Technologies for Proteome-Wide Discovery of Extracellular Host-Pathogen Interactions

Martinez-Martin, Nadia

Classical biochemical and biophysical approaches are particularly suitable for detection of high affinity host-pathogen interactions, such as those mediated by a viral capsid protein and a host cell surface receptor, or a pathogen-encoded glycoprotein and a secreted host factor. Typically, these approaches have relied on the utilization of recombinant pathogen proteins as baits to probe for host binding partners, followed by immunoprecipitation and MS, or biophysical techniques for analysis of PPI such as surface plasmon resonance (SPR). SPR requires prior knowledge of the possible interacting partners and is therefore unsuitable for unbiased PPI discovery, whereas immunoprecipitation and MS approaches usually fail to detect weak interactions, which often characterize ePPIs, particularly those that take place on the cell surface. Notwithstanding, the identification of the receptors for some of the most prominent pathogens, such as the severe acute respiratory syndrome coronavirus (SARS-CoV) or the New World Arenaviruses, was made utilizing standard immunoprecipitation techniques [56, 57] . Despite their inherent limitations, biochemical approaches continue to provide relevant insights into host-pathogen interactions, such as the discovery of the dipeptidyl peptidase 4 (DPP4) as the receptor for the MERS-CoV just within a few months after the emergence of this virus [58] . In addition, more recently Kabanova and colleagues identified the cell surface receptor for the trimeric entry complex gHgLgO encoded by human cytomegalovirus (hCMV) [21] . Several studies have shown that the gHgLgO trimer is involved in the infection of fibroblasts, whereas the gHgLUL128L pentameric complex is required for entry into endothelial, epithelial, and myeloid cells [59] [60] [61] . In this work, both the trimeric and the pentameric CMV protein complexes were generated as recombinant products and used as baits to perform binding experiments on biotinylated cell surfaces, followed by immunoprecipitation and MS identification of bait-cell receptor complexes. Using this approach, the authors identified the cell surface protein PDGFR as a high affinity receptor for the gHgLgO trimer and demonstrated that this interaction was required for infection of fibroblasts. Interestingly, in the case of the pentameric CMV complex, multiple bands were detected upon protein immunoprecipitation from epithelial cells, suggesting the existence of multiple receptors on these cells, which so far remain unknown [21] (Table 1) . Different biophysical techniques for detection of PPI, in particular SPR, have also proven valuable in the field of host-pathogen interactions. SPR offers the advantage of label-free, sensitive detection of interactions between a diversity of ligands in real time, thus allowing calculation of kinetic parameters. SPR has been widely utilized to monitor antibody binding to a variety of pathogen antigens, information that has informed vaccine development [62] [63] [64] . The SPR technology has also been exploited for discovery of ePPIs. For example, Viejo-Borbolla and coworkers utilized SPR to screen several secreted and membrane-expressed glycoproteins encoded by herpes simplex viruses (HSVs) for binding to chemoattractant cytokines (the chemokine family) and were able to identify a subset of human chemokines that bound to HSV glycoprotein G with high affinity [22] . Recently, Day and colleagues utilized a combination of glycan arrays and SPR and identified o