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, 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.

Comparison of type 5d autotransporter phospholipases demonstrates a correlation between high activity and intracellular pathogenic lifestyle

“…The correctness of the constructs was determined by Sanger sequencing. Plasmids encoding the FplA lipase domain have been described before (10). All plasmids used in this study are summarized in Table 1.…”

“…Phospholipases are usually secreted or membrane-associated proteins and are, in many cases, connected to virulence in a wide range of extracellular, vacuolar and intracellular pathogens. The actual role in infection can be manifold, ranging from membrane disruption as a means for competition, colonization benefits, generating nutrients, phagosomal escape or infection establishment to the formation of bioactive molecules or membrane remodeling (4)(5)(6)(7)(8) Type 5 secretion systems (T5SSs) are the most widespread secretion systems in bacteria (9) membrane-associated phospholipase and thoroughly investigated in regard to enzymatic efficiency, inhibitors of lipase activity as well as lipid binding specificity (10). The Pseudomonas aeruginosa type 5d secretion system (T5dSS), called patatin-like protein D (PlpD), belongs to the family of patatin-like lipolytic enzymes (11).…”

<i>Fusobacterium nucleatum</i> Infection in Colorectal Cancer: Linking Inflammation, DNA Mismatch Repair and Genetic and Epigenetic Alterations

“…Thus, FadA on the surface of Fn stimulates tumor growth through bacterial attachment but also triggers the inflammatory response through bacterial invasion. There are many other uncharacterized Fn proteins that may control host-pathogen interactions (64). Further studies are required to identify and characterize these proteins in relation to CRC carcinogenesis.…”

Advances in the Assembly Model of Bacterial Type IVB Secretion Systems

“…It is proposed that these virulence factors are autonomous secretion systems [62,63]. Currently, T5SSs are classified into five classes (Types Va-Ve) based on their domain architecture [64,65]. The genes for T6SS are often arranged together to form a gene cluster, which encode 13 to 25 proteins that form core and auxiliary components [66].…”