Marcelo Jacobs-Lorena, PhD - US grants

The eukaryotic ribosome is composed of equimolar amounts of RNAs and proteins encoded by over 70 genes transcribed by three different polymerases. A major goal of this proposal is to elucidate mechanisms that coordinate the expression of some of these genes. Our recent observations suggest that specific translational regulation of ribosomal-protein mRNAs (rp-mRNAs) plays a major role in modulating ribosomal gene expression during early embryogenesis of Drosophila, when rRNA synthesis is undetectable. To study the interdependence of rRNA synthesis and rp-mRNA translation, bobbed mutants deficient in rRNA genes will be used to genetically reduce rRNA synthesis in the ovary (a site of intense ribosome synthesis) and investigate its effects on the expression of r-protein genes. A complementary series of experiments will address the issue of balanced r-protein gene expression by introducing cloned rp-genes into the embryo and examining at which level (e.g. rp-mRNA abundance, rp-mRNA translation, r-protein turnover) the embryo compensates for this excess dosage. To identify sequences on rp-mRNAs that confer their unique pattern of translation during early development, "hybrid genes" composed of varying portions of rp-mRNAs (whose translation is strongly regulated in early embryos) and actin mRNA (whose translation is not regulated) will be constructed. These "hybrid genes" will be introduced into early embryos and the association of the corresponding "hybrid mRNAs" with polysomes will be measured. These experiments should indicate which portion of the rp-mRNA confers upon it the ability to be translationally regulated. To study rp-genes by a genetic approach, a screen has been devised to isolate multiple mutants in individual r-protein genes. Each mutant will be analyzed at the molecular level by DNA sequencing. This combined genetic and molecular approach will constitute a first step in the identification of sequences in r-protein genes that are important for their expression and function. As a whole, these studies may serve as a model for other sets of genes that need to be coordinately regulated during development and cell differentiation.

In most insects, the peritrophic matrix is thought to play a major role in food digestion and absorption. The peritrophic matrix is an acellular sheath that lines the entire intestine and is secreted by the cardia, an organ located at the Drosophila foregut/midgut junction. One aim of this proposal is to study the function of genes that encode peritrophic matrix components. Another aim is to study organogenesis of the cardia during embryonic development. Genes that are expressed specifically in the adult cardia were isolated in Dr. Jacobs-Lorena's laboratory and will be characterized to determine patterns of expression, DNA sequence, and gene function. To identify genes that play a determinative role in the genesis of the cardia, he will use a newly developed P-element/beta-galactosidase vector in a screen for genes that are expressed specifically in embryonic cardia precursor cells. These studies will be complemented by cell ablation experiments that will assess the importance of cell-cell interactions and should define the contribution of different cell types to the formation of the peritrophic matrix. %%% In insects that transmit human diseases, the peritrophic matrix interacts directly with parasites and modulates their transmission. Understanding how the organ that secretes this matrix is formed and functions will have practical implications as well as adding to our knowledge of organogenesis.

In spite of its importance as a potential target for disease control, the insect gut has received little attention. Insect-borne parasites of human disease frequently initiate their development in the insect by penetrating its gut. The gut of hematophagous insects is also the site of blood digestion, a process that triggers egg production and ultimately influences insect fitness and reproduction. This proposal is to develop molecular approaches for the study of the Simulium gut, focusing on the above-mentioned processes. Simulium (blackfly) is the vector for onchocerciasis (river blindness), a debilitating disease that affects millions of people in Africa and Latin America. The parasite is ingested by the blackfly with the blood meal from an infected individual. In order to develop, the parasite must penetrate the blackfly gut. Penetration is largely but not entirely hindered by the secretion by the gut epithelium of a thick peritrophic matrix (PM) that surrounds the blood meal. There are reasons to believe that inhibition of PM formation would result in excessive parasite burden which causes fly lethality. One goal of the proposed research is to clone the genes encoding the two major PM components. The structural organization of these genes, including essential promoter elements, Will be defined and factors required for the gut-specific regulation of gene expression will be investigated. A second focus of this research is the investigation of genes which function in the digestion of the blood meal. Two genes which were previously isolated in this laboratory and encode putative proteolytic enzymes, will be similarly characterized. Finally, antibodies to Simulium gut proteins will be produced and used to explore the feasibility of developing immunological approaches to alter the fitness of the fly or to modify its vector capacity. The ultimate goal of these experiments is to provide the means for devising rational approaches of vector and disease control.

