Mechanism of action of novel Anopheles active toxins

 描述(申请人提供)：控制疾病传播媒介的项目通常使用化学和生物杀虫剂。因此，芽孢杆菌和溶杆菌属。已经在世界各地使用了40多年，成功地控制了蚊子和黑蝇。但一种新的菌株马来西亚双酵梭菌(Clostridium bifermentans，CB)是对按蚊毒性最大的生物杀虫剂，尽管是厌氧菌，但尚未使用；它更容易用最少的设备进行培养。由于缺乏对马来西亚CB中的毒素的了解，阻碍了对其的使用。因此，我们的长期目标是了解CB Malaysia的杀蚊作用，促进其用于控制按蚊幼虫，从而减少成年蚊子数量和疟疾传播。目前提案的总体目标是确定 按蚊的活性毒素并阐明其作用机制。为了鉴定所涉及的灭蚊毒素，我们产生了一个功能缺失的CB马来西亚突变体。对该突变体CBM-77、野生型CB Malaysia株和非杀蚊型CB株的基因组进行了测序。获得的数据表明，在CBM-77突变体中丢失巨质粒或在CB中缺失巨质粒会导致完全丧失杀蚊活性。因此， 我们假设灭蚊活性是由该质粒编码的，该质粒中的特定基因是导致CB马来西亚对按蚊的高毒性的原因。质谱学数据证实，巨质粒编码了对按蚊有毒性的蛋白质，这些蛋白质形成了一个复合体。该质粒有两个编码毒素的基因座，它们分别是CRY和CTOX；前者由一个操纵子组成，而第二个基因含有一个CpP(杀蚊蛋白)操纵子和另外两个基因p47和ha41。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基因座中关键的按蚊活性毒素。由于这个位点编码的蛋白质与其他梭状芽孢杆菌产生的蛋白质相似，我们推测HA41蛋白参与了中肠膜的结合，而具有内肽酶基序的CMP是一种活性毒素，可以裂解囊泡释放所需的圈套复合体。初步证据支持这两种假设。我们将在接下来的两个目标中检验这些假设：b)。描述马来西亚CB毒素与中肠膜的结合情况；以及c)。确定CB马来西亚毒素的细胞内靶点。这项提案的完成将表明这种新型细菌的用途，因为它易于使用。其新的毒素作用模式也是对B.T.毒素的补充。Israelensis和L.sphaericus，便于将其用作防治按蚊的生防剂。其新的毒性机制将拓宽现有按蚊活性生物杀虫剂的范围，并有助于减弱抗药性的发展。

 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.