Progress 01/01/00 to 12/31/04
Outputs
Today, 300-500 million people are at risk for malaria and 2-3 million die from the infection each year. Our goal is to identify genes and gene products in the Anopheles mosquito that adversely affect parasite development, with the hope that manipulation of these genes could lead to the production of parasite-resistant mosquitoes. Our goal has been to identify and characterize anti-malaria parasite resistance genes in anopheline mosquitoes, vectors of human malaria. Mosquito nitric oxide synthase (NOS) is induced in response to parasite infection and catalyzes the synthesis of the free radical gas NO to limit parasite development. This response is important in the field (Kenya). We hypothesized that NO synthesis results in the formation of reactive nitrogen intermediates (RNIs) in the mosquito that are toxic to malaria parasites. We have determined that the higher oxides of nitrogen are the predominant midgut RNIs. We can now test candidate RNIs against mosquito-stage
parasites to evaluate toxicity. Because NO is equally toxic to the host, mosquito tissue may be damaged by RNIs. We have characterized a novel gene product that protects mosquito cells from RNI damage. We have also studied regulation of mosquito NOS. Toward this end, we have identified a parasite molecule that can induce NOS expression in mosquito cells. These findings are of interest because the same parasite factor induces NOS expression in mammalian cells. We have also discovered that human TGF-beta1, which is ingested during bloodfeeding by the mosquito, is recognized by mosquito cells and can regulate mosquito NOS and malaria parasite development. We have shown that mosquitoes also produce TGF-beta-like proteins, receptors and signaling molecules that can transduce signals from mosquito and mammalian TGF-betas, establishing a signaling architecture for immunological crosstalk that impacts mosquito immunity to malaria parasites.
Impacts
We are the first to discover that mammalian immunity to malaria parasites influences parasite development in the mosquito. This interaction occurs at the interface of bloodfeeding. We are also the first to discover that malaria parasite signaling of mosquito cells and mammalian cells is conserved.
Publications
- Lieber MJ, Luckhart S. 2004. Transforming growth factor-betas and related gene products in mosquito vectors of human malaria parasites: signaling architecture for immunological crosstalk. Molecular Immunology 41:965-77.
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Progress 10/01/02 to 09/30/03
Outputs
Our goal is to identify and characterize anti-malaria parasite resistance genes in anopheline mosquitoes, the primary insect vectors of human malaria. We are currently focused on nitric oxide synthase (NOS) and 60A, a transforming growth factor (TGF)-beta homolog. Mosquito NOS is induced in response to malaria parasite infection and catalyzes the synthesis of the free radical gas NO; this response limits parasite development. Based on published reports in other systems, we hypothesized that NO synthesis results in the generation of reactive nitrogen intermediates (RNIs) in the mosquito that are toxic to malaria parasites. In the past year, we have nearly completed our identification of the RNIs formed in the mosquito midgut following parasite infection. We have determined that the higher oxides of nitrogen are likely to represent the predominant RNIs in the midgut. With this information, we can now test candidate RNIs against mosquito-stage parasites to determine if
these compounds are capable of inhibiting parasite development at levels comparable to those detected in the mosquito. Because NO is equally toxic to the host mosquito, mosquito tissue damage can occur following this defense response. In the last year, we have more extensively characterized a novel gene product in anopheline mosquitoes that protects host cells from nitrosative stress. In addition to understanding how NO synthesis limits parasite development, we are also interested in how inducible NO synthesis is regulated. Recently, we have determined that a parasite cell surface protein-anchoring molecule can induce NOS expression in mosquito cells and this signal appears to be transduced through insulin signaling and inflammatory signaling pathways in mosquito cells. These findings are of interest because the same parasite factor induces NOS expression in mammalian cells through similar pathways. We have characterized mosquito 60A and demonstrated that this gene product has
features characteristic of an inflammatory cytokine. While we have not yet demonstrated that mosquito 60A can regulate mosquito NOS expression, we have made the discovery that mammalian TGF-beta, a distant relative of mosquito 60A and ingested during bloodfeeding by the mosquito, persists in the mosquito midgut for at least 48h. Mammalian TGF-beta is recognized by mosquito cells and can regulate the endogenous mosquito NOS response and malaria parasite development. This discovery demonstrates that mammalian and mosquito immune factors interface during the process of bloodfeeding to regulate parasite development in the mosquito. A current global effort is directed toward the release of transgenic mosquitoes that are resistant to malaria parasite development. Expression of transgenes and activity of transgene products related to mosquito immunity may be influenced by mammalian factors that remain active in the midgut environment following ingestion. Accordingly, prudent experimental
designs would include assessments of parasite development in transgenic mosquitoes following exposure to a variety of hosts, maintained under physiological conditions and stresses typical in malaria-endemic regions.
