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

Martinez-Martin, Nadia

Microarrays. One of the main limitations of any protein microarray platform is the lower protein coverage relative to other genome-wide methods for PPI identification, due to the costs and difficulties for generation of comprehensive protein libraries to be deposited onto the microarrays. In an attempt to address this caveat, Ramachandran and colleagues developed a method called nucleic-acid programmable protein array (NAPPA), in which DNAs are directly deposited onto the array followed by protein synthesis in situ using an in vitro transcription and translation (IVTT) system, thus avoiding the need for protein purification [88] . Although this promising approach has proven superior in generating transmembrane-containing molecules as soluble proteins, it still remains to be systematically addressed if the extracellular human proteins produced in this manner present the folding and posttranslational modifications necessary for protein functionality. Nevertheless, emerging data support the utility of NAPPA as a useful tool for the study of bacterial proteins. For example, Montor and colleagues used a bioinformatics approach to predict the Pseudomonas aeruginosa proteins that reside in the outer membrane of the bacteria or are secreted to the extracellular environment of the infected cell [29] . In this work, the authors utilized the NAPPA approach to screen all predicted extracellular gene products for interaction with sera from cystic fibrosis patients, where P. aeruginosa establish a life-threatening lung infection. From 266 bacterial proteins initially selected, 12 proteins were recognized by antibodies in the sera, indicating that these bacterial proteins represent major antigens that trigger adaptive immune responses in humans. Interestingly, robust antibody responses against three previously uncharacterized proteins were detected, suggesting this approach could help identify new extracellular proteins that exert unknown functions during the infection [29] . These results confirmed the utility of the microarrays to detect immune responses against membrane proteins encoded by pathogens, and supported the use of this methodology for diagnosis applications. In this regard, several groups have developed microarrays composed of pathogen-encoded proteins [30, [89] [90] [91] [92] [93] . Such pathogen protein arrays have so far being exploited mainly for diagnosis purposes, to allow screening of antibodies present in patient sera for binding to extracellular bacterial or viral antigens on the array. Nevertheless, their inherent high throughput and compatibility with multivalent bait approaches makes them a powerful tool for ePPI discovery. For example, Margarit et al. developed a Streptococcus microarray to find novel microbial proteins capable of binding to the human proteins fibronectin, fibrinogen, and C4BP and were able to identify a set of streptococcal proteins that interacted with these factors [31] . Nevertheless, despite such pathogen protein-based arrays offering great promise, this methodology remains to be systematically analyzed for ePPI discovery.