The ultimate goal of the experiments in this proposal is to define components that play a role in regulating the stability and translation of "pattern-determining" mRNAs, and to understand the importance of these post-transcriptional events for embryonic development. Unlike the typical housekeeping gene, genes involved in the determination of the Drosophila body plan are frequently regulated at multiple levels to attain their temporal and spatial pattern of expression. For instance, accumulation of bicoid protein is inhibited during oogenesis, presumably to avoid premature initiation of the embryonic developmental program. The bicoid mRNA is stable and is translated during the first few hours of embryogenesis establishing a gradient of bicoid protein that activates downstream genes. At the onset of gastrulation this mRNA is quickly degraded, presumably to turn off the genes it had earlier activated. The fushi tarazu (ftz) mRNA is unstable in the embryo, even at times when bicoid mRNA is stable, indicating that the stability of ftz and bicoid are regulated by different mechanisms. The ftz mRNA instability is believed to be essential for establishment of its striped pattern of expression. The experiments have the following objectives. i) Previous work from this laboratory indicated that a gene (termed gene R) located in chromosome 3R is required zygotically for the destabilization of bicoid mRNA around the time of gastrulation. A major goal of the present experiments is to genetically identify gene R by mutational analysis. ii) Gene R will be cloned and this will be followed by experiments to investigate the mechanism of its action. iii) Cis-acting sequences that signal destabilization of bicoid mRNA will be identified. iv) Mechanisms that prevent bicoid protein from accumulating during oogenesis will be investigated by studying the expression of structurally altered bicoid genes. v) Sequences that serve as targets of ftz mRNA instability will be identified and the effects of mutationally stabilizing ftz mRNA on its spatial pattern of expression and on embryonic development will be assessed. Unstable mRNAs often encode products that are required in a temporally and/or spatially restricted manner. The proposed studies address the importance of post-transcriptional regulatory events for pattern formation during embryonic development. These findings may be of general relevance for understanding developmental mechanisms in other organisms, including mammals.

In spite of its importance as a potential target for disease control, the insect gut has received little attention. Insect-borne parasites of human disease frequently initiate their development in the insect by penetrating its gut. The gut of hematophagous insects is also the site of blood digestion, a process that triggers egg production and ultimately influences insect fitness and reproduction. This proposal is to develop molecular approaches for the study of the Simulium gut, focusing on the above-mentioned processes. Simulium (blackfly) is the vector for onchocerciasis (river blindness), a debilitating disease that affects millions of people in Africa and Latin America. The parasite is ingested by the blackfly with the blood meal from an infected individual. In order to develop, the parasite must penetrate the blackfly gut. Penetration is largely but not entirely hindered by the secretion by the gut epithelium of a thick peritrophic matrix (PM) that surrounds the blood meal. There are reasons to believe that inhibition of PM formation would result in excessive parasite burden which causes fly lethality. One goal of the proposed research is to clone the genes encoding the two major PM components. The structural organization of these genes, including essential promoter elements, Will be defined and factors required for the gut-specific regulation of gene expression will be investigated. A second focus of this research is the investigation of genes which function in the digestion of the blood meal. Two genes which were previously isolated in this laboratory and encode putative proteolytic enzymes, will be similarly characterized. Finally, antibodies to Simulium gut proteins will be produced and used to explore the feasibility of developing immunological approaches to alter the fitness of the fly or to modify its vector capacity. The ultimate goal of these experiments is to provide the means for devising rational approaches of vector and disease control.

Despite the great importance of hematophagous insects as disease vectors, they have received relatively little attention from investigators utilizing modern molecular biological techniques. This proposal focuses on the insect gut, the first site of interaction between the insect and the parasites it transmits. The project has two broad objectives: (i) to develop means for interfering with the capacity of insects to transmit disease, and (ii) to gain a better understanding of the mechanisms that regulate gene expression in the insect gut. Specifically, we propose the following research plan. I) Functional characterization of a gut-specific promoter. Recently, we found that putative promoter sequences from the black fly carboxypeptidase gene can drive gut-specific expression of a reporter when transformed into Drosophila. In a separate set of experiments, we also found that a recombinant retrovirus can transfect Anopheles gut epithelial cells in vivo. While the transgenic Drosophila system is useful to assay tissue-specific gene expression, transfection of retroviruses into Anopheles is useful to measure induction of gene expression by a blood meal. To identify cis-acting promoter sequences required for gut-specific expression and expression induced by blood feeding, we will construct a series of promoter deletions, and assay for function by the two aforementioned in vivo assays. Interaction between promoter sequences and gut nuclear proteins will be investigated by the use of DNA mobility-shift assays. DNA-protein interactions will be further characterized by DNase I footprinting experiments. Cloning of genes encoding DNA-binding proteins will be accomplished by one of three approaches: direct screening of an expression library with a DNA probe, the yeast "one-hybrid" genetic screen, or biochemical purification of the binding protein by DNA affinity chromatography. These experiments may inform on how genes with potential for blocking parasite transmission, can be expressed in the mosquito gut. II) Identification of genes expressed early after a blood meal. Following a blood meal, transcription of the major digestive enzymes is activated after a lag of 6-8 hours. There is reason to believe that induction of the late enzymes depends on the activation of intermediate gene products, including transcription factors, during the lag time. To identify genes whose transcription is activated early after the blood meal, we will compare gut RNAs isolated from mosquitoes prior to, and 2-4 hours after a blood meal, by use of the PCR-based "differential display" technique. PCR fragments originating from the blood-induced mRNAs will be cloned, and the corresponding genes will be characterized by conventional approaches. The peritrophic matrix (PM), which forms late after a blood meal, constitutes a physical barrier for the diffusion of secreted products into the blood meal. The promoters of the "early genes" identified by these experiments, may be used to drive the expression of gene products with anti-parasite activity, prior to PM formation.

