Automated Image Analysis of the Host-Pathogen Interaction between Phagocytes and Aspergillus fumigatus

Abstract

Aspergillus fumigatus is a ubiquitous airborne fungus and opportunistic human pathogen. In immunocompromised hosts, the fungus can cause life-threatening diseases like invasive pulmonary aspergillosis. Since the incidence of fungal systemic infections drastically increased over the last years, it is a major goal to investigate the pathobiology of A. fumigatus and in particular the interactions of A. fumigatus conidia with immune cells. Many of these studies include the activity of immune effector cells, in particular of macrophages, when they are confronted with conidia of A. fumigus wild-type and mutant strains. Here, we report the development of an automated analysis of confocal laser scanning microscopy images from macrophages coincubated with different A. fumigatus strains. At present, microscopy images are often analysed manually, including cell counting and determination of interrelations between cells, which is very time consuming and error-prone. Automation of this process overcomes these disadvantages and standardises the analysis, which is a prerequisite for further systems biological studies including mathematical modeling of the infection process. For this purpose, the cells in our experimental setup were differentially stained and monitored by confocal laser scanning microscopy. To perform the image analysis in an automatic fashion, we developed a ruleset that is generally applicable to phagocytosis assays and in the present case was processed by the software Definiens Developer XD. As a result of a complete image analysis we obtained features such as size, shape, number of cells and cell-cell contacts. The analysis reported here, reveals that different mutants of A. fumigatus have a major influence on the ability of macrophages to adhere and to phagocytose the respective conidia. In particular, we observe that the phagocytosis ratio and the aggregation behaviour of pksP mutant compared to wild-type conidia are both significantly increased.

Facing the challenges of multiscale modelling of bacterial and fungal pathogen–host interactions

“…Although the automated analysis of image and video data from HPI remains a challenging task [66–68], it holds great potential because it automatically extracts important parameters such as velocity or turning angles for individual cells. Moreover, automatic analysis identifies interactions between individual host and pathogen cells, such as touching events, adherence or phagocytosis.…”

The Aspergillus fumigatus sitA Phosphatase Homologue Is Important for Adhesion, Cell Wall Integrity, Biofilm Formation, and Virulence

“…The AMs were counted in a hemocytometer. The phagocytic assay was performed according to the methods of Dinamarco et al and Mech et al (44,45), with some changes. Briefly, in a 24-well plate containing one 15-mm-diameter coverslip per well, about 2 ϫ 10 4 macrophages were incubated with 1 ml of RPMI-FBS at 37°C with 5% CO 2 for 1 h. Afterward, the wells were washed with 1 ml of assay medium to remove nonadherent cells.…”

Molecular Characterization of the Putative Transcription Factor SebA Involved in Virulence in Aspergillus fumigatus

“…Cells were counted in a hemocytometer. The phagocytic assay was per- formed according to Mech and coworkers (38) with some changes. Briefly, in a 24-well plate containing one 15-mm-diameter coverslip per well, 5 ϫ 10 4 macrophages were incubated with 1 ml of RPMI 1640 -10% FCS (assay medium) at 37°C with 5% CO 2 for 1 h. Afterward, the wells were washed with 1 ml of assay medium to remove nonadherent cells.…”

“…After 10 min at room temperature, samples were removed from the plate and serially diluted in sterile water. The dilutions were plated on A. fumigatus complete medium (38) and incubated at 37°C for 2 days. The percentage of conidial killing was calculated by comparing CFU numbers from samples incubated with macrophages to CFU numbers from those incubated without macrophages.…”