TY - JOUR
T1 - A chemical and biological toolbox for Type Vd secretion
T2 - Characterization of the phospholipase A1 autotransporter FplA from Fusobacterium nucleatum
AU - Casasanta, Michael A.
AU - Yoo, Christopher C.
AU - Smith, Hans B.
AU - Duncan, Alison J.
AU - Cochrane, Kyla
AU - Varano, Ann C.
AU - Allen-Vercoe, Emma
AU - Slade, Daniel J.
N1 - Funding Information:
This work was supported in part by Virginia Tech new faculty start-up funds and a seed grant from the Institute for Critical Technology and Applied Science (ICTAS, Virginia Tech) (to D. J. S.). This work was also supported in part through an Innovation grant from the Canadian Cancer Society Research Institute (to E. A. V.). The authors declare that they have no conflicts of interest with the contents of this article. We thank the following individuals for help and guidance with these studies: S. Melville (Virginia Tech) for critical insight into bacterial mutagenesis and for providing the pJIR750 plasmid; C. Caswell (Virginia Tech) for critical conversations regarding bacterial genetics; W. Lewis (Washington University, St. Louis, MO) for help with the Fusobacterium electroporation protocol; and M. Klemba (Virginia Tech) for reagents and critical phospholipase insights.
Funding Information:
This work was supported in part by Virginia Tech new faculty start-up funds and a seed grant from the Institute for Critical Technology and Applied Science (ICTAS, Virginia Tech) (to D. J. S.). This work was also supported in part through an Innovation grant from the Canadian Cancer Society Research Institute (to E. A. V.). The authors declare that they have no con-flicts of interest with the contents of this article.
Publisher Copyright:
© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2017/12/8
Y1 - 2017/12/8
N2 - 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, multi-protein 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.
AB - 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, multi-protein 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.
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U2 - 10.1074/jbc.M117.819144
DO - 10.1074/jbc.M117.819144
M3 - Article
C2 - 29021252
AN - SCOPUS:85037524846
SN - 0021-9258
VL - 292
SP - 20240
EP - 20254
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 49
ER -