Mosquito-transmitted diseases are a major cause of suffering and death in the tropical world. Malaria alone, kills one to two million people every year. The urgency for developing new control strategies is underscored by the development of resistance by parasites to previously effective drugs, by the resistance of mosquitoes to a variety of insecticides, and by the lack of an effective vaccine. Inhibition of the parasite's life cycle in the mosquito is a strategy that requires more attention. This proposal focuses on the gut of the human malaria vector, Anopheles gambiae. The gut is the first site of interaction between Plasmodium and the mosquito. Ingestion of blood by the adult mosquito triggers the secretion of a peritrophic matrix (PM), which is a thick extra-cellular sheath that completely surrounds the blood meal and any ingested parasites. The PM poses a partial barrier for malaria parasite invasion. Modifications of the PM may lead to a more complete barrier to infection. To devise such strategies will require a thorough molecular characterization of the PM, and a more complete understanding of its structure and function. The major objectives of this proposal are to isolate and characterize genes encoding PM proteins, to investigate how PM proteins interact for the assembly of the PM, and to probe into the physiological role of the PM. Specifically, PM genes will be cloned by screening expression libraries with anti-PM antibodies or based on amino acid sequence of fractionated PM proteins. Interaction between PM proteins will be measured in vivo using the yeast two-hybrid system or in vitro using affinity blotting techniques. Antibodies to recombinant PM proteins will be used to determine if PM proteins are stored in secretion vesicles. The thickness and porosity of the PM will be experimentally altered to measure the effects on digestion and on the ability of parasites to traverse the PM. Genetic transformation of Ae. aegypti is already possible and transformation of An. gambiae is likely to become available in the near future. The characterization of PM protein genes and an understanding of their function may lead to novel strategies for malaria control.

Despite the great importance of hematophagous insects as disease vectors, they have received relatively little attention from investigators utilizing modern molecular biological techniques. This proposal focuses on the insect gut, the first site of interaction between the insect and the parasites it transmits. The project has two broad objectives: (i) to develop means for interfering with the capacity of insects to transmit disease, and (ii) to gain a better understanding of the mechanisms that regulate gene expression in the insect gut. Specifically, we propose the following research plan. I) Functional characterization of a gut-specific promoter. Recently, we found that putative promoter sequences from the black fly carboxypeptidase gene can drive gut-specific expression of a reporter when transformed into Drosophila. In a separate set of experiments, we also found that a recombinant retrovirus can transfect Anopheles gut epithelial cells in vivo. While the transgenic Drosophila system is useful to assay tissue-specific gene expression, transfection of retroviruses into Anopheles is useful to measure induction of gene expression by a blood meal. To identify cis-acting promoter sequences required for gut-specific expression and expression induced by blood feeding, we will construct a series of promoter deletions, and assay for function by the two aforementioned in vivo assays. Interaction between promoter sequences and gut nuclear proteins will be investigated by the use of DNA mobility-shift assays. DNA-protein interactions will be further characterized by DNase I footprinting experiments. Cloning of genes encoding DNA-binding proteins will be accomplished by one of three approaches: direct screening of an expression library with a DNA probe, the yeast "one-hybrid" genetic screen, or biochemical purification of the binding protein by DNA affinity chromatography. These experiments may inform on how genes with potential for blocking parasite transmission, can be expressed in the mosquito gut. II) Identification of genes expressed early after a blood meal. Following a blood meal, transcription of the major digestive enzymes is activated after a lag of 6-8 hours. There is reason to believe that induction of the late enzymes depends on the activation of intermediate gene products, including transcription factors, during the lag time. To identify genes whose transcription is activated early after the blood meal, we will compare gut RNAs isolated from mosquitoes prior to, and 2-4 hours after a blood meal, by use of the PCR-based "differential display" technique. PCR fragments originating from the blood-induced mRNAs will be cloned, and the corresponding genes will be characterized by conventional approaches. The peritrophic matrix (PM), which forms late after a blood meal, constitutes a physical barrier for the diffusion of secreted products into the blood meal. The promoters of the "early genes" identified by these experiments, may be used to drive the expression of gene products with anti-parasite activity, prior to PM formation.

