Mechanism of action of novel Anopheles active toxins

 描述（由申请人提供）：控制疾病传播媒介的程序通常使用化学和生物杀虫剂。因此，芽孢杆菌属和赖氨酸芽孢杆菌属已经在世界范围内使用了四十多年，成功地控制了蚊子和黑蝇。但一种新的菌株，双酶梭菌（Cb）马来西亚，最有毒的生物杀虫剂按蚊尚未使用，即使是一个厌氧菌;它是更容易培养与最少的设备。缺乏知识的毒素是什么在Cb马来西亚阻止其利用。因此，我们的长期目标是了解Cb malaysia灭蚊作用，促进其用于控制按蚊幼虫蚊子，从而减少成虫种群和疟疾传播。本提案的总体目标是确定 按蚊活性毒素，并阐明其作用机制。为了鉴定所涉及的杀蚊毒素，我们产生了功能丧失的Cb马来西亚突变体。对该突变体Cbm-77、野生型Cb马来西亚株和非杀蚊型菌株Cb的基因组进行了测序。所获得的数据表明，在Cbm-77突变体中大质粒的损失，或在Cb中其缺失，导致杀蚊活性的完全损失。因此，我们认为， 我们假设杀蚊活性是由质粒编码的，并且该质粒中的特定基因是造成马来西亚衣原体对按蚊的高毒性的原因。质谱数据证实，按蚊毒性蛋白质由大质粒编码，并且这些蛋白质形成复合物。该质粒有两个毒素编码基因座，cry和ctox;前者由单个操纵子组成，而第二个基因座有一个cmp（梭菌杀蚊蛋白）操纵子和两个额外的基因，p47和ha 41。因此，在第一个目标中，我们将通过以下方式验证我们的初步结果：a）鉴定ctox基因座中的关键按蚊活性毒素。由于该位点编码的蛋白与其他梭菌属菌株产生的蛋白相似，我们推测HA 41蛋白参与中肠膜结合，而具有内肽酶基序的CMP是可以切割囊泡释放所需的SNARE复合物的活性毒素。初步证据支持这两种假设。我们将在以下两个目标中检验这些假设：B）。表征Cb马来西亚毒素与中肠膜的结合;和c）.确定Cb马来西亚毒素的细胞内靶位点。该提案的完成将显示这种新型细菌的实用性，因为它易于使用。其新的毒素作用模式也补充了B. t. israelensis和L. sphaericus，促进其用作按蚊控制的生物控制剂。其新的毒性机制将拓宽可用的按蚊活性生物杀虫剂的谱，并且还将有助于减弱抗性的发展。

 DESCRIPTION (provided by applicant): Programs that control disease-transmitting vectors often use insecticides, both chemical and biological. Thus Bacillus and Lysinibacillus sp. have been used worldwide for over four decades for successful control of mosquitoes and blackflies. But a novel strain, Clostridium bifermentans (Cb) malaysia, the most toxic bioinsecticide to Anopheles has not been used, even though being an anaerobe; it is much easier to culture with minimal equipment. The lack of knowledge of what the toxins are in Cb malaysia prevents its utilization. Thus our long-term goal is to understand Cb malaysia mosquitocidal action, facilitating its use for the control of anopheles larval mosquitoes, thereby attenuating adult populations and malarial transmission. The overall objective of the current proposal is to identify the anopheline active toxins and elucidate their mechanism of action. To identify the mosquitocidal toxins involved, we generated a loss of function Cb malaysia mutant. Genomes of this mutant, Cbm-77, together with that of wild-type Cb malaysia and of the non-mosquitocidal type strain Cb were sequenced. The data obtained demonstrated that a loss of a megaplasmid in the Cbm-77 mutant, or its absence in Cb, results in total loss of mosquitocidal activity. Hence, we hypothesize the mosquitocidal activity is encoded by the plasmid, and specific genes in this plasmid are responsible for high Cb malaysia toxicity to anophelines. Mass spectrometry data confirmed that the anopheline-toxic proteins were encoded by the megaplasmid, and these proteins form a complex. The plasmid has two toxin encoding loci, cry and ctox; the former consists of a single operon, while the second locus has a cmp (clostridal mosquitocidal protein) operon and two additional genes, p47 and ha41. Therefore in the first aim we will validate our preliminary results by: a) identifying the critical anopheline active toxins in the ctox locus. Sine this locus encodes proteins that are similar to those produced by other Clostridium strains we hypothesize that the HA41 protein is involved in midgut membrane binding, while the CMP, which has an endopeptidase motif, is the active toxin that could cleave SNARE complexes required for vesicular release. Preliminary evidence supports both hypotheses. We will test these hypotheses in the next two aims: b). Characterize the binding of the Cb malaysia toxins to midgut membranes; and c). Define the intracellular target site for the Cb malaysia toxins. Completion of this proposal will show the utility of this novel bacterium because of its ease of use. Its novel mode of toxin action also complements that of toxins from B.t. israelensis and L. sphaericus, facilitating its use as a biocontrol agent for anopheline control. Its novel mechanism of toxicity would broaden the spectrum of available anopheline-active bioinsecticides, and will also help attenuate resistance development.