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

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

Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles stephensi Mosquitoes

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity

The role of central memory CD4+ T cells in Leishmania major infection

This thesis characterizes and explores the role of central memory CD4 + T cells generated in response to Leishmania major infection. Central memory CD4+ T (Tcm) cells provide a pool of lymph node-homing, antigen-experienced cells that are capable of responding rapidly after a secondary infection. We have previously described a population of Tcm cells in L. major infected mice that were capable of mediating immunity to a secondary infection. The first part of this thesis focuses on characterizing the Tcm cells generated in response to L. major infection. Our results suggest that this population is non-polarized and requires IL-12 in order to differentiate into Th1 effector cells. IL-12 is a heterodimeric cytokine composed of two subunits, p35 and p40. However, the p40 subunit can also associate with a distinct p19 subunit to form the cytokine IL-23. The main role of IL-23 appears to be the maintenance of Th17 effector cells. Th17 cells can increase inflammation by increasing the recruitment of immune cells to site of infection. The second part of this thesis focuses on expanding our understanding of the role of IL-23 in L. major infection. We show that IL-23 is necessary for the increased inflammation observed following high-dose L. major infection and that this inflammation is most likely being mediated by a population of IL-17 producing effector cells. In the third part of this thesis we use attenuated Ipg2- mutant parasites to examine the cell populations involved in immunity. Lpg2- mutant parasites persist in mice without inducing any overt pathology. These parasites were generated by the deletion of the LPG2 gene and as a result fail to synthesize LPG and other surface and secreted phosphoglycans and proteophosphoglycans. We have previously shown that Ipg2- mutant parasites are capable of conferring complete protection against virulent L. major challenge. The data shown here suggests that the protection induced by Ipg2- parasites is mediated in part by a population of CD4 + and CD8+ central memory T cells. The work of this thesis provides insight into the generation of central memory CD4+ T cells and their requirements for providing protection against L. major infection

The role of central memory CD4+ T cells in Leishmania major infection

This thesis characterizes and explores the role of central memory CD4 + T cells generated in response to Leishmania major infection. Central memory CD4+ T (Tcm) cells provide a pool of lymph node-homing, antigen-experienced cells that are capable of responding rapidly after a secondary infection. We have previously described a population of Tcm cells in L. major infected mice that were capable of mediating immunity to a secondary infection. The first part of this thesis focuses on characterizing the Tcm cells generated in response to L. major infection. Our results suggest that this population is non-polarized and requires IL-12 in order to differentiate into Th1 effector cells. IL-12 is a heterodimeric cytokine composed of two subunits, p35 and p40. However, the p40 subunit can also associate with a distinct p19 subunit to form the cytokine IL-23. The main role of IL-23 appears to be the maintenance of Th17 effector cells. Th17 cells can increase inflammation by increasing the recruitment of immune cells to site of infection. The second part of this thesis focuses on expanding our understanding of the role of IL-23 in L. major infection. We show that IL-23 is necessary for the increased inflammation observed following high-dose L. major infection and that this inflammation is most likely being mediated by a population of IL-17 producing effector cells. In the third part of this thesis we use attenuated Ipg2- mutant parasites to examine the cell populations involved in immunity. Lpg2- mutant parasites persist in mice without inducing any overt pathology. These parasites were generated by the deletion of the LPG2 gene and as a result fail to synthesize LPG and other surface and secreted phosphoglycans and proteophosphoglycans. We have previously shown that Ipg2- mutant parasites are capable of conferring complete protection against virulent L. major challenge. The data shown here suggests that the protection induced by Ipg2- parasites is mediated in part by a population of CD4 + and CD8+ central memory T cells. The work of this thesis provides insight into the generation of central memory CD4+ T cells and their requirements for providing protection against L. major infection