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

Michael A. Casasanta, Christopher C. Yoo, Hans B. Smith, Alison J. Duncan, Kyla Cochrane, Cameron Varano, Emma Allen-Vercoe, Daniel J. Slade

Research output: Contribution to journalArticle

6 Citations (Scopus)

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, 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.

Original languageEnglish (US)
Pages (from-to)20240-20254
Number of pages15
JournalJournal of Biological Chemistry
Volume292
Issue number49
DOIs
StatePublished - Jan 1 2017

Fingerprint

Phospholipases A1
Fusobacterium
Fusobacterium nucleatum
Phospholipases
Bacteria
Gram-Negative Bacteria
Genes
Lipids
Gene Knockout Techniques
Enzyme Assays
Biofilms
Phosphatidylinositols
Type V Secretion Systems
Pathogens
Virulence
Colorectal Neoplasms
Membrane Proteins
Assays
Membranes

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Casasanta, Michael A. ; Yoo, Christopher C. ; Smith, Hans B. ; Duncan, Alison J. ; Cochrane, Kyla ; Varano, Cameron ; Allen-Vercoe, Emma ; Slade, Daniel J. / A chemical and biological toolbox for Type Vd secretion : Characterization of the phospholipase A1 autotransporter FplA from Fusobacterium nucleatum. In: Journal of Biological Chemistry. 2017 ; Vol. 292, No. 49. pp. 20240-20254.
@article{76848362b15a466e87fc469bdc7dda72,
title = "A chemical and biological toolbox for Type Vd secretion: Characterization of the phospholipase A1 autotransporter FplA from Fusobacterium nucleatum",
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, 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.",
author = "Casasanta, {Michael A.} and Yoo, {Christopher C.} and Smith, {Hans B.} and Duncan, {Alison J.} and Kyla Cochrane and Cameron Varano and Emma Allen-Vercoe and Slade, {Daniel J.}",
year = "2017",
month = "1",
day = "1",
doi = "10.1074/jbc.M117.819144",
language = "English (US)",
volume = "292",
pages = "20240--20254",
journal = "Journal of Biological Chemistry",
issn = "0021-9258",
publisher = "American Society for Biochemistry and Molecular Biology Inc.",
number = "49",

}

A chemical and biological toolbox for Type Vd secretion : Characterization of the phospholipase A1 autotransporter FplA from Fusobacterium nucleatum. / Casasanta, Michael A.; Yoo, Christopher C.; Smith, Hans B.; Duncan, Alison J.; Cochrane, Kyla; Varano, Cameron; Allen-Vercoe, Emma; Slade, Daniel J.

In: Journal of Biological Chemistry, Vol. 292, No. 49, 01.01.2017, p. 20240-20254.

Research output: Contribution to journalArticle

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, Cameron

AU - Allen-Vercoe, Emma

AU - Slade, Daniel J.

PY - 2017/1/1

Y1 - 2017/1/1

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.

UR - http://www.scopus.com/inward/record.url?scp=85037524846&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85037524846&partnerID=8YFLogxK

U2 - 10.1074/jbc.M117.819144

DO - 10.1074/jbc.M117.819144

M3 - Article

C2 - 29021252

AN - SCOPUS:85037524846

VL - 292

SP - 20240

EP - 20254

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 49

ER -