Our protocol investigates fungal infection from both the host and pathogen perspectives to identify interactions between the biological systems that are critical for a disease. We can detect both fungal and host proteins in a single experiment and identify fungal proteins only produced during infection, representing novel infection-associated proteins. Although we focus on treating fungal infections by uncovering novel anti-virulent strategies for treatment, our proteomics and bioinformatics pipeline is universal and could be applied to many biological systems.
Our approach lays the foundation for studies of different pathogens and numerous immune responses and can be used to provide new insights into the relationship between cryptococcus and the innate immune system. The macrophage response to pathogens and detecting enough fungal proteins are important. Therefore testing and optimizing MOIs and time points for each species are recommended.
As macrophage and fungal cell culture can be challenging, knowing what to expect after coculture is important. Visualization provides the researcher with confidence in implementing the procedure. To prepare fungal cells for a macrophage infection, collect and centrifuge C neoformans cells from a mid long phase culture, followed by three gentle washes with one milliliter of room temperature PBS under the same centrifuge conditions.
After the last wash, resuspend the fungal cells at a 1.2 times 10 to the eighth cells per milliliter of antibiotic free cell culture, medium concentration and add one milliliter of fungal cell suspension to each of four wells of a 70%to 80%confluent six well macrophage culture plate. Then, place the plate in the cell culture incubator for three hours. Carefully tilt the plate to allow each well to be gently washed three times with one milliliter of PBS per well per wash.
To measure the infection proficiency after the last wash, add one milliliter of antibiotic free medium to each well of the six well plate, and place the plate in the cell culture incubator. At the appropriate experimental time points, collect the supernatant from each well and measure the amount of LDH in each well according to standard protocols. At the same time points, lyse the uninfected macrophage cells to determine the value for maximum cytotoxicity.
The cytotoxicity can then be calculated using the formula as indicated. For uninfected macrophage collection and co-culture, add one milliliter of cold PBS to each well of uninfected macrophages. After one minute, gently tap the plate to detach the cells from the bottoms of the wells and pool the cell suspensions into a single 15 milliliter tube.
Collect the cells by centrifugation and completely remove the supernatant to allow immediate flash freezing of the pellet in liquid nitrogen for minus 80 degree storage. Principal component analysis of the process to datasets reveals that, as expected, the largest component of separation among the data is infected versus non-infected samples. The second distinguishing feature of the samples is their biological variability.
Combining a Pearson correlation with hierarchical clustering by Euclidean distance shows a distinct clustering of infected versus non-infected samples with a replicate reproducibility ranging from 95%to 96%Students'T test corrected for multiple hypothesis testing using a Benjamini-Hochberg False Discovery Rate can be performed to identify proteins with significant differences in abundance during infection compared to non-infected controls. In this analysis, 117 host proteins demonstrating a significant change in expression upon infection were identified. Notably, significant increases in the abundance of fungal proteins, as expected during infection, were also observed.
It is important to handle the macrophages gently during coculturing, washing, and sample collection. By defining how the pathogen adapts to the host and how the host defends itself from infection, new insights are acquired into the fields of microbiology and immunology.
