Detoxification functionality of lactic acid bacteria

在我们目前的NSERC发现资助中已经表明，乳酸杆菌可以对重金属，黄曲霉毒素和农药进行解毒，我们现在希望确定减轻农药影响的机制。这对于对抗蜜蜂中的群体崩溃障碍很重要。没有什么比失去这些授粉者更能危及全球粮食供应了。我们的初步研究结果表明，常用的杀虫剂会损害昆虫的免疫力，使它们更容易受到各种病原体的影响。 假设我们假设乳酸杆菌属的成员具有解毒危及蜜蜂和其他生命形式的农药的能力。 长期目标本研究计划的长期目标是推进我们对一种重要细菌属的认识，该细菌属在食品和补充剂中具有长期的安全使用历史，并利用对抗环境化学物质和改善传粉者生存的属性。 研究目的1. 1.确定乳酸杆菌如何减轻农药的免疫抑制作用和对病原体的敏感性2.确定乳酸杆菌对农业相关外源性物质的解毒功能。我们将使用黑腹果蝇作为蜜蜂的成熟和强预测模型，以确定乳酸杆菌如何影响对农药和/或病原体的先天免疫（在广泛的分类范围内高度保守）的共调节反应，因为它们主要适用于蜜蜂，但也适用于其他宿主。我们将利用我们的RNAi介导的DUOX和Imd通路的敲低和过表达突变体的储备，并将这些敲除与Imd和DUOX通路相关基因表达的基于qPCR的测量和免疫荧光染色相关免疫蛋白偶联。我们将让苍蝇接触不同种类的杀虫剂：类烟碱、有机磷酸酯、氨基甲酸酯、农达（草甘膦）、乙酰甲胺磷、？三氟氯氰菊酯、杀线威、四氟醚唑。鼠李糖乳杆菌GR-1、L. plantarum Lp39，L. kunkeei BR1，L. reuteri RC-14，L.鼠李糖杆菌GG和非洲蜜蜂的菌株将用于对抗杀虫剂，并确定它们是否真的能降解化学物质。我们将比较来自健康蜂巢的蜜蜂与感染了P.幼虫、Melissococcus plutonius、N. apis，新单胞菌N. cerebellum、Ascosphaera apis、Crithidia bombi或Varroa destructor，将在体外检查乳杆菌抑制它们的能力的病原体。果蝇模型也将用于测试各种乳酸杆菌调节解毒途径的能力。最终结果将是了解乳酸杆菌菌株如何通过降解，以及通过调节昆虫免疫反应，微生物群恢复力和对抗关键蜜蜂病原体来对抗最广泛使用的农药。

Having shown in our current NSERC Discovery Grant that lactobacilli can perform detoxification of heavy metals, aflatoxins and pesticides, we now wish to identify the mechanisms involved in mitigating the pesticide effects. This is important to counter colony collapse disorder in honey bees. Nothing endangers the global food supply more than the loss of these pollinators. Our preliminary findings suggest that commonly used pesticides impair insect immunity making them more susceptible to a variety of pathogens. Hypothesis We hypothesize that members of the genus Lactobacillus harbor the ability to detoxify pesticides that endanger honey bees and other life forms. Long term goals The long-term goal of this research program is to advance our knowledge of an important bacterial genus with a long history of safe use in foods and supplements, and to exploit attributes that counter environmental chemicals and improve the survival of pollinators. Research objectives 1.1.Characterize how lactobacilli mitigates the immunosuppressive effects of pesticides and susceptibility to pathogens 2.Determine the functional capacity of lactobacilli to detoxify agriculturally-relevant xenobiotics. Methodological approach We will use Drosophila melanogaster as a well-established and strongly predictive model of the honey bee to determine how lactobacilli affect the co-modulatory response of innate immunity (highly conserved across broad taxonomic range) towards pesticides and/or pathogens as they apply primarily to honeybees, but also other hosts. We will utilize our stocks of RNAi-mediated knockdown and overexpression mutants of the DUOX and Imd pathways, and couple these knockouts with qPCR-based measurement of Imd and DUOX pathway-related gene expression and immunofluorescent stain relevant immune proteins. We will expose flies to different classes of pesticides: neonicotinoids, organophosphates, carbamates, Roundup (glyphosate), acephate, ?-cyhalothrin, oxamyl, tetraconazole administered at different concentrations. Lactobacillus rhamnosus GR-1, L. plantarum Lp39, L. kunkeei BR1, L. reuteri RC-14, L. rhamnosus GG and strains from African bees will be used to counter the pesticides and determine if they can actually degrade the chemicals. We will compare the microbiota in bees from healthy hives to that of hives infected with P. larvae, Melissococcus plutonius, N. apis, N. ceranae, Ascosphaera apis, Crithidia bombi or Varroa destructor, pathogens that will be examined in vitro for the ability of the lactobacilli to inhibit them. The Drosophila model will also be used to test various lactobacilli for their ability to modulate detoxification pathways. Significance The net result will be to develop an understanding of how lactobacilli strains can counter the most widely used pesticides through their degradation, and by modulation of insect immune responses, microbiota resilience, and countering key honey bee pathogens.