Kristin Michel, Ph.D. - US grants

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Despite ongoing control and prevention efforts, malaria remains one of the most devastating infectious diseases, world-wide. Female Anopheles mosquitoes are the vectors for human malaria. When a mosquito bites an infected individual, it takes up infectious forms of the parasite along with blood. The parasite undergoes a complex journey through its mosquito vector, before it can subsequently be injected into the next human host via mosquito bite. As part of this journey, the parasite must cross two epithelia, the mosquitoes'midgut and the salivary glands This study aims to characterize a common mosquito epithelial immune response against malaria parasite invasion, which is characterized by the serine protease inhibitor serpin-6 (SRPN6). Our aims are to (a) explore the regulation of SRPN6 during the invasion of the midgut and salivary glands by the parasite and (b) to measure any potential effect of SRPN6 on the passage of the parasite through these epithelia. The results of this study can provide important clues to the potential contribution of epithelial immunity to parasite transmission by mosquito vectors in a disease-relevant mosquito/parasite combination.

DESCRIPTION (provided by applicant): Malaria control and eradication strategies continue to primarily rely on vector control that is stymied by insecticide resistance in vector populations. In addition to resistance management, novel insecticides with new modes of action - particularly for which efficacy is not affected by existing resistance mechanisms - are required. The long-term goal is to develop a malaria vector control methodology based on a new insecticide target, the serine protease inhibitor serpin (SRPN)2 from the African malaria mosquito, Anopheles gambiae s.s.. SRPN2 is a key negative regulator in the extracellular proteinase cascade that controls activation of prophenoloxidase (proPO) and thus melanization - a powerful, arthropod-specific innate immune response. SRPN2 depletion from the hemolymph of adult female mosquitoes significantly reduces longevity with escalating daily mortality nine days after treatment. The objectives in this application are to identify the interactions of SRPN2 with its proteinase targets and to evaluate the malaria control potential of a SRPN2 inhibitor. The rationale for the proposed research is that detailed information on SRPN2's biological proteinase targets and mode of action will ultimately allow the design of small molecule inhibitors of SRPN2 that could act as an insecticide for mosquito vector control. Guided by our preliminary data, the following three specific aims will be pursued: (1) Determine the molecular targets of SRPN2 inhibitory function;(2) Determine the molecular interface between SRPN2 and its target proteinase(s);and (3) Model the impact of SRPN2 depletion on disease transmission. Under the first aim, a combination of biochemical and genetic approaches that have been used successfully in the applicant's laboratory will be used to identify serine proteinase targets of SRPN2 based on their ability to (a) form covalent complexes with the serpin and (b) revert the serpin's depletion phenotype. Under the second aim, the molecular interactions between SRPN2 and one of its target proteinases, CLIPB9, will be identified by protein crystallography and mutational analysis. Under the third aim, the potential effect of SRPN2 inhibition on malaria transmission and resistance development against a potential SRPN2 inhibitor will be assessed by mathematical modeling. The proposed research is innovative, as it will for the first time evaluate the mosquito immune system as a physiological target for novel insecticides. Additionally, this project will use microfluidics as a highly innovative approach to the study of mosquito innate immunity, which if successful will be transformative to the field of insect biochemistry. This project is significant as it will provide fundamental knowledge of the nature of serine proteinase cascades that regulate melanization in mosquitoes. Ultimately, it has the potential to advance the development of new a generation of insecticide targets for malaria control. PUBLIC HEALTH RELEVANCE: Vector-borne diseases, especially malaria, continue to be a major public health threat world-wide, with roughly half of the world population at risk of malaria. The proposed project is relevant to public health because the identification of novel insecticide targets in mosquitoes will allow the development of new vector control measures for malaria control. Therefore, the proposed project is relevant to the NIH-NIAID's mission in relation to the understanding and prevention of re-emerging infectious diseases.

DESCRIPTION (provided by applicant): This application requests funds to support vector biologists to attend the 8th annual Arthropod Genomics Symposium (AGS) to be held at the University of Illinois campus June 12 -14, 2014. The AGS is the international forum for arthropod genomics research and fosters technology and knowledge exchange between bioinformatics, genomics, and fundamental and applied arthropod research. While the philosophy of the symposium is to cover a broad range of phylogenetic arthropod groups, a substantial focus has been on arthropod vectors of human disease. The 2014 symposium will be an open meeting of approximately 230 participants. The meeting will be preceded by a one day RNAseq Workshop that concludes in time for the AGC Symposium. The meeting agenda will offer a keynote speaker and invited speakers presenting in sessions the areas of i5K/1kite, Vector Genomics, Social Insects, Population genomics, Comparative Genomics, and Microbiomes. The program will be augmented by shorter talks chosen from submitted poster abstracts, and two poster sessions for which anticipate 60-70 posters based on past participation. Our over-arching goal is to increase genomics research in the field of vector biology. Progress towards this goal will be achieved through the following specific aims: (1) supplementing genomics training for early stage vector biologists; (2) Recruiting arthropod genomics researchers to the vector biology field; and (3) exposing non- vector arthropod genomics researchers to the field of vector biology we propose to support the travel of accomplished vector biologists working in the genomics field to present their work. The significance of this application lays in the key function of genomic information to modern vector biology research. Its innovation is the AGS's unique ability to bring together experts in vector biology, genomics, bioinformatics, and genomics technology developers. The meeting is public health relevant as research capacity building in cutting edge genomics research is crucial for the development of novel tools to combat and prevent vector-borne diseases.