DESCRIPTION (provide by applicant): The mosquito is an obligatory vector for the transmission of malaria, a disease that kills about 2 million people every year. The urgency for developing new control strategies is underscored by the development of resistance by parasites to previously effective drugs, by the resistance of mosquitoes to a variety of insecticides, and by the lack of an effective vaccine. Inhibition of the parasite's life cycle in the mosquito is a strategy that needs to be explored. This proposal focuses on the molecular interactions between Plasmodium, the causative agent of malaria, and the mosquito vector. To complete its life cycle in the mosquito, Plasmodium must invade and traverse two different epithelia: the midgut and the salivary gland. A major objective of this proposal is to gain insights on the mechanisms utilized by Plasmodium to traverse these epithelia. Extensive use will be made of phage display libraries, which consist of large numbers of bacteriophages each displaying on their surface a different peptide or protein domain. By incubating the mosquito epithelia with a high-titer stock of the library, phages can be selected that bind with high affinity to surface ligands. Initial results suggest that at least one phage that has been selected by this approach displays a peptide that inhibits Plasmodium invasion of the salivary gland. A transgenic mosquito line that secretes this peptide into the hemolymph will be produced. These genetically modified mosquitoes are expected to be impaired in their ability to transmit the parasite to a vertebrate host. Mosquito salivary gland and midgut surface ligands that are recognized by the selected phages will be isolated and characterized. A phage library that displays Plasmodium proteins will be used to screen for parasite proteins that interact with the mosquito epithelia. A phage display library will be used to investigate the basis for resistance of a selected An. stephensi strain to transmit Plasmodium falciparum. Techniques for the genetic transformation of Ae. aegypti and An. stephensi are already available and transformation of An. gambiae is likely to become available in the near future. A better understanding of how Plasmodium develops in the mosquito may lead to novel strategies for malaria control.

DESCRIPTION: (provided by the applicant): Malaria kills over one million persons every year. Because current intervention procedures with drugs, insecticides and vaccines are ineffective, new approaches for malaria control are urgently needed. Malaria transmission depends on the ability of the Plasmodium parasite to develop in the mosquito vector. However, at the molecular level, very little is known about the complex developmental program that governs Plasmodium differentiation in the mosquito. The proposed project seeks to reduce this knowledge gap. Initial experiments conducted in this laboratory have assembled many of the required tools for the project. Four cDNA subtraction libraries have been constructed that are enriched for sequences expressed at different stages of Plasmodium development in the mosquito. Micro-arrays containing 4,000 clones are being produced and over 2,000 inserts are being sequenced. Using these tools, Plasmodium genes that are preferentially expressed at specific stages of development in the mosquito (and not in the vertebrate host) will be identified and selected for further study. Particular attention will be devoted to genes encoding proteins with transmembrane domains (candidate receptors and transmission-blocking antigens) or that have motifs suggesting involvement in regulatory functions (e.g., signal transduction, kinase domains, similarity to transcription factors). Temporal expression of these genes will be investigated and antibodies will be produced to determine cellular localization. The possible ability of these antibodies to block development of the parasite in the mosquito will be assessed. Knockout mutations will be produced and changes of gene expression caused by these mutations will be measured by use of the micro-arrays.

