Lalita Gupta

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.

The STAT Pathway Mediates Late-Phase Immunity against Plasmodium in the Mosquito Anopheles gambiae

The STAT family of transcription factors activates expression of immune system genes in vertebrates. The ancestral STAT gene (AgSTAT-A) appears to have duplicated in the mosquito Anopheles gambiae, giving rise to a second intronless STAT gene (AgSTAT-B), which we show regulates AgSTAT-A expression in adult females. AgSTAT-A participates in the transcriptional activation of nitric oxide synthase (NOS) in response to bacterial and plasmodial infection. Activation of this pathway, however, is not essential for mosquitoes to survive a bacterial challenge. AgSTAT-A silencing reduces the number of early Plasmodium oocysts in the midgut, but nevertheless enhances the overall infection by increasing oocyst survival. Silencing of SOCS, a STAT suppressor, has the opposite effect, reducing Plasmodium infection by increasing NOS expression. Chemical inhibition of mosquito NOS activity after oocyte formation increases oocyte survival. Thus, the AgSTAT-A pathway mediates a late-phase antiplasmodial response that reduces oocyst survival in A. gambiae.

Inducible Peroxidases Mediate Nitration of Anopheles Midgut Cells Undergoing Apoptosis in Response to Plasmodium Invasion

Plasmodium berghei invasion of Anopheles stephensi midgut cells causes severe damage, induces expression of nitric-oxide synthase, and leads to apoptosis. The present study indicates that invasion results in tyrosine nitration, catalyzed as a two-step reaction in which nitric-oxide synthase induction is followed by increased peroxidase activity. Ookinete invasion induced localized expression of peroxidase enzymes, which catalyzed protein nitration in vitro in the presence of nitrite and H2O2. Histochemical stainings revealed that when a parasite migrates laterally and invades more than one cell, the pattern of induced peroxidase activity is similar to that observed for tyrosine nitration. In Anopheles gambiae, ookinete invasion elicited similar responses; it induced expression of 5 of the 16 peroxidase genes predicted by the genome sequence and decreased mRNA levels of one of them. One of these inducible peroxidases has a C-terminal oxidase domain homologous to the catalytic moiety of phagocyte NADPH oxidase and could provide high local levels of superoxide anion (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}\overline{_{{\dot{2}}}}\) \end{document}), that when dismutated would generate the local increase in H2O2 required for nitration. Chemically induced apoptosis of midgut cells also activated expression of four ookinete-induced peroxidase genes, suggesting their involvement in general apoptotic responses. The two-step nitration reaction provides a mechanism to precisely localize and circumscribe the toxic products generated by defense reactions involving nitration. The present study furthers our understanding of the biochemistry of midgut defense reactions to parasite invasion and how these may influence the efficiency of malaria transmission by anopheline mosquitoes.

Reactive oxygen species detoxification by catalase is a major determinant of fecundity in the mosquito Anopheles gambiae

The mosquito Anopheles gambiae is a primary vector of Plasmodium parasites in Africa. The effect of aging on reproductive output in A. gambiae females from three strains that differ in their ability to melanize Plasmodium and in their systemic levels of hydrogen peroxide (H2O2), a reactive oxygen species (ROS), was analyzed. The number of eggs oviposited after the first blood meal decreases with age in all strains; however, this decline was much more pronounced in the G3 (unselected) and R (refractory to Plasmodium infection) strains than in the S (highly susceptible to Plasmodium) strain. Reduction of ROS levels in G3 and R females by administration of antioxidants reversed this age-related decline in fecundity. The S and G3 strains were fixed for two functionally different catalase alleles that differ at the second amino acid position (Ser2Trp). Biochemical analysis of recombinant proteins revealed that the Trp isoform has lower specific activity and higher Km than the Ser isoform, indicating that the former is a less efficient enzyme. The Trp-for-Ser substitution appears to destabilize the functional tetrameric form of the enzyme. Both alleles are present in the R strain, and Ser/Ser females had significantly higher fecundity than Trp/Trp females. Finally, a systemic reduction in catalase activity by dsRNA-mediated knockdown significantly reduced the reproductive output of mosquito females, indicating that catalase plays a central role in protecting the oocyte and early embryo from ROS damage.

Apolipophorin-III Mediates Antiplasmodial Epithelial Responses in Anopheles gambiae (G3) Mosquitoes

Background Apolipophorin-III (ApoLp-III) is known to play an important role in lipid transport and innate immunity in lepidopteran insects. However, there is no evidence of involvement of ApoLp-IIIs in the immune responses of dipteran insects such as Drosophila and mosquitoes. Methodology/Principal Findings We report the molecular and functional characterization of An. gambiae apolipophorin-III (AgApoLp-III). Mosquito ApoLp-IIIs have diverged extensively from those of lepidopteran insects; however, the predicted tertiary structure of AgApoLp-III is similar to that of Manduca sexta (tobacco hornworm). We found that AgApoLp-III mRNA expression is strongly induced in the midgut of An. gambiae (G3 strain) mosquitoes in response to Plasmodium berghei infection. Furthermore, immunofluorescence stainings revealed that high levels of AgApoLp-III protein accumulate in the cytoplasm of Plasmodium-invaded cells and AgApoLp-III silencing increases the intensity of P. berghei infection by five fold. Conclusion There are broad differences in the midgut epithelial responses to Plasmodium invasion between An. gambiae strains. In the G3 strain of An. gambiae AgApoLp-III participates in midgut epithelial defense responses that limit Plasmodium infection.

