Visualizing Bacteria in Nematodes using Fluorescent Microscopy
Visualizing Bacteria in Nematodes using Fluorescent Microscopy
复制标题
DOI：
10.3791/4298
复制
发表时间：
2012-10-01
影响因子：
1.2
通讯作者：
Goodrich-Blair, Heidi
中科院分区：
文献类型：
--
作者：
Murfin, Kristen E.;Chaston, John;Goodrich-Blair, Heidi
共生，即两个或更多有机体共同生活在一起，广泛存在于所有的生命王国中。作为地球上最普遍的两种生物，线虫和细菌形成了广泛的共生关系，从有益的到致病的(1-3)。其中一个关联是杆菌和斯氏线虫之间的互惠关系，这种关系已经成为共生的模式系统(4)。斯氏线虫是昆虫性的，利用它们的细菌共生体杀死昆虫(5)。为了在昆虫宿主之间传播，细菌在线虫感染幼虫阶段的肠道中定居(6-8)。最近，其他几种线虫已被证明利用细菌来杀死昆虫(9-13)，研究已经开始研究这些系统中线虫和细菌之间的相互作用(9)。我们描述了一种方法，利用线虫在显微镜下观察时的光学透明性，显示线虫宿主内或宿主上的细菌共生体。这些细菌被改造成表达一种荧光蛋白，从而可以通过荧光显微镜进行可视化。有许多质粒携带编码不同波长(即绿色或红色)荧光的蛋白质的基因，而将来自供体大肠杆菌菌株的质粒连接到受体细菌共生体对广泛的细菌来说是成功的。所描述的方法是为了研究肉质斯氏线虫和嗜线虫之间的联系(14)。类似的方法也被用来研究其他线虫-细菌联合(9，15-18)，因此该方法普遍适用。该方法允许表征不同发育阶段线虫内细菌的存在和定位，提供对联合的性质和定植过程的洞察(14，16，19)。显微镜分析揭示了细菌在种群内的定植频率和宿主组织的细菌定位(14，16，19-21)。与其他监测线虫种群内细菌的方法相比，这是一种优势，例如超声波(22)或研磨(23)，后者可以提供平均的定殖率，但例如不能区分低共生菌负载频率高的种群和低频率高共生菌负载的种群。在筛选或表征细菌的定植表型突变株时，区分定植细菌的频率和负载可能特别重要(21，24)。事实上，荧光显微镜已经被用于高通量筛选细菌突变体的定植缺陷(17，18)，并且比其他方法更省力，包括超声波(22，25-27)和单个线虫解剖(28，29)。
Symbioses, the living together of two or more organisms, are widespread throughout all kingdoms of life. As two of the most ubiquitous organisms on earth, nematodes and bacteria form a wide array of symbiotic associations that range from beneficial to pathogenic (1-3). One such association is the mutually beneficial relationship between Xenorhabdus bacteria and Steinernema nematodes, which has emerged as a model system of symbiosis (4). Steinernema nematodes are entomopathogenic, using their bacterial symbiont to kill insects (5). For transmission between insect hosts, the bacteria colonize the intestine of the nematode's infective juvenile stage (6-8). Recently, several other nematode species have been shown to utilize bacteria to kill insects (9-13), and investigations have begun examining the interactions between the nematodes and bacteria in these systems (9).We describe a method for visualization of a bacterial symbiont within or on a nematode host, taking advantage of the optical transparency of nematodes when viewed by microscopy. The bacteria are engineered to express a fluorescent protein, allowing their visualization by fluorescence microscopy. Many plasmids are available that carry genes encoding proteins that fluoresce at different wavelengths (i.e. green or red), and conjugation of plasmids from a donor Escherichia coli strain into a recipient bacterial symbiont is successful for a broad range of bacteria. The methods described were developed to investigate the association between Steinernema carpocapsae and Xenorhabdus nematophila (14). Similar methods have been used to investigate other nematode-bacterium associations (9,15-18)and the approach therefore is generally applicable.The method allows characterization of bacterial presence and localization within nematodes at different stages of development, providing insights into the nature of the association and the process of colonization (14,16,19). Microscopic analysis reveals both colonization frequency within a population and localization of bacteria to host tissues (14,16,19-21). This is an advantage over other methods of monitoring bacteria within nematode populations, such as sonication (22)or grinding (23), which can provide average levels of colonization, but may not, for example, discriminate populations with a high frequency of low symbiont loads from populations with a low frequency of high symbiont loads. Discriminating the frequency and load of colonizing bacteria can be especially important when screening or characterizing bacterial mutants for colonization phenotypes (21,24). Indeed, fluorescence microscopy has been used in high throughput screening of bacterial mutants for defects in colonization (17,18), and is less laborious than other methods, including sonication (22,25-27)and individual nematode dissection (28,29).