[unreadable] DESCRIPTION (provided by applicant): Malaria is one of the most serious infectious diseases in the world. The number of cases is increasing, in part because of insufficient means to fight the disease. Malaria is caused by the protozoan parasite Plasmodium, which multiplies within human red blood cells. Two forms of the parasite circulate in the human blood: asexually dividing parasites (approximately 95%) and gametocytes (male and female; approximately 5%). Once ingested by the mosquito, the gametocytes develop into forms that infect the insect vector while the asexual parasites die. Transmission of Plasmodium from one person to the next strictly depends on formation of gametocytes (gametocytogenesis). Little is known about genes that are involved in the differentiation from the Plasmodium asexual to the sexual stages (gametocyte). We plan to use a genome-wide genetic screen of P. falciparum, the deadliest of the human parasites, by use of transposon-mediated insertional mutagenesis combined with a screen for mutants incapable to form gametocytes. This approach is feasible because gametocyte formation is not required for parasite survival in culture (the desired mutants are viable) and takes advantage of the fact that transposons molecularly mark the affected gene, facilitating its identification. With almost half of the world's population at risk of malaria infection, it is imperative to find new control strategies. A better understanding of P. falciparum gametocytogenesis may lead to the development of means to prevent gametocyte formation and thus, block transmission. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Around 46% of the World's population lives in areas where mosquito-borne diseases including malaria, filariasis, viral encephalitides, dengue and yellow fever are endemic. It was recently established that the insect pathogenic fungus Metarhizium anisopliae has the potential to control adult mosquitoes in an urban setting, but only if its potency is increased. Our goal is to produce one or more fungal products which can deplete anopheline and Plasmodium populations to the extent that marked reductions in malaria prevalence are achieved. We have already shown that M. anisopliae is a very effective delivery system for the insect-selective scorpion toxin AaIT, and that expressing AaIT produced a 9-fold reduction in effective spore doses against mosquitoes. This was very significant but kill times remain too slow for adequate protection. In this application, we propose experiments to compare several strategies for optimizing M. anisopliae's ability to curtail disease transmission. Mosquitoes infected with fungi showed a significant reduction in the number of sporozoites on salivary glands, but the mechanism responsible is unknown. We will carry out a detailed analysis of the interactions between Plasmodium, mosquitoes and M. anisopliae. This will include testing an attenuated strain of M. anisopliae that elicits a hyperimmune response to determine whether M. anisopliae can be used to immunize mosquitoes from Plasmodium. Further, we will compare mortality and sporozoite prevalence in mosquitoes infected with M. anisopliae strains expressing different combinations of insecticidal and anti-plasmodial proteins. It will be determined if these can be used synergistically to achieve effective reductions in transmission potential. Based on these results, we will also test the efficacy of using M. anisopliae to express synthetic multifunctional genes that are hybrids of different activities and that could, for example, target both the insect and the Plasmodium. The current proposal will: 1) explore the mosquito immune system;2) develop tools and genetically engineered fungi that have the potential to greatly reduce malaria prevalence, and 3) develop M. anisopliae as a tractable model system that can be used to screen novel effectors. We envisage that after screening, the most potent effectors could be delivered against mosquitoes or Plasmodium by expression in M. anisopliae and/or in alternative pathogens, commensals or via transgenic mosquitoes. PUBLIC HEALTH RELEVANCE This project aims to design, construct and evaluate recombinant fungal pathogens that target adult mosquitoes and the malaria parasite. The most significant possible outcome of producing an optimized fungal pathogen will be a reduction of human disease as a result of interrupting transmission of the target parasite.

DESCRIPTION (provided by applicant): Malaria kills an estimated 1-2 million people (mostly children) every year. For transmission to occur, Plasmodium, the causative agent of malaria, has to complete a complex developmental cycle in the mosquito. Only a small proportion of the parasites survive the entire cycle. Thus, the mosquito is a potential weak link that can be exploited for disease control. Invasion of the mosquito midgut by Plasmodium ookinetes is a crucial step, yet little is known about the molecular mechanisms that operate at this stage. We made two unexpected observations during the current grant period: 1) The surface of Plasmodium ookinetes (the form that invades the midgut) is lined with an enolase-like protein and 2) ookinetes can invade the midgut by more than one pathway, one that can be blocked by the SM1 peptide and another that cannot. One aim of this proposal is to investigate, at the molecular level, the mechanism of midgut invasion. Our first aim will address the following working hypothesis. Enolase expressed on the surface of midgut ookinetes captures plasminogen from the surrounding blood meal. A mosquito type II annexin on the surface of the midgut epithelium binds to both tissue type plasminogen activator (tPA) from the blood meal and to ookinete surface enolase. We hypothesize that this bridge facilitates both ookinete docking to the surface of the midgut epithelium and tPA activation of plasminogen into plasmin (a protease). The combination of these two separate but intimately entwined events results in successful midgut invasion. Our second aim is to identify P. berghei ookinete genes that are responsible for the different invasion pathways. This is the first comprehensive study of the mechanisms of Plasmodium invasion of the midgut epithelium. Knowledge generated by these studies may have important implications for the development of multivalent transmission-blocking vaccines. PUBLIC HEALTH RELEVANCE Malaria, AIDS and tuberculosis are three infectious diseases that cause the largest numbers of deaths worldwide. Of these, only malaria requires an intermediate vector for transmission to occur. Therefore, the mosquito vector is a potential weak point in the transmission cycle. A better understanding of parasite development in the mosquito may translate in the discovery of new strategies to fight this deadly disease.

