A chemical and biological toolbox for Type Vd secretion: Characterization of the phospholipase A1 autotransporter FplA from .

is an oral pathogen that is linked to multiple human infections and colorectal cancer. Strikingly, achieves virulence in the absence of large, multiprotein secretion systems (Types I, II, III, IV, and VI), which are widely used by Gram-negative bacteria for pathogenesis. By contrast, strains contain genomic expansions of Type V secreted effectors (autotransporters) that are critical for host cell adherence, invasion, and biofilm formation. Here, we present the first characterization of an Type Vd phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby rename phospholipase autotransporter (FplA). Biochemical analysis of multiple strains revealed that 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. Whereas the role of Type Vd secretion in bacteria remains unidentified, we show that FplA binds with high affinity to host phosphoinositide-signaling lipids, revealing a potential role for this enzyme in establishing an intracellular niche. To further analyze the role of FplA, we developed an gene knock-out strain, which will guide future studies to determine its potential role in pathogenesis. In summary, using recombinant FplA constructs, we have identified a biochemical toolbox that includes lipid substrates for enzymatic assays, potent inhibitors, and chemical probes to detect, track, and characterize the role of Type Vd secreted phospholipases in Gram-negative bacteria.