Impacts
We are the first to discover that mammalian immunity to malaria parasites may influence parasite development in the mosquito. This interaction occurs at the interface of bloodfeeding. We are also the first to discover that detection of malaria parasites by both mosquito and mammalian immune cells may be evolutionarily conserved. Together, these findings establish new paradigms for innate immunity to malaria parasites and caution that studies of parasite transmission by mosquitoes in endemic areas should include the potential impact of mammalian immunity.
Publications
- Vodovotz, Y., Zamora, R., Lieber, M.J. and Luckhart, S. 2003. Cross-talk between nitric oxide and transforming growth factor-beta1 in malaria. Current Molecular Medicine (in press).
- Luckhart, S., Crampton, A.L., Zamora, R., Lieber, M.J., Dos Santos, P.C., Peterson, T.M.L., Lim, J., Emmith, N., Wink, D.A. and Vodovotz, Y. 2003. Mammalian TGF-beta1, activated after ingestion by Anopheles stephensi, modulates mosquito immunity. Infection and Immunity 71:3000-3009.
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Progress 10/01/01 to 09/30/02
Outputs
Our goal is to identify and characterize anti-malaria parasite resistance genes in anopheline mosquitoes, the primary insect vectors of human malaria. We are currently focused on nitric oxide synthase (NOS) and 60A, a transforming growth factor (TGF)-beta homolog. Mosquito NOS is induced in response to malaria parasite infection and catalyzes the synthesis of the free radical gas NO; this response limits parasite development. Based on published reports in other systems, we hypothesized that NO synthesis results in the generation of reactive nitrogen intermediates (RNIs) in the mosquito that are toxic to malaria parasites. Since October 2001, we have worked to identify the RNIs formed in the mosquito following parasite infection. We have determined that the higher oxides of nitrogen are likely to represent the predominant RNIs in the mosquito. With this information, we can now test candidate RNIs against mosquito-stage parasites to determine if these compounds are
capable of inhibiting parasite development at levels comparable to those detected in the mosquito. Because NO is equally toxic to the host mosquito, mosquito tissue damage can occur following this defense response. In the last year, we have characterized a novel gene product in anopheline mosquitoes that may protect mosquito cells from nitrosative stress. In addition to understanding how NO synthesis limits parasite development, we are also interested in how inducible NO synthesis is regulated. Recently, we have determined that a parasite cell surface protein-anchoring molecule can induce NOS expression in mosquito cells and this signal appears to be transduced through a pathway that depends on tyrosine phosphorylation. These findings are interesting because the same parasite factor induces NOS expression in mammalian cells through similar pathways. We have characterized mosquito 60A and demonstrated that this gene product has features characteristic of an inflammatory cytokine. While
we have not yet demonstrated that mosquito 60A can regulate mosquito NOS expression, we have made the discovery that mammalian TGF-beta, a distant relative of mosquito 60A and ingested during bloodfeeding by the mosquito, persists in the mosquito midgut for at least 48h. Mammalian TGF-beta is recognized by mosquito cells and can regulate the endogenous mosquito NOS response and malaria parasite development. This discovery demonstrates that mammalian and mosquito immune factors interface during the process of bloodfeeding to regulate parasite development in the mosquito. A current global effort is directed toward the release of transgenic mosquitoes that are resistant to malaria parasite development. Expression of transgenes and activity of transgene products related to mosquito immunity may be influenced by mammalian factors that remain active in the midgut environment following ingestion. Accordingly, prudent experimental designs would include assessments of parasite development in
transgenic mosquitoes following exposure to a variety of hosts, maintained under physiological conditions and stresses typical in malaria-endemic regions.
Impacts
Today, 300-500 million people are at risk for malaria and 1-2 million die from the infection each year. Novel control methods include a strategy to release genetically engineered mosquitoes that cannot transmit malaria parasites. Our goal is to identify genes and gene products in anopheline that adversely affect parasite development, with the hope that manipulation of these genes could lead to the production of parasite-resistant mosquitoes.
