Challenges and Strategies for Proteome Analysis of the Interaction of Human Pathogenic Fungi with Host Immune Cells

Abstract

Opportunistic human pathogenic fungi including the saprotrophic mold Aspergillus fumigatus and the human commensal Candida albicans can cause severe fungal infections in immunocompromised or critically ill patients. The first line of defense against opportunistic fungal pathogens is the innate immune system. Phagocytes such as macrophages, neutrophils and dendritic cells are an important pillar of the innate immune response and have evolved versatile defense strategies against microbial pathogens. On the other hand, human-pathogenic fungi have sophisticated virulence strategies to counteract the innate immune defense. In this context, proteomic approaches can provide deeper insights into the molecular mechanisms of the interaction of host immune cells with fungal pathogens. This is crucial for the identification of both diagnostic biomarkers for fungal infections and therapeutic targets. Studying host-fungal interactions at the protein level is a challenging endeavor, yet there are few studies that have been undertaken. This review draws attention to proteomic techniques and their application to fungal pathogens and to challenges, difficulties, and limitations that may arise in the course of simultaneous dual proteome analysis of host immune cells interacting with diverse morphotypes of fungal pathogens. On this basis, we discuss strategies to overcome these multifaceted experimental and analytical challenges including the viability of immune cells during co-cultivation, the increased and heterogeneous protein complexity of the host proteome dynamically interacting with the fungal proteome, and the demands on normalization strategies in terms of relative quantitative proteome analysis.

Aspergillus fumigatus and Aspergillosis in 2019

“…Annotation is complicated in that A. fumigatus is rich in protein families which have different functions in spite of similar sequences and in that the redundancy of certain gene families requires multiple deletions to assess a relevant phenotype (e.g., chitin synthase gene family) (165,166). Transcriptome (167)(168)(169)(170) and proteome (171)(172)(173)(174)(175) databases are becoming available and will be used to compare omics data obtained under different conditions. It should be noted that the genetic background of the parental strain is important and may complicate the omics studies, since parental fungal strains may differ in virulence (176).…”

Dissecting Candida albicans Infection from the Perspective of C. albicans Virulence and Omics Approaches on Host–Pathogen Interaction: A Review

“…Krüger et al [132] addressed some of the challenges that need to be considered when conducting proteomic analysis of the direct interaction between phagocytic immune effector cells and fungi, including the enormous complexity of proteins that include expanded dynamic ranges, selection of suitable blood donors for the study, selection of an appropriate multiplicity of infection (MOI) to avoid downgrading of the result of relative quantitative analysis, the demands on isolation procedures for immune effector cells, the demands on culture conditions for immune effector cells, and the homogeneity of the cell suspensions used for inoculation [132]. …”

Modulation of Immune Responses by Particle Size and Shape

“…A major class of PRRs are Toll-like receptors (TLR), which bind to many different MAMPs, such as TLR4 which binds to LPS. The engagement of different receptors, such as dectin-1 and TLRs, enables immune phagocytes to differentiate between different morphological states of fungi ( 14 , 15 ). For example, compared to the hyphae, the swollen conidia form of the pathogenic fungus Aspergillus fumigatus has increased levels of surface-exposed β-glucans and triggers stronger inflammatory responses ( 16 ).…”