The fungal pathogens Aspergillus fumigatus and Candida albicans are major health threats for immune-compromised patients. Normally, macrophages and neutrophil granulocytes phagocytose inhaled Aspergillus conidia in the two-dimensional (2-D) environment of the alveolar lumen or Candida growing in tissue microabscesses, which are composed of a three-dimensional (3-D) extracellular matrix. However, neither the cellular dynamics, the per-cell efficiency, the outcome of this interaction, nor the environmental impact on this process are known. Live imaging shows that the interaction of phagocytes with Aspergillus or Candida in 2-D liquid cultures or 3-D collagen environments is a dynamic process that includes phagocytosis, dragging, or the mere touching of fungal elements. Neutrophils and alveolar macrophages efficiently phagocytosed or dragged Aspergillus conidia in 2-D, while in 3-D their function was severely impaired. The reverse was found for phagocytosis of Candida. The phagocytosis rate was very low in 2-D, while in 3-D most neutrophils internalized multiple yeasts. In competitive assays, neutrophils primarily incorporated Aspergillus conidia in 2-D and Candida yeasts in 3-D despite frequent touching of the other pathogen. Thus, phagocytes show activity best in the environment where a pathogen is naturally encountered. This could explain why “delocalized” Aspergillus infections such as hematogeneous spread are almost uncontrollable diseases, even in immunocompetent individuals.

The pathogenic fungus Candida albicans is able to cover its most potent proinflammatory cell wall molecules, the b-glucans, underneath a dense mannan layer, so that the pathogen becomes partly invisible for immune cells such as phagocytes. As the C. albicans histidine kinases Chk1p, Cos1p and CaSln1p had been reported to be involved in virulence and cell wall biosynthesis, we investigated whether deletion of the respective genes influences the activity of phagocytes against C. albicans. We found that among all histidine kinase genes, CHK1 plays a prominent role in phagocyte activation. Uptake of the deletion mutant Dchk1 as well as the acidification of Dchk1-carrying phagosomes was significantly increased compared with the parental strain. These improved activities could be correlated with an enhanced accessibility of the mutant b-1,3-glucans for immunolabelling. In addition, any inhibition of b-1,3-glucan-mediated phagocytosis resulted in a reduced uptake of Dchk1, while ingestion of the parental strain was hardly affected. Moreover, deletion of CHK1 caused an enhanced release of interleukins 6 and 10, indicating a stronger activation of the b-1,3-glucan receptor dectin-1. In conclusion, the Chk1p protein is likely to be involved in masking b-1,3-glucans from immune recognition. As there are no homologues of fungal histidine kinases in mammals, Chk1p has to be considered as a promising target for new antifungal agents.

Phagocytosis is the first step of defence against infections from the innate immune system, as it is the process of internalization of pathogens by cells with phagocytic activity, such as macrophages, which is followed by pathogen killing and destruction. Thus, phagocytosis assays are used as assays for one function of the innate immune system. As fungal infections are of increasing relevance and phagocytic mechanisms are dependent on the pathogenic organism and its viability, we established a microtiter plate phagocytosis assay based on viable, fluorescence-labelled Candida albicans. The distinction between internalized yeast cells and cells attached to macrophages was done via quenching of FITC-fluorescence by trypan blue, and the remaining fluorescence was quantified and used as indicator of the phagocytosis efficiency. As a proof of principle we showed that compounds acting on the dynamics of the actin cytoskeleton of the macrophages reduced the phagocytosis efficiency in a concentration dependent manner.