Exploring calcium homeostasis and signalling in Aspergillus nidulans by using a calcium auxotrophy and a calcium-responsive reporter

通过使用钙营养缺陷型和钙响应报告基因探索构巢曲霉中的钙稳态和信号传导

• 批准号: BB/D521781/1
• 负责人: Herbert Arst
• 金额: $ 27.69万
• 依托单位: Imperial College London
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2006
• 资助国家: 英国
• 起止时间: 2006 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FD521781%2F1
• 关键词: Exploring; calcium; homeostasis; signalling; Aspergillus

Calcium is an essential nutrient for most, if not all forms of life. For example, among its roles in mammals, it is essential for muscle contraction, the proper functioning of the nervous system, the ability of cells to divide, fertilisation and the formation of teeth and bones. Among its roles in plants, it is essential for responses to temperature changes and the availability of water and the symbiotic relationship which enables nitrogen gas in the atmosphere to be incorporated into organic molecules needed for growth and the maintenance of life. In bacteria and fungi calcium plays roles in the structure of the organisms and in formation of various forms such as spores. It is also likely to play a role in the ability of bacteria and fungi to infect animals and plants. By learning more about the various roles of calcium in fungi we not only add to basic knowledge about an ubiquitous and important group of organisms but potentially identify ways in which we can prevent fungal growth. For example, if we can identify roles that calcium plays in fungi but does not play in humans then potentially drugs can be found that can treat fungal infections in humans. There is a considerable need for drugs of this sort because very few currently exist and there is a danger because of mutant fungi that these might lose their effectiveness. Similarly, identifying roles that calcium plays in fungi but not in plants can aid in the design of fungicides able to protect plants of agricultural or horticultural importance. It might also point to new ways of preventing spoilage of foods and other commodities by fungi. This proposal involves the use of a soil-borne fungus, Aspergillus nidulans, which is virtually non-pathogenic, to both animals and plants, but which is very easy to manipulate using both traditional genetic methods and modern genetic modification methods. For this work I have obtained two rather unique tools which should prove extremely useful in identifying factors responsible for the distribution of calcium between the environment and the fungus, its distribution within fungal cells and its roles within the fungus. One of these is a combination of mutations which results in a requirement for adding calcium to obtain normal growth of the fungus. Normally, the amount of calcium required for growth is so tiny that calcium contamination of water and the other chemicals required for growth suffices. The behaviour of this calcium-requiring organism is of interest in its own right but, in addition, it can be used to obtain further mutations affecting calcium responses by treating with a DNA damaging agent (mutagen) and seeking derivative organisms which no longer require addition of calcium for growth. The other tool involves a gene made in such a way that a blue colour can be produced in the presence of a certain chemical. The response of this gene, as determined by whether a blue colour is formed and, if so, how intense it is, is influenced by added calcium and by an unfulfilled calcium requirement. By treating an organism containing this gene with a mutagen, we can obtain mutants which differ from their parent in blue colour formation or its intensity in the presence or absence of added calcium. Finally, we will use a system in which a jellyfish gene is inserted into the fungus. The protein made from this gene responds to calcium by emitting blue light. This provides us with a means of following the behaviour of calcium in response to treatments which affect its behaviour in living mutant organisms whose response to calcium is altered as determined by one or both of the above two tools.

钙是大多数(如果不是所有形式的)生命所必需的营养物质。例如，在哺乳动物中，它对肌肉收缩、神经系统的正常运作、细胞分裂、受精以及牙齿和骨骼的形成都是必不可少的。在它在植物中的作用中，它对温度变化和水的可用性以及共生关系至关重要，这种共生关系使大气中的氮气能够并入生长和维持生命所需的有机分子中。在细菌和真菌中，钙在生物体的结构和各种形式的形成如孢子的形成中起作用。它还可能在细菌和真菌感染动植物的能力中发挥作用。通过更多地了解钙在真菌中的各种作用，我们不仅增加了关于一组无处不在的重要生物体的基本知识，而且潜在地确定了我们可以防止真菌生长的方法。例如，如果我们能确定钙在真菌中发挥的作用，但在人类中不起作用，那么就可能找到治疗人类真菌感染的药物。这类药物有相当大的需求，因为目前存在的药物很少，而且由于突变真菌的存在，这些药物可能会失去效力。同样，确定钙在真菌中扮演的角色，而不是在植物中扮演的角色，可以帮助设计能够保护农业或园艺重要植物的杀菌剂。它还可能指出防止食品和其他商品被真菌变质的新方法。这项建议涉及使用一种土传真菌--尼杜拉曲霉，它对动物和植物几乎都没有致病作用，但使用传统的遗传方法和现代的遗传修改方法都很容易操纵。对于这项工作，我获得了两个相当独特的工具，它们应该被证明在识别负责环境和真菌之间钙分布的因素、它在真菌细胞中的分布以及它在真菌中的作用方面非常有用。其中之一是突变的组合，这导致需要添加钙来获得真菌的正常生长。正常情况下，生长所需的钙量是如此之少，以至于水中的钙污染和生长所需的其他化学物质就足够了。这种需要钙的生物的行为本身是有意义的，但此外，它还可以用来获得进一步影响钙反应的突变，方法是用DNA损伤剂(诱变剂)处理，并寻找不再需要添加钙来生长的衍生生物。另一种工具涉及一种基因，这种基因可以在某种化学物质存在的情况下产生蓝色。这种基因的反应取决于是否形成蓝色，如果是的话，它的强度受到添加的钙和未满足的钙需求的影响。通过用诱变剂处理含有该基因的有机体，我们可以获得在有或没有添加钙的情况下在蓝色形成或其强度方面与其亲本不同的突变体。最后，我们将使用一个将水母基因插入真菌的系统。由该基因产生的蛋白质通过发出蓝光对钙作出反应。这为我们提供了一种方法来跟踪钙在影响其行为的治疗中的行为，在活的突变生物体中，其对钙的反应由上述两种工具中的一种或两种来确定。

项目成果

Rescue of Aspergillus nidulans severely debilitating null mutations in ESCRT-0, I, II and III genes by inactivation of a salt-tolerance pathway allows examination of ESCRT gene roles in pH signalling.

• DOI: 10.1242/jcs.088344
• 发表时间: 2011-12-01
• 期刊: Journal of cell science
• 影响因子: 4
• 作者: Calcagno-Pizarelli AM;Hervás-Aguilar A;Galindo A;Abenza JF;Peñalva MA;Arst HN Jr
• 通讯作者: Arst HN Jr

An ordered pathway for the assembly of fungal ESCRT-containing ambient pH signalling complexes at the plasma membrane.

• DOI: 10.1242/jcs.098897
• 发表时间: 2012-04-01
• 期刊: Journal of cell science
• 影响因子: 4
• 作者: Galindo A;Calcagno-Pizarelli AM;Arst HN Jr;Peñalva MÁ
• 通讯作者: Peñalva MÁ

Analysis of a novel calcium auxotrophy in Aspergillus nidulans.

• DOI: 10.1016/j.fgb.2010.04.002
• 发表时间: 2010-07
• 期刊: FUNGAL GENETICS AND BIOLOGY
• 影响因子: 3
• 作者: Findon, Helen;Calcagno-Pizarelli, Ana-Maria;Martinez, Jose L.;Spielvogel, Anja;Markina-Inarrairaegui, Ane;Indrakumar, Tanya;Ramos, Jose;Penalva, Miguel A.;Espeso, Eduardo A.;Arst, Herbert N., Jr.
• 通讯作者: Arst, Herbert N., Jr.