Center for Global Health, Department of Internal Medicine, University of New Mexico, New Mexico, USA.
Parasit Vectors. 2011 May 19;4:82. doi: 10.1186/1756-3305-4-82.
Leishmania donovani is transmitted by the bite of the sand fly, Phlebotomus argentipes. This parasite is the agent of visceral leishmaniasis (VL), an endemic disease in Bihar, India, where prevention has relied mainly on DDT spraying. Pesticide resistance in sand fly populations, environmental toxicity, and limited resources confound this approach. A novel paratransgenic strategy aimed at control of vectorial transmission of L. donovani is presented using Bacillus subtilis, a commensal bacterium isolated from the sand fly gut. In this work, B. subtilis expressing Green Fluorescent Protein (GFP) was added to sterilized larval chow. Control pots contained larval chow spiked either with untransformed B. subtilis or phosphate-buffered saline. Fourth-instar P. argentipes larvae were transferred into the media and allowed to mature. The number of bacterial colony forming units, relative abundance and the mean microbial load were determined per developmental stage.
Addition of B. subtilis to larval chow did not affect sand fly emergence rates. B. cereus and Lys fusiformis were identified at each developmental stage, revealing transstadial passage of endogenous microbes. Larvae exposed to an exogenous bolus of B. subtilis harbored significantly larger numbers of bacteria. Bacterial load decreased to a range comparable to sand flies from control pots, suggesting an upper limit to the number of bacteria harbored. Emerging flies reared in larval chow containing transformed B. subtilis carried large numbers of these bacteria in their gut lumens. Strong GFP expression was detected in these paratransgenic flies with no spread of transformed bacteria to other compartments of the insects. This is the first demonstration of paratransgenic manipulation of P. argentipes.
Paratransgenic manipulation of P. argentipes appears feasible. Expression of leishmanicidal molecules via commensal bacteria commonly found at breeding sites of P. argentipes could render adult sand flies refractory to L. donovani infection.
利什曼原虫由沙蝇叮咬传播，沙蝇是 Phlebotomus argentipes。这种寄生虫是内脏利什曼病（VL）的病原体，VL 是印度比哈尔邦的地方性疾病，其预防主要依赖于滴滴涕喷洒。沙蝇种群的抗药性、环境毒性和有限的资源使这种方法变得复杂。本研究提出了一种新的共生转基因策略，旨在控制利什曼原虫的媒介传播，该策略使用枯草芽孢杆菌作为工具，枯草芽孢杆菌是从沙蝇肠道中分离出来的共生细菌。在这项工作中，表达绿色荧光蛋白（GFP）的枯草芽孢杆菌被添加到消毒的幼虫饲料中。对照盆中含有添加未转化枯草芽孢杆菌或磷酸盐缓冲盐水的幼虫饲料。第四龄期的 P. argentipes 幼虫被转移到培养基中并允许成熟。每个发育阶段都确定了细菌集落形成单位的数量、相对丰度和平均微生物负荷。
向幼虫饲料中添加枯草芽孢杆菌不会影响沙蝇的出蛹率。在每个发育阶段都鉴定出了蜡样芽孢杆菌和赖氨酸梭菌，这表明内源性微生物的转节期通过。暴露于枯草芽孢杆菌外源菌的幼虫携带的细菌数量明显更多。细菌负荷降低到与对照盆中的沙蝇相当的范围，表明携带的细菌数量存在上限。在含有转化枯草芽孢杆菌的幼虫饲料中饲养的出蛹苍蝇在其肠道腔中携带大量这些细菌。在这些共生转基因苍蝇中检测到强烈的 GFP 表达，没有转化细菌传播到昆虫的其他部位。这是对 P. argentipes 进行共生转基因操作的首次证明。
对 P. argentipes 的共生转基因操作似乎是可行的。通过在 P. argentipes 繁殖地常见的共生细菌表达杀利什曼原虫分子，可以使成年沙蝇对利什曼原虫感染产生抗性。