Publications
- Luckhart, S., Li, K., Dunton, R., Lewis, E.E., Crampton, A.L., Ryan, J.R., and Rosenberg, R. 2003. Anopheles gambiae immune gene variants associated with natural Plasmodium infection. Molecular and Biochemical Parasitology.(in press)
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Progress 10/01/00 to 09/30/01
Outputs
Our goal is to identify and characterize anti-malaria parasite resistance genes in anopheline mosquitoes, the primary insect vectors of human malaria. Since October 2000, we have worked to characterize two target genes, nitric oxide synthase (NOS) and 60A. Mosquito NOS is induced by malaria parasite infection. This enzyme catalyzes the synthesis of NO gas which inhibits parasite development. Intriguingly, humans also produce NOS and NO in response to parasite infection, indicating that evolutionarily diverse hosts of the malaria parasite share a conserved defense response. We have characterized the molecular basis of mosquito NOS expression and how mosquito NO is delivered to the parasite. Because NO is equally toxic to the host mosquito, some mosquito tissue damage occurs following this response. We have determined that the mosquito may, in fact, protect itself from this unintentional tissue damage. Unfortunately, the complex molecular basis of NOS expression likely
precludes direct genetic manipulation of NOS to enhance mosquito resistance. Mosquito 60A, however, is also induced by parasite development and may regulate the expression and synthesis of NOS and NO. Mosquito 60A has a less complex molecular basis of expression and, therefore, may be more amenable than NOS to genetic manipulation to increase mosquito anti-parasite resistance. We have also applied our insights to field studies of malaria parasite transmission by mosquitoes. The mosquito NOS gene contains numerous small DNA sequences that differ slightly from one mosquito to another. We have determined that, from analyses of more than 1300 mosquitoes that a specific subset of these genetic differences tends to be more associated with malaria parasite infection than does an alternate subset. Because malaria parasite infection prevalence in African mosquitoes is low (less than 5 percent), finding parasites to understand transmission is difficult. Our "genetic fingerprint" for malaria
parasite infection in mosquitoes may allow us to predict which mosquitoes are at risk for transmission by analyzing many fewer mosquitoes than would be required for current standard techniques.
Impacts
Today, more than 40 percent of the global population is at risk for infection with malaria, a mosquito-borne disease. Drug-resistant parasites and insecticide-resistant mosquitoes have allowed malaria to return to areas that were once malaria-free. These developments have led to interest in novel control strategies to interrupt malaria parasite transmission, including the development of transgenic mosquitoes that kill parasites rather than transmit them to humans.
Publications
- Crampton, A.L. and Luckhart, S. 2001. The role of As60A, a TGF-beta homolog, in Anopheles stephensi innate immunity and defense against Plasmodium infection. Infect. Genet. Evol. 14:1 - 11.
- Crampton, A.L. and Luckhart, S. 2001. Isolation and characterization of As60A, a transforming growth factor-beta gene, from the malaria vector Anopheles stephensi. Cytokine 13:65-74.
- Luckhart, S. and Li, K. 2001. Transcriptional complexity of the Anopheles stephensi nitric oxide synthase gene. Insect Biochem. Mol. Biol. 31:249-256.
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Progress 10/01/99 to 09/29/00
Outputs
New strategies for control of mosquito-borne malaria transmission have been proposed in the face of rapidly re-emerging disease throughout the world. One strategy involves genetic engineering of mosquitoes for resistance to parasite development. We are focused on characterizing mosquito gene targets for manipulation. These include the immune effector nitric oxide synthase (AsNOS), an enzyme that catalyzes synthesis of free radical NO gas that limits parasite development, and a potential NOS regulator, transforming growth factor-beta (As60A). AsNOS gene expression in the mosquito is complex and strikingly similar to that observed for human NOS genes. The potential regulator As60A, a homolog of the human cytokine TGF-beta, is expressed in the same mosquito tissues, under the same treatments, and temporal patterns as have been observed for AsNOS. Current studies include assays to define the AsNOS promoter, analysis of As60A involvement in direct regulation of AsNOS
transcription, and biochemical and cellular assays of the effects of NO synthesis on the mosquito and the parasite. In related field work, some immune gene polymorphs appear to be correlated with malaria parasite infection in field-collected African mosquitoes, indicating that these markers may be useful in predicting parasite infection in natural insect populations.
Impacts
Malaria is a re-emerging disease with 3-4 million deaths/yr. One novel strategy for control involves release of mosquitoes that are genetically resistant to the parasite. This strategy requires identification of: (1) suitable anti-parasite resistance genes, (2) techniques to transform mosquitoes, and (3) genetic "drive" mechanisms that allow spread of resistance genes in targeted mosquito populations. Our work contributes to step (1) of this novel strategy.
Publications
- Luckhart, S. and Rosenberg, R. 1999. Gene structure and polymorphism of an invertebrate nitric oxide synthase gene. Gene 232: 25-32.
- Luckhart, S. and Li, K. 2000. Transcriptional complexity of the Anopheles stephensi nitric oxide synthase gene. Insect Biochemistry and Molecular Biology (in press).
- Crampton, A.L. and Luckhart, S. 2000. Isolation and characterization of As60A, a transforming growth factor-beta gene, from the malaria vector Anopheles stephensi. Cytokine (in press).
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