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

Martinez-Martin, Nadia

The Wright lab developed a novel method for detection of low affinity ePPIs, termed avidity-based extracellular interaction screen (AVEXIS) [2] . In brief, AVEXIS consists of the expression of the extracellular domain (ECD) of the bait of interest as a recombinant protein, which retains its binding properties while removing the insoluble transmembrane region of the protein. These ECDs are tagged with a coiled-coil sequence from the rat cartilage oligomeric matrix protein to allow for pentamerization of the bait and therefore increased binding avidity, alongside a -lactamase tag for detection of baitprey interactions upon incubation with the colorimetric substrate nitrocefin. This multivalent strategy has been used for medium-scale screens, allowing detection of weak interactions between human receptors with low false-positive rates [2] . Notably, Crosnier and colleagues applied AVEXIS to search for the plasma membrane receptor responsible for Plasmodium falciparum infection of erythrocytes [27] . The authors compiled a library consisting of most secreted or cell surface-expressed proteins in erythrocytes and systematically assayed more than 40 red blood cell proteins for binding to P. falciparum protein PfRh5, a parasite protein essential for blood stage growth, expressed as an AVEXIS pentameric bait. Notably, the Ok blood group antigen BASIGIN was identified as a unique receptor for PfRh5, and inhibition of this interaction was shown to be sufficient to block parasite invasion of the erythrocyte, findings that may importantly inform antimalarial therapies [27] . In later studies, AVEXIS was miniaturized making this approach compatible with the protein microarray format, thus permitting more comprehensive and lower resource-intensive screenings [67] . Although this technique should allow for high throughout and sensitive determination of ePPIs, this approach has not yet been applied to elucidation of pathogen-host interactions.