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Interspecies protein-protein interactions are essential mediators of infection. While bacterial proteins required for host cell invasion and infection can be identified through bacterial mutant library screens, information about host target proteins and interspecies complex structures has been more difficult to acquire. Using an unbiased chemical crosslinking/mass spectrometry approach, we identified interspecies protein-protein interactions in human lung epithelial cells infected with Acinetobacter baumannii. These efforts resulted in identification of 3,076 crosslinked peptide pairs and 46 interspecies protein-protein interactions. Most notably, the key A. baumannii virulence factor, OmpA, was identified as crosslinked to host proteins involved in desmosomes, specialized structures that mediate host cell-to-cell adhesion. Co-immunoprecipitation and transposon mutant experiments were used to verify these interactions …