Molecular characterization of Aspergillus fumigatus TcsC, a characteristic type III hybrid histidine kinase of filamentous fungi harboring six HAMP domains.

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.