DESCRIPTION (provided by applicant): Malaria is one of the deadliest infectious diseases and kills an estimated 2 million persons every year. Even though a considerable body of knowledge exists on the parasite cycle, our understanding of how the parasite infects its vertebrate host is incomplete. Infection is initiated when an infected mosquito delivers sporozoites at the time of blood feeding. The sporozoites find their way to the circulation and of all organs through which they transit, they specifically target and infect the liver. Previous work has established that sporozoites attach to highly sulfated, liver-specific glycosaminoglycans (GAGs) that protrude the fenestrated walls of the liver blood vessels, called sinusoids. Two cell types line the sinusoids: endothelial cells and specialized macrophages, termed Kupffer cells. It is known that to reach the hepatocytes, sporozoites invade Kupffer cells, not endothelial cells, indicating that sporozoite-Kupffer recognition takes place. Despite its importance for the outcome of infection, the molecular basis for this recognition step remains largely unknown. In preliminary work we have identified three peptides from a phage display library that bind specifically to Kupffer cells causing an inhibition of sporozoite invasion. By crosslinking the peptides to their target protein on the Kupffer cells, we will identify and characterize candidate Kupffer cell receptors for sporozoite invasion. We hypothesize that the peptides mimic the conformation of sporozoite proteins that interact with the Kupffer cells. We will produce antibodies against each of the peptides and use these antibodies to identify and characterize the sporozoite proteins that presumably interact with the Kupffer cells during invasion. Such proteins have the potential of becoming candidates for development of a malaria vaccine that prevents liver infection. PUBLIC HEALTH RELEVANCE: Malaria is one of the deadliest infectious diseases and kills an estimated 2 million persons every year. After an infected mosquito delivers Plasmodium sporozoites to its host it enters the circulation and specifically recognize and invade liver macrophages (Kupffer cells). This project is to identify and characterize the proteins (Kupffer receptors and sporozoite ligands) involved in sporozoite invasion of liver Kupffer cells.

DESCRIPTION (provided by applicant): The fight against the intolerable burden of malaria is restricted to the use of insecticides that kill the mosquito vector and drugs that kill the parasite in humans. Considering that recent estimates place the number humans infected with malaria at over 500 million (nearly 1 in 12 humans), the introduction of new means to counter the disease is urgently needed. We propose to develop a novel strategy to prevent the spread of malaria parasites by anopheline mosquitoes, based on genetic modification of bacteria that inhabit the gut of these insects. We will evaluate in detail the ability of two candidate bacterial species (Pantoea agglomerans and Asaia sp.) to colonize larval and adult mosquitoes and their ability to be transmitted vertically from one generation to the next. We will develop robust methods to secrete antimalarial effector proteins from each bacterial species to ensure that the effector proteins will reach the intended targets on the parasite or on the midgut epithelium. Different bacterial strains that secrete effector proteins will be evaluated for their efficacy to interfere with parasite development in the mosquito. Using the data obtained from engineered bacterial strains producing single effectors, we will create an optimal combination of strains that combine multiple effectors for maximum efficacy. Given that this funding mechanism is restricted in its time frame, no field trials are proposed although discussions with the relevant regulatory agency (US-EPA) are already underway. This research is expected to lead to the development of a novel weapon that can be used in combination with traditional control strategies (drugs, insecticides, vaccines) to combat malaria. PUBLIC HEALTH RELEVANCE: Malaria is one of the deadliest infectious diseases and kills an estimated 2 million persons every year. The mosquito is the obligatory vector for transmission. This project will devise new ways to interfere with the mosquito capacity to transmit the parasite, by genetically modifying bacteria that live in the mosquito midgut.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise;2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues;and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): This proposal requests partial support for a meeting on "Tropical Infectious Diseases: from bench to field" as part of the Gordon Research Conference series to be held in Galveston, Texas, March 13-18, 2010. The broad and long-term goal of the conference is to increase our understanding of host-pathogen-vector interactions with the aim of developing new insights for controlling Tropical Infectious Diseases (TIDs). The specific aims of this meeting are to convene 33 speakers representing critical areas of neglected tropical disease research with a total of 150 participants for a five-day conference in a relatively isolated, relaxed setting. The program will open with two keynote presentations addressing present global needs for infectious disease research. The conference will focus on the diseases for which most significant recent progress is being made and on those of the highest medical importance. The sessions will cover arboviral diseases, cholera, filariases, leishmaniasis, malaria, rickettsioses and trypanosomiasis. Each session will consider recent basic discoveries in the areas of pathogen genomics, cell biology and immunology in the context of the most current findings from field based epidemiologic studies and intervention trials. The conference will close with two keynote presentations addressing the priorities for future TID research, emphasizing how parasite biology can be explored for disease control and the challenges of vaccine development. Two afternoon poster sessions will permit all participants to contribute to these topics. The significance of this application is that the Gordon Research Conference on TIDs provides a forum that brings together multidisciplinary members of the international research community on topical diseases. The intent is to promote discussions from basic to applied questions and to generate 'cross-fertilization'among different specialties and fields of knowledge while establishing a community of scientists interested in TIDs. The health relatedness of this application is that the discussions will define the important questions on basic science as they relate to the development of new strategies to control the most important tropical diseases. PUBLIC HEALTH RELEVANCE: Approximately 1 billion people - one sixth of the world's population - suffer from one or more tropical infectious diseases. This Tropical Infectious Diseases (TIDs) Gordon Research Conference will bring together scientists working on the most prevalent TIDs and cover topics ranging from laboratory research to field-based applications. The health relatedness of this application is that the gathering of top scientists working in different but related fields will stimulate the generation of new ideas and foster cross-discipline collaboration with the aim of accelerating progress toward the control and elimination of TIDs.

