LiveFunPath

Despite the growing burden of opportunistic fungal infections, there are still few corresponding drugs. In an effort to uncover new treatments the fungal pathogenicity mechanisms and antifungal immunity are in focus.

The aim is to visualize and understand these processes through live-cell analysis. "With the new live-cell imaging system, we can study and visualize host pathogen interactions in real-time," explains Mark Gresnigt, head of the DFG-funded Emmy Noether junior research group Adaptive Pathogenicity Strategies. This is important because these infectious diseases are highly dynamic processes. For example, a single spore or yeast cell can be eaten by the immune system’s phagocytes, but long hyphae cannot. Cell death can also be specifically monitored with this specific microscope. "We investigate this in human cells which we have infected with a fungus," explains Gresnigt. "Over 24 hours we can track how the fungus destroys the tissue and how this process is slowed down after adding a molecule that inhibits a fungal toxin.”

The high-throughput capacity of up to six 96-well plates that can be examined simultaneously allows to test far more samples than before and thus makes the system ideal for screening novel drug candidates identified by the Transfer-Group Antiinfectives and the group Robotic-assisted Discovery of Antiinfectivesat the Leibniz-HKI.

A special feature of the new live-cell analysis system is the lack of a separate incubator unit. Instead, the microscope itself is positioned in an incubator. As a result, the conditions for the cells are much more stable and correspond exactly to those of other cell culture experiments. By filtering out the cell-toxic blue light in this device, cell damage is minimized. Another advantage is that the plates with the cells to be observed are not constantly moved, but the objective of the microscope is maneuvered around them. This means that the biological processes cannot be influenced by vibrations.

"We are confident that live-cell analysis will enable us to gain new insights into the immunology of fungal infections that have so far remained hidden from us due to technical limitations. This should also allow us to take a significant step forward in the search for targets for host-directed treatment of severe fungal infections," says Mark Gresnigt.

Kidney cells are infected with Candida albicans in the presence of a non-cell permeable DNA stain. When the kidney cells are severely damaged by the infection the dye enters the cell and the nucleus is stained. When the infection is performed in the presence of the toxin-neutralizing host protein albumin, it can be appreciated that kidney damage is being reduced. Source: Sophie Austermeier / Leibniz-HKI

A macrophage has taken up four fungal spores (dark dots) and is beginning to digest them, recognizable by phagolysosome markers in red coloring. It moves towards two new fungal cells and ingests them. To initiate their digestion, molecules are recruited to the phagosome, which can be seen as a green ring around the fungus. Source: Mark Gresnigt / Leibniz-HKI

Funding

This project is funded by the Free State of Thuringia with means of the European Regional Development Fund.