ABSTRACTFusobacterium nucleatumis a pathogenic oral bacterium that is linked to multiple human infections and colorectal cancer. While most Gram-negative pathogens utilize secretion systems for cellular invasion and infection,F. nucleatumlacks Type I, II, III, IV, and VI secretion. By contrast,F. nucleatumstrains are enriched in Type V secreted autotransporters, which are Gram-negative bacterial virulence factors critical for binding and entry into host cells. Here we present the first biochemical characterization of aF. nucleatumType Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby renameFusobacteriumphospholipase autotransporter (FplA). FplA is expressed as a full-length 85 kDa outer membrane embedded protein, or as a truncated phospholipase domain that remains associated with the outer membrane. Using multiple FplA constructs we characterized lipid substrate specificity, potent inhibitors, and chemical probes to detect and track this enzyme family. While the role of FplA is undetermined inF. nucleatumvirulence, homologous phospholipases from intracellular pathogens are critical for vacuole escape, altered host signaling, and intracellular survival. We hypothesize that upon intracellular invasion of the host, FplA could play a role in phagosomal escape, subversion of autophagy, or eicosanoid-mediated inflammatory signaling, as we show that FplA binds with high affinity to host phosphoinositide signaling lipids critical to these processes. Our identification of substrates, inhibitors, and chemical probes for FplA, in combination with anfplAgene deletion strain, encompass a powerful set of tools for the future analysis of FplAin vivo. In addition, these studies will guide the biochemical characterization of additional Type Vd autotransporter phospholipases.IMPORTANCEF. nucleatumis an emerging pathogen that is linked to the pathogenesis of colorectal cancer, yet there is a critical knowledge gap in the mechanisms used by this bacterium to elicit changes in the host for intracellular entry and survival. As phospholipases are critical virulence factors for intracellular bacteria to initiate vacuole lysis, cell-to-cell spread, and evasion of autophagy, we set out to characterize a unique Type Vd secreted phospholipase A1 enzyme fromF. nucleatum. Our results show a potential role for modulating host signaling pathways through cleavage of phosphoinositide dependent signaling lipids. These studies open the door for further characterization of this unique enzyme family in bacterial virulence, host-pathogen interactions, and forF. nucleatum, in colorectal carcinogenesis.