anopheles gambiae</it>, the primary vector species in Africa.</p> <p>Methods</p> <p>To begin to understand the importance of these associations under natural conditions, an association mapping approach was adopted to determine whether single nucleotide polymorphisms (SNPs) in selected immune signaling genes in <it>A. gambiae </it>collected in Mali were associated with the phenotype of <it>Plasmodium falciparum </it>infection.</p> <p>Results</p> <p>Three SNPs were identified in field-collected mosquitoes that were associated with parasite infection in molecular form-dependent patterns: two were detected in the <it>Toll5B </it>gene and one was detected in the gene encoding insulin-like peptide 3 precursor. In addition, one infection-associated <it>Toll5B </it>SNP was in linkage disequilibrium with a SNP in sequence encoding a mitogen-activated protein kinase that has been associated with Toll signaling in mammalian cells. Both <it>Toll5B </it>SNPs showed divergence from Hardy-Weinberg equilibrium, suggesting that selection pressure(s) are acting on these loci.</p> <p>Conclusions</p> <p>Seven of these eight infection-associated and linked SNPs alter codon frequency or introduce non-synonymous changes that would be predicted to alter protein structure and, hence, function, suggesting that these SNPs could alter immune signaling and responsiveness to parasite infection.</p

The mitogen-activated protein kinome from Anopheles gambiae: identification, phylogeny and functional characterization of the ERK, JNK and p38 MAP kinases

<p>Abstract</p> <p>Background</p> <p><it>Anopheles gambiae </it>is the primary mosquito vector of human malaria parasites in sub-Saharan Africa. To date, three innate immune signaling pathways, including the nuclear factor (NF)-kappaB-dependent Toll and immune deficient (IMD) pathways and the Janus kinase/signal transducers and activators of transcription (Jak-STAT) pathway, have been extensively characterized in <it>An. gambiae</it>. However, in addition to NF-kappaB-dependent signaling, three mitogen-activated protein kinase (MAPK) pathways regulated by JNK, ERK and p38 MAPK are critical mediators of innate immunity in other invertebrates and in mammals. Our understanding of the roles of the MAPK signaling cascades in anopheline innate immunity is limited, so identification of the encoded complement of these proteins, their upstream activators, and phosphorylation profiles in response to relevant immune signals was warranted.</p> <p>Results</p> <p>In this study, we present the orthologs and phylogeny of 17 <it>An. gambiae </it>MAPKs, two of which were previously unknown and two others that were incompletely annotated. We also provide detailed temporal activation profiles for ERK, JNK, and p38 MAPK in <it>An. gambiae </it>cells <it>in vitro </it>to immune signals that are relevant to malaria parasite infection (human insulin, human transforming growth factor-beta1, hydrogen peroxide) and to bacterial lipopolysaccharide. These activation profiles and possible upstream regulatory pathways are interpreted in light of known MAPK signaling cascades.</p> <p>Conclusions</p> <p>The establishment of a MAPK "road map" based on the most advanced mosquito genome annotation can accelerate our understanding of host-pathogen interactions and broader physiology of <it>An. gambiae </it>and other mosquito species. Further, future efforts to develop predictive models of anopheline cell signaling responses, based on iterative construction and refinement of data-based and literature-based knowledge of the MAP kinase cascades and other networked pathways will facilitate identification of the "master signaling regulators" in biomedically important mosquito species.</p

Mast Cell Chymase/Mcpt4 Suppresses the Host Immune Response to Plasmodium yoelii, Limits Malaria-Associated Disruption of Intestinal Barrier Integrity and Reduces Parasite Transmission to Anopheles stephensi

An increase in mast cells (MCs) and MCs mediators has been observed in malaria-associated bacteremia, however, the role of these granulocytes in malarial immunity is poorly understood. Herein, we studied the role of mouse MC protease (Mcpt) 4, an ortholog of human MC chymase, in malaria-induced bacteremia using Mcpt4 knockout (Mcpt4(-/-)) mice and Mcpt4(+/+) C57BL/6J controls, and the non-lethal mouse parasite Plasmodium yoelii yoelii 17XNL. Significantly lower parasitemia was observed in Mcpt4(-/-) mice compared with Mcpt4(+/+) controls by day 10 post infection (PI). Although bacterial 16S DNA levels in blood were not different between groups, increased intestinal permeability to FITC-dextran and altered ileal adherens junction E-cadherin were observed in Mcpt4(-/-) mice. Relative to infected Mcpt4(+/+) mice, ileal MC accumulation in Mcpt4(-/-) mice occurred two days earlier and IgE levels were higher by days 8-10 PI. Increased levels of circulating myeloperoxidase were observed at 6 and 10 days PI in Mcpt4(+/+) but not Mcpt4(-/-) mice, affirming a role for neutrophil activation that was not predictive of parasitemia or bacterial 16S copies in blood. In contrast, early increased plasma levels of TNF-alpha, IL-12p40 and IL-3 were observed in Mcpt4(-/-) mice, while levels of IL-2, IL-10 and MIP1 beta (CCL4) were increased over the same period in Mcpt4(+/+) mice, suggesting that the host response to infection was skewed toward a type-1 immune response in Mcpt4(-/-) mice and type-2 response in Mcpt4(+/+) mice. Spearman analysis revealed an early (day 4 PI) correlation of Mcpt4(-/-) parasitemia with TNF-alpha and IFN-gamma, inflammatory cytokines known for their roles in pathogen clearance, a pattern that was observed in Mcpt4(+/+) mice much later (day 10 PI). Transmission success of P. y. yoelii 17XNL to Anopheles stephensi was significantly higher from infected Mcpt4(-/-) mice compared with infected Mcpt4(+/+) mice, suggesting that Mcpt4 also impacts transmissibility of sexual stage parasites. Together, these results suggest that early MCs activation and release of Mcpt4 suppresses the host immune response to P. y. yoelii 17XNL, perhaps via degradation of TNF-alpha and promotion of a type-2 immune response that concordantly protects epithelial barrier integrity, while limiting the systemic response to bacteremia and parasite transmissibility