Spadinger Anja, Ebel Frank
Institute for Infectious Diseases and Zoonoses, Ludwig-Maximilians-University, Munich, Germany.
Institute for Infectious Diseases and Zoonoses, Ludwig-Maximilians-University, Munich, Germany.
Int J Med Microbiol. 2017 Jun;307(4-5):200-208. doi: 10.1016/j.ijmm.2017.05.002. Epub 2017 May 11.
The type III hybrid histidine kinase (HHK) TcsC enables the pathogenic mold Aspergillus fumigatus to thrive under hyperosmotic conditions. It is, moreover, of particular interest, since it is the target of certain antifungal agents, such as fludioxonil. This study was aimed at a functional characterization of the domains that constitute the sensing and the kinase module of TcsC. The sensing module consists of six HAMP domains, an architecture that is commonly found in type III HHKs of filamentous fungi. To dissect the functional role of the individual domains, we have analyzed a set of truncated derivatives of TcsC with respect to their impact on fungal growth and their ability to respond to hyperosmotic stress and fludioxonil. Our data demonstrate that the TcsC kinase module per se is constitutively active and under the control of the sensing module. We furthermore found that the sixth HAMP domain alone is sufficient to arrest the kinase module in an inactive state. This effect can be partially lifted by the presence of the fifth HAMP domain. Constructs harboring more than these two HAMP domains are per se inactive and all six HAMP domains are required to enable a response to fludioxonil or hyperosmotic stress. When expressed in an A. fumigatus wild type strain, the construct harboring only the sixth HAMP domain exerts a strong dominant negative effect on the native TcsC. This effect is successively reduced in other constructs harboring increasing numbers of HAMP domains. To our knowledge, this is the first molecular characterization of a type III HHK containing six HAMP domains. Our data strongly suggest that TcsC is a positive regulator of its MAPK SakA and thereby differs fundamentally from the prototypic yeast type III HHK DhNik1 of Debaryomyces hansenii, which harbors only five HAMP domains and acts as a negative regulator of its MAPK.
III型杂合组氨酸激酶（HHK）TcsC使致病霉菌烟曲霉能够在高渗条件下生长。此外，它特别引人关注，因为它是某些抗真菌剂（如咯菌腈）的作用靶点。本研究旨在对构成TcsC传感和激酶模块的结构域进行功能表征。传感模块由六个HAMP结构域组成，这种结构在丝状真菌的III型HHK中普遍存在。为了剖析各个结构域的功能作用，我们分析了一组TcsC截短衍生物对真菌生长的影响以及它们对高渗胁迫和咯菌腈的响应能力。我们的数据表明，TcsC激酶模块本身具有组成型活性，并受传感模块的控制。我们还发现，仅第六个HAMP结构域就足以使激酶模块处于无活性状态。第五个HAMP结构域的存在可以部分解除这种作用。含有这两个以上HAMP结构域的构建体本身无活性，所有六个HAMP结构域都是对咯菌腈或高渗胁迫作出响应所必需的。当在烟曲霉野生型菌株中表达时，仅含有第六个HAMP结构域的构建体对天然TcsC产生强烈的显性负效应。在含有越来越多HAMP结构域的其他构建体中，这种效应会逐渐减弱。据我们所知，这是对含有六个HAMP结构域的III型HHK的首次分子表征。我们的数据强烈表明，TcsC是其丝裂原活化蛋白激酶（MAPK）SakA的正调节因子，因此与汉逊德巴利酵母的原型酵母III型HHK DhNik1有根本区别，后者仅含有五个HAMP结构域，并且作为其MAPK的负调节因子发挥作用。
