1. Academic Validation
  2. A chemical and biological toolbox for Type Vd secretion: Characterization of the phospholipase A1 autotransporter FplA from Fusobacterium nucleatum

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

  • J Biol Chem. 2017 Dec 8;292(49):20240-20254. doi: 10.1074/jbc.M117.819144.
Michael A Casasanta 1 Christopher C Yoo 1 Hans B Smith 1 Alison J Duncan 1 Kyla Cochrane 2 Ann C Varano 3 Emma Allen-Vercoe 4 Daniel J Slade 5
Affiliations

Affiliations

  • 1 Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061.
  • 2 Canada's Michael Smith Genome Sciences Centre, British Columbia Cancer Agency, Vancouver, British Columbia V5Z 4S6, Canada; Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada; Department of Biochemistry and Molecular Biology, Simon Fraser University, Vancouver, British Columbia V5A 1S6, Canada.
  • 3 Virginia Tech Carilion Research Institute, Roanoke, Virginia 24016.
  • 4 Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario N1G 2W1, Canada.
  • 5 Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061. Electronic address: dslade@vt.edu.
Abstract

Fusobacterium nucleatum is an oral pathogen that is linked to multiple human infections and colorectal Cancer. Strikingly, F. nucleatum 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, F. nucleatum 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 F. nucleatum Type Vd Phospholipase class A1 autotransporter (strain ATCC 25586, gene FN1704) that we hereby rename Fusobacterium Phospholipase autotransporter (FplA). Biochemical analysis of multiple Fusobacterium 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 F. nucleatum intracellular niche. To further analyze the role of FplA, we developed an fplA gene knock-out strain, which will guide future in vivo studies to determine its potential role in F. nucleatum 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.

Keywords

Fusobacterium; autotransporter; chemical biology; colorectal cancer; host-pathogen interaction; phospholipase A; protein secretion.

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