FAD-I, a Fusobacterium nucleatum Cell Wall-Associated Diacylated Lipoprotein That Mediates Human Beta Defensin 2 Induction through Toll-Like Receptor-1/2 (TLR-1/2) and TLR-2/6

Abstract

We previously identified a cell wall-associated protein from Fusobacterium nucleatum, a Gram-negative bacterium of the oral cavity, that induces human beta defensin 2 (hBD-2) in primary human oral epithelial cells (HOECs) and designated it FAD-I (Fusobacterium-associated defensin inducer). Here, we report differential induction of hBD-2 by different strains of F. nucleatum; ATCC 25586 and ATCC 23726 induce significantly more hBD-2 mRNA than ATCC 10953. Heterologous expression of plasmid-borne fadI from the highly hBD-2-inducing strains in a ⌬fadI mutant of ATCC 10953 resulted in hBD-2 induction to levels comparable to those of the highly inducing strains, indicating that FAD-I is the principal F. nucleatum agent for hBD-2 induction in HOECs. Moreover, anti-FAD-I antibodies blocked F. nucleatum induction of hBD-2 by more than 80%. Recombinant FAD-I (rFAD-I) expressed in Escherichia coli triggered levels of hBD-2 transcription and peptide release in HOECs similar to those of native FAD-I (nFAD-I) isolated from F. nucleatum ATCC 25586. Tandem mass spectrometry revealed a diacylglycerol modification at the cysteine residue in position 16 for both nFAD-I and rFAD-I. Cysteine-to-alanine substitution abrogated FAD-I's ability to induce hBD-2. Finally, FAD-I activation of hBD-2 expression was mediated via both Toll-like receptor-1/2 (TLR-1/2) and TLR-2/6 heterodimerization. Microbial molecules like FAD-I may be utilized in novel therapeutic ways to bolster the host innate immune response at mucosal surfaces.T he epithelial surfaces of the oral cavity are sites of active bacterial colonization. While colonizing, certain bacteria promote activation of human beta defensin (hBD) expression in the oral mucosa (1-3). By virtue of their antimicrobial and immunoregulatory properties, these epithelial-cell-derived innate response peptides contribute to the homeostasis between the bacterium and the host (4). Human beta defensin 2 (hBD-2) and hBD-3 are the two inducible members of the hBD peptide family that we and others have described in the oral cavity (1, 5-10). Interestingly, while hBD-3 is associated with the highly proliferating, nondifferentiated stratum basale of the oral mucosa, hBD-2 is compartmentalized in the more superficial stratum spinosum and stratum granulosum; i.e., nonproliferating yet differentiating regions of the oral mucosa (11,12). This, along with other results showing that hBD-2 is induced as a result of inflammation via MAPK or NF〉 (5) while hBD-3 is activated through epidermal growth factor receptor (13, 14), strongly suggests that the latter is more involved in wound healing while the former plays a more active role in inhibiting microbial invasion during mucosal-barrier disruption (15-17). Moreover, in addition to their antimicrobial properties (18, 19), both peptides have been shown to act as chemokines in recruiting lymphoid and myeloid cells from the bloodstream (20).Fusobacterium nucleatum, a ubiquitous Gram-negative bacterium of the human oral cavity, has been extensively studied for its propert...

Role of FAD-I in Fusobacterial Interspecies Interaction and Biofilm Formation

“…Sequence and transcriptional analyses revealed that RadD is encoded by the last gene of a four-gene operon, which is conserved across all four subspecies of F. nucleatum [15]. The gene directly upstream of radD, previously described as fad-I, encodes the lipoprotein FAD-I, which is characterized by its ability to induce human β-defensin 2 (hBD-2) in oral epithelial cells in a subspecies-dependent manner [21,22]. FAD-I of F. nucleatum ssp nucleatum, type strains ATCC 25586 and ATCC 23726, induce expression of hBD-2, while FAD-I of F. nucleatum ssp polymorphum, type strain ATCC 10953, fails to do so.…”

Reevaluating the Fusobacterium Virulence Factor Landscape

“…Not analyzed in this study, Fad-I is an outer membrane protein that was shown to induce human beta defensin 2 (hBD-2) through a toll-like receptor mediated host response. The immune modulation induced by Fad-I in F. nucleatum strains 23726 and 25586 was far more potent than that seen in F. nucleatum 10953 (70) . As with all protein families, this is a great example of how small sequence variations in key proteins could account for altered virulence between phylogenetically similar strains of Fusobacterium .…”

Toll-like receptor 2-mediated induction of avian β -defensin 9 by Lactobacillus rhamnosus and its cellular components in chicken intestinal epithelial cells

“…Specifically some heat-killed strains exhibited β-defensin induction comparable to viable bacteria (Rizzo et al, 2013;Wehkamp et al, 2004). Induction of β-defensin expression in epithelial cells by bacterial components from probiotics or commensal microorganisms has also been previously reported (Bhattacharyya et al, 2016;Ghadimi et al, 2011;Gupta et al, 2010;Krisanaprakornkit, Kimball, & Dale, 2002;Li et al, 2013;Schlee et al, 2007;Schlee et al, 2008;Schmitt et al, 2015;Zhang, Jin, & Yang, 2019). Probiotics are generally viable beneficial microbes that confer a health benefit on the host.…”

“…Thus, the interactions between TLR2 and its ligands lead to immediate innate immune responses, such as production of antimicrobial peptides, pro-inflammatory cytokines, and chemokines, preventing the spread of infection (Brownlie & Allan, 2011;St Paul et al, 2013). The role of TLR2 in the regulation of β-defensin induction in response to probiotic bacteria, commensal microorganisms, and bacterial cell wall components, as well as pathogen infection, has been demonstrated in various epithelial cells, including human intestinal epithelial cells (Paolillo, Carratelli, Sorrentino, Mazