The overall objective our research is to develop more effective and environmentally safe bacteria for controlling the mosquito vectors of major human diseases including malaria, filariasis, dengue, and the viral encephalitides. In the present application, we propose to continue our basic studies that have already enabled us to develop recombinant bacteria based on Bacillus thuringiensis subsp. israelensis (Bti) and Bacillus sphaericus (Bs) that are significantly more efficacious than the wild type strains of these species currently used as mosquito larvicides for vector control. In addition, as shown in our progress report, our recombinant bacteria have the capacity to delay resistance significantly, which has been a major problem with B. sphaericus preparations. To support the sustainable use of bacterial larvicides in vector control, we propose three major objectives, to (1) continue our basic studies of endotoxin synthesis and parasporal body assembly in Bti, (2) strengthen our knowledge of the molecular biology and genetics of the Cyt1A protein and its ability to delay mosquito resistance, and (3) test the recent hypothesis that bacterial larvicides used at sublethal doses may be capable of reducing the adult life span of female mosquitoes, thereby reducing their vectorial capacity significantly. The studies of endotoxin synthesis and parasporal body assembly are aimed primarily at elucidating the molecular mechanisms by which mosquitocidal proteins are trafficked to and deposited in the parasporal envelope of Bti, as well as attempting to define key genes involved in assembly of the PB envelope. We will use a combination of recombinant DNA technology and analysis of mutants to identify both key proteins and/or regions of the endotoxin proteins involved in transport, as well as the proteins important to envelope assembly. The results of these studies should enable us to further improve upon the recombinant bacteria already developed, for example by targeting the Bs Bin protein to the Bti parasporal body. Our additional studies of Cyt1A, other endotoxins, and the genetics of resistance should produce valuable information for further improving resistance management programs. And the studies of the effect of Bti on adult longevity will yield a model that can be used to test whether any larvicide can be used to decrease vector longevity and thus pathogen transmission. Combined, these studies will strengthen the basis for using larvicides as component of integrated vector management programs for controlling species belonging to the most important vector genera, namely, Anopheles, Aedes, and Culex.

Public Health Relevance

The results of our studies described in the Progress Report show clearly that recombinant bacteria can be constructed that are much more efficacious than wild type bacteria, and importantly are much less prone to the evolution of resistance. Further improvement and commercial development of recombinant mosquitocidal bacteria based on Bti as a host cell should enable these to be used cost- effectively as components of many integrated vector control programs, both in developed countries as well as developing countries. In some habitats, at certain times of the year, these recombinant bacteria could be a major component of IVC programs. Aside from improved efficacy, use of these strains should greatly reduce the need for synthetic chemical insecticides. Moreover, these novel larvicides should be useful in IVC management programs even after strategies to genetically engineer vectors to reduce pathogen transmission are developed or other strategies are developed. As these new types of bacteria are recombinant organisms, they will require more safety testing than wild type mosquitocidal bacteria. However, we already have approval from the U.S. Environmental Protection Agency, and the corresponding state agencies in California and Florida to proceed with field trial. Within the next two years, pending receipt of funding and approval for such studies, we anticipate trials in Africa, likely Kenya, Tanzania, or Nigeria, against major local anopheline vectors of malaria. Because fermentations of our existing recombinants, especially the Bti strain that produces the Bs Bin toxin already look very promising, this is a technology, pending regulatory approvals, that could be operational for malaria control within the next five years. This is unlikely to be true for any of the other molecular technologies for vector control and disease reduction under development.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Vector Biology Study Section (VB)
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Costero, Adriana
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University of California Riverside
Schools of Earth Sciences/Natur
United States
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Bideshi, Dennis K; Park, Hyun-Woo; Hice, Robert H et al. (2017) Highly Effective Broad Spectrum Chimeric Larvicide That Targets Vector Mosquitoes Using a Lipophilic Protein. Sci Rep 7:11282
Sakano, Yuko; Park, Hyun-Woo; Bideshi, Dennis K et al. (2017) Contributions of 5'-UTR and 3'-UTR cis elements to Cyt1Aa synthesis in Bacillus thuringiensis subsp. israelensis. J Invertebr Pathol 149:66-75
Park, Hyun-Woo; Hice, Robert H; Federici, Brian A (2016) Effect of Promoters and Plasmid Copy Number on Cyt1A Synthesis and Crystal Assembly in Bacillus thuringiensis. Curr Microbiol 72:33-40
Colletier, Jacques-Philippe; Sawaya, Michael R; Gingery, Mari et al. (2016) De novo phasing with X-ray laser reveals mosquito larvicide BinAB structure. Nature 539:43-47
Wirth, Margaret C; Walton, William E; Federici, Brian A (2015) Evolution of Resistance in Culex quinquefasciatus (Say) Selected With a Recombinant Bacillus thuringiensis Strain-Producing Cyt1Aa and Cry11Ba, and the Binary Toxin, Bin, From Lysinibacillus sphaericus. J Med Entomol 52:1028-35
Sawaya, Michael R; Cascio, Duilio; Gingery, Mari et al. (2014) Protein crystal structure obtained at 2.9 Å resolution from injecting bacterial cells into an X-ray free-electron laser beam. Proc Natl Acad Sci U S A 111:12769-74
Wirth, Margaret C; Berry, Colin; Walton, William E et al. (2014) Mtx toxins from Lysinibacillus sphaericus enhance mosquitocidal cry-toxin activity and suppress cry-resistance in Culex quinquefasciatus. J Invertebr Pathol 115:62-7
Bideshi, Dennis K; Waldrop, Greer; Fernandez-Luna, Maria Teresa et al. (2013) Intermolecular interaction between Cry2Aa and Cyt1Aa and its effect on larvicidal activity against Culex quinquefasciatus. J Microbiol Biotechnol 23:1107-15
Park, Hyun-Woo; Pino, Brent C; Kozervanich-Chong, Switzerlyna et al. (2013) Cyt1Aa from Bacillus thuringiensis subsp. israelensis enhances mosquitocidal activity of B. thuringiensis subsp. kurstaki HD-1 against Aedes aegypti but not Culex quinquefasciatus. J Microbiol Biotechnol 23:88-91
Diaz-Mendoza, Mercedes; Bideshi, Dennis K; Federici, Brian A (2012) A 54-kilodalton protein encoded by pBtoxis is required for parasporal body structural integrity in Bacillus thuringiensis subsp. israelensis. J Bacteriol 194:1562-71

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