DESCRIPTION (provided by applicant): Continued training in The Molecular and Cellular Bases of Infectious Diseases (MCBID) is proposed for 8 PhD students and 3 postdoctoral fellows selected from large pools of highly qualified applicants. The training program is uniquely situated in the Molecular Microbiology and Immunology Department (MMI) within the Johns Hopkins Bloomberg School of Public Health. The 29 training faculty have a wide range of experience and expertise in viruses, bacteria and parasites causing human disease and in the vectors and environmental factors associated with emergence and transmission of these pathogens. The training program has been funded since 1994 and has produced scientists working in many areas of academia and government on problems related to infectious diseases, vaccine development and the public's health. The goal of the MCBID training program is to provide students with both a firm foundation in the basic disciplines necessary for the study of infectious diseases and a perspective that will enable them to apply their knowledge creatively to public health problems. Each student is expected to complete 1) a series of required courses in the basic disciplines of cell and molecular biology, biochemistry, and immunology, 2) courses in virology, bacteriology, parasitology, and disease ecology, 3) courses in research ethics and public health perspectives, and 4) elective courses relevant to thesis topic and long-term career goals. Elective courses are chosen from among courses available in MMI, other departments in the School of Public Health, or in other Divisions of the University. Students will also complete 3 11-week laboratory rotations during the first year. Student progress is monitored by a Thesis Advisory Committee and the Graduate Program Committee. The goals of the postdoctoral training program are 1) to provide focused training in those areas of the molecular and cellular basis of infectious diseases in which program faculty have special expertise; 2) to provide an opportunity for doctoral degree holders trained in more traditional environments to broaden their exposure to problems of public health importance and to evaluate their career goals in terms of public health issues; and 3) to prepare the PDF for an independent career in the biological sciences. RELEVANCE : This program is highly relevant to national interests in the areas of emerging infectious diseases, as it trains students and postdoctoral fellows broadly not only in both the molecular aspects of pathogen biology and disease pathogenesis, but also in the ecology of disease emergence and the role of vectors in pathogen transmission.

DESCRIPTION (provided by applicant): This proposal requests partial support for a meeting on Tropical Infectious Diseases: from bench to field as part of the Gordon Research Conference series to be held in Galveston, Texas, Feb. 10-15, 2013. The broad and long-term goal of the conference is to increase our understanding of host-pathogen-vector interactions with the aim of developing new insights for controlling Tropical Infectious Diseases (TIDs). The specific aims of this meeting are to convene 33 speakers representing critical areas of tropical disease research with a total of 150 participants for a five-day conference in a relatively isolate, relaxed setting. The program will open with two keynote presentations addressing present global needs for infectious disease research. The conference will focus on the diseases for which most significant recent progress is being made and on those of the highest medical importance. The sessions will cover arboviral and enteric diseases, filariasis, leishmaniasis, malaria, tuberculosis, and vector-pathogen interactions. Each session will consider recent basic discoveries in the areas of pathogen genomics, cell biology, and immunology in the context of the most current findings from field-based epidemiologic studies and intervention trials. The conference will close with two keynote presentations that provide outstanding examples of basic and applied research, emphasizing how pathogen biology can be explored for disease control and the challenges of vaccine development. Four afternoon poster sessions will permit all participants to contribute to these topics. The significance of this application is that the Gordon Research Conference on TIDs provides a forum that brings together multidisciplinary members of the international research community on tropical diseases. The intent is to promote discussions from basic to applied questions and to generate cross-fertilization among different specialties and fields of knowledge while establishing a community of scientists interested in TIDs. The health relatedness of this application is that the discussions will define the important questions on basic science as they relate to the development of new strategies to control the most important tropical diseases affecting over one-sixth of the world's population.