? DESCRIPTION (provided by the applicant): This application requests funds to support vector biologists to attend the 9th annual Arthropod Genomics Symposium (AGS) hosted at the Kansas State campus in Manhattan, KS from June 17 -19, 2015. The AGS is the international forum for arthropod genomics research and fosters technology and knowledge exchange between bioinformatics, genomics, and fundamental and applied arthropod research. While the philosophy of the symposium is to cover a broad range of phylogenetic arthropod groups, a substantial focus has been on arthropod vectors of human disease. The 2015 symposium will be an open meeting of approximately 250 participants. The meeting agenda offers a keynote speaker, Dr. David O'Brochta, U. Maryland and invited speakers presenting in sessions the areas of i5K/Emerging Genomes, Medical Vector Genomics, Agricultural Vector Genomics, Population genomics, Epigenomics, and Microbiomes. The program will be augmented by shorter talks chosen from submitted poster abstracts, and two poster sessions for which we anticipate 60-70 posters based on past participation. WE will provide two workshops, one on Insect Genetic Tools with the participation of the IGTRCN representatives (http://igtrcn.org/) and a workshop on bioinformatics tools with leading experts in the field to train attendees, particularly young scientists and established investigators moving into the insect genetics and genomics field. Our over-arching goal remains to increase genomics research in the field of vector biology. Progress towards this goal will continued to be made through the following specific aims: (1) supplementing genomics training for early stage vector biologists; (2) Recruiting arthropod genomics researchers to the vector biology field; and (3) exposing nonvector arthropod genomics researchers to the field of vector biology we propose to support the travel of accomplished vector biologists working in the genomics field to present their work. The significance of this application lays in the key function of genomic information to modern vector biology research. Its innovation is the AGS's unique ability to bring together experts in vector biology, genomics, bioinformatics, and genomics technology developers. The meeting is public health relevant as research capacity building in cutting edge genomics research is crucial for the development of novel tools to combat and prevent vector-borne diseases.

PROJECT SUMMARY The innate immune system of mosquitoes is a critical determinant of their vector competence. This includes the ability to support development and transmission of the protozoan parasite species in the genus Plasmodium by Anopheles mosquitoes, the principal vectors of human malaria world-wide. Insight into the regulation of innate immune effector mechanisms remains incomplete, but is vitally important to our fundamental understanding of host-pathogen interactions in this most important human vector-borne disease. The long-term goal is to understand immune system regulation in An. gambiae to inform current and future vector control strategies. The objective of this application is to globally identify mechanisms of immune system regulation by determining the interactions within the extracellular protease network that activate and link opsonization to melanization in the context of distinct microbial infections. The rationale for the proposed research is that detailed information on the protease network that regulate mosquito immunity could be employed to predict long-term efficacy of novel vector control strategies that employ microbial agents, and manipulate infection outcome. Guided by our preliminary data, the following three specific aims will be pursued: (1) Determine the interactions of proteases and their homologs that are critical for mosquito immunity; (2) Assess the impact of the protease network on immunity and mosquito fitness; and (3) Visualize the immunoregulatory network in mosquitoes using network science. Under the first aim, we will test the hypothesis that clip-serine proteinases and their homologs form functional modules that are required for optimal immune responses by defining their cleavage patterns, genetic interactions, and precise biochemi-cal function. Under the second aim, the potential effect of the protease network on pathogen resistance and tolerance as well as mosquito fitness will be assessed using common microbial challenge models and life table analyses. Under the third aim standard network science approaches will be used to visualize all protease interactions in the system as a static multilayered network and to analyze this network to infer proteolytic flow through that links opsonization and melanization and to identify the key molecules that control immunity. The proposed research is innovative, as it will for the first time evaluate protease cascades as a single, integrated network that controls mosquito humoral immunity during diverse immune challenges. Additionally, this project will use network science as a highly innovative approach to the study of mosquito innate immunity, which if successful will be transformative to the field of insect immunology. This project is significant as it will provide comprehensive understanding of the contribution of the protease network to mosquito health as well as the limitations of the system in overcoming infection. Ultimately, this knowledge could be employed to manipulate infection outcome and thus inform the development of new disease control strategies that aim at disrupting malaria parasite development in its vector.