New tools to identify var sequence tags and clone full-length genes using type-specific primers to Duffy binding-like domains

Cytoadherence of infected erythrocytes is a hallmark of Plasmodium falciparum infection and a key determinant in the particular virulence of this species. Infected erythrocytes bind a variety of host receptors but certain adhesion traits are associated with more severe disease. A large, diverse protein family named P. falciparum erythrocyte membrane protein 1 (PfEMP1) is responsible for sequestration of mature stage infected erythrocytes and orchestrates parasite binding tropism. To better understand the molecular basis for malaria disease, more study is needed to identify the subset of PfEMP1 variants that contribute to basic disease phenotypes. PfEMP1 proteins have multiple receptor-like domains that group into different homology types based upon sequence similarity. Universal primers have been developed that recognize some, but not all PfEMP1 adhesion domain types. In this study, we designed and validated a new series of type-discriminatory primers to the DBL-beta, -gamma, and -delta adhesion types for epidemiological profiling. In addition, we used new primers to the var upstream region and exon 2 to demonstrate how the strategic placement of primers throughout the gene structure can be exploited to efficiently clone the var gene coding region. These new approaches provide valuable tools to gain novel insights into cytoadherence and malaria pathogenesis.

Ambivalent Outcomes of Cell Apoptosis: A Barrier or Blessing in Malaria Progression.

The life cycle of Plasmodium in two evolutionary distant hosts, mosquito, and human, is a complex process. It is regulated at various stages of developments by a number of diverged mechanisms that ultimately determine the outcome of the disease. During the development processes, Plasmodium invades a variety of cells in two hosts. The invaded cells tend to undergo apoptosis and are subsequently removed from the system. This process also eliminates numerous parasites along with these apoptotic cells as a part of innate defense against the invaders. Plasmodium should escape the invaded cell before it undergoes apoptosis or it should manipulate host cell apoptosis for its survival. Interestingly, both these phenomena are evident in Plasmodium at different stages of development. In addition, the parasite also exhibits altruistic behavior and triggers its own killing for the selection of the best ‘fit’ progeny, removal of the ‘unfit’ parasites to conserve the nutrients and to support the host survival. Thus, the outcomes of cell apoptosis are ambivalent, favorable as well as unfavorable during malaria progression. Here we discuss that the manipulation of host cell apoptosis might be helpful in the regulation of Plasmodium development and will open new frontiers in the field of malaria research.

Molecular characterization of SOCS gene and its expression analysis on Plasmodium berghei infection in Anopheles culicifacies.

Anopheles culicifacies mosquitoes are able to transmit both falciparum and vivax malaria in India. More than 65% of malaria cases reported annually spread through this vector. Despite the fact that it poses major vectorial burden in India, the molecular basis of its immune role against Plasmodium development has not been explored intensively. Here, we characterized An. culicifacies SOCS (suppressor of cytokine signaling) gene, a regulator of STAT pathway and its expression analysis upon Plasmodium infection. Our analysis has demonstrated that An. culicifacies SOCS gene shares strikingly high level of sequence similarity in SH2 domain and SOCS box region with other mosquito species. However, its N-terminal identity is limited to Anophelines mosquito only, suggesting its genus specific role. SOCS mRNA is expressed in all developmental stages of mosquito and its expression is higher in male than female adults. SOCS mRNA is significantly induced after Plasmodium infection in midgut tissue indicating its involvement in the immune defense responses. This is the first evidence of involvement of SOCS as an immune gene in Indian malaria vector An. culicifacies.

Identification of an Anopheles Lineage-Specific Unique Heme Peroxidase HPX15: A Plausible Candidate for Arresting Malaria Parasite Development

Background: Human malaria parasite Plasmodium falciparum is transmitted by several species of Anopheles mosquito. The advancement of drug-resistant parasites and insecticide resistance in mosquito vectors are major hurdles in the malaria control. Alternatively, the manipulation of mosquito immunity is also an ideal way to block Plasmodium development inside the insect host. This approach demands the identification of key mosquito molecules that regulate anti-plasmodial immunity. Our previous findings revealed that the silencing of Anopheles gambiae heme peroxidase 15 (AgHPX15, AGAP013327) induced mosquito innate immunity and drastically suppressed the development of human and rodent malaria parasites. Further, we aim to characterize HPX15 orthologs in Indian malaria vectors and other worldwide-distributed anophelines to understand the novelty of this molecule as a plausible target to block Plasmodium development. Method: AgHPX15 orthologs were cloned from major Indian malaria vectors A. stephensi and A. culicifacies and their conserve domains were determined by CDD search tool. The sequence homology and phylogenetic relationship of these clones with other heme peroxidases was analysed using Mega5.2 software. Results and conclusion: We found that A. stephensi AsHPX15 and A. culicifacies AcHPX15 clones are close orthologs of A. gambiae AgHPX15. The phylogenetic relationship of these anopheline HPX15 with other animal and plant heme peroxidases revealed that they form a separate lineage-specific cluster and their orthologs are not found in human, nematodes or other related arthropods such as, Drosophila, Aedes and Culex mosquitoes. However, their putative orthologs are present in 16 other globally distributed anophelines and exhibit a highly conserved amino acids identity in the range of 70-99%. Based on these findings we propose that the anopheline-specific and evolutionary conserved heme peroxidase HPX15 may serve as a unique target for designing transmission-blocking strategies to block Plasmodium-mosquito cycle. These findings will generate new frontiers in the field of malaria research and disease control.