? DESCRIPTION (provided by applicant): CM is a serious complication of Plasmodium falciparum infection, and has a profoundly devastating effect especially on children, non-immune travelers and military personnel. Clinically, CM can result in several neurological problems, which include seizures, reversible coma and is associated with a high mortality of up to 30%, with the highest rate in children. Acute neurological symptoms include impaired consciousness, coma, delirium, seizures, and increased intracranial hypertension. The hallmark of CM pathology is the intra-vascular sequestration of parasitized red blood cells (PRBC) inside high endothelial venules throughout the brain. PRBC bind to the blood brain barrier (BBB) endothelium in both WM and gray matter (GM) but do not invade into the brain. Interestingly, the PRBC sequestration in blood vessels leads to a distinctly different pathology between WM and GM. Recent postmortem studies reveal a clear hemorrhagic pathology within WM. Our previous data showed highly inflammatory responses associated with GM endothelium. Yet, little is known of the factors that cause these differences of the brain endothelium residing in GM versus WM and how any potential differences could relate to divergent responses in CM. Therefore, we hypothesize that, due to differences in the direct physiological environment of GM and WM (e.g. astrocyte-neuronal versus pericyte-oligodendrocytes), the vessel endothelium in these different brain tissues exhibit differing properties. In combination with a specific var-gene expressing Plasmodium binding pattern, these different endothelial properties result in diverging CM pathologies. Here, we propose to study the underlying WM versus GM endothelial differences and responses to PRBC by using in vitro models of human BBB, and compare these differences to in situ human brain samples and vascular responses in an in vivo experimental CM model. This application is response to RFA-HL-15-023 Vascular Dysfunction in the Pathogenesis of Severe Malaria (R01). These studies will provide an improved understanding of the BBB and could provide new understandings of the molecular mechanisms of the brain vessels differentiation in WM versus GM that may also be implicated in other neurological diseases.

? DESCRIPTION (provided by applicant): Malaria remains one of the most devastating infectious diseases. It kills around a million people every year while causing immense suffering and economic losses worldwide. The two main weapons to fight the disease - insecticides that kill the mosquito vector and drugs that kill the parasites in infected individuals - significantly contribute to contain the disease. However, it is imperative that additional measures, such as vaccines, be developed for the elimination and eventual eradication of the disease. Human infection is initiated when during a bite by an infected mosquito, sporozoites are deposited in the skin of the bitten person. Next, sporozoites enter the circulation and must leave it in the liver t continue their cycle. Liver vessels are lined by two cell types - endothelial cells and Kupffer cels - and sporozoites exit the vessels by preferentially traversing Kupffer cells. Using a phage peptide display library we have identified three peptides - P39, P61 and P52 - that bind specifically to Kupffer cells and by doing so, inhibit sporozoite traversal both in vitro (Kupffer ell cultures) and in vivo (live mice). Further work determined that the peptides bind to the CD68 Kupffer cell surface receptor protein. Moreover, the peptides structurally mimic domains of Plasmodium berghei glyceraldehyde 3-phosphate dehydrogenase (PbGAPDH), a protein present on the surface of sporozoites. In addition, we have shown that the CD68 and PbGAPDH proteins interact directly. This project aims at identifying domains of the PbGAPDH protein that mediate interactions with the CD68 receptor. These protein domains will be used as antigens to immunize mice. It is expected that the antibodies of immune mice will interfere with sporozoite traversal of Kupffer cells and in this way, thwart liver infection. The antigens identified in this study may conceivably be used to enhance the effectiveness of the RTS,S pre-erythrocytic vaccine, which in phase III trials has proven to be partially effective.

ABSTRACT Malaria remains one of the most devastating infectious diseases. It kills over a million people every year while causing immense suffering and economic losses worldwide. Whereas much progress has been made in understanding the life cycle of the parasite in the human host and in the mosquito vector, significant gaps of knowledge remain. Fertilization of malaria parasites is a poorly understood process that takes place in the lumen of the mosquito gut. This process is important because survival in nature is completely dependent on the ability of the parasite to undergo sexual reproduction. The proposed research aims to identify molecular interactions that take place during fertilization of malaria parasites. This project is based on an unorthodox approach (identification of peptides that bind to the gamete surface) made possible by the recent development of an important tool, a transgenic parasite that produces red-fluorescent female gametes and green-fluorescent male gametes. Pure populations of malaria female and male gametes from this transgenic parasite were isolated by cell sorting and then used to screen a phage display library for peptides that recognize molecules on the gamete surfaces. A peptide (FG1) that binds to female gametes and another peptide (MG1) that binds to male gametes were identified. Importantly, when added to a malaria infectious blood meal, each of these peptides blocked parasite fertilization, suggesting that the peptides bound to a receptor and prevented its interaction with a ligand on the gamete of the opposite sex. Our working hypothesis is that the peptide structurally mimics the gamete ligand and that peptide and ligand compete for binding to the corresponding receptor. This proposal lays out a research plan to identify the receptor to which the FG1 and MG1 peptides bind and the ligands on the gametes of the opposite sex that the two peptides structurally mimic. An additional aim is to characterize the proteome on the surface of female gametes before and after fertilization, to gain insights on additional proteins involved in fertilization and possible block to polyspermy. Elucidation of mechanisms of fertilization is important not only for understanding the basic biology of malaria and other parasitic diseases but could also lead to the identification of new targets for blocking transmission and the spread of disease. Moreover, should the mechanisms be conserved, our findings could be extended to the biology of fertilization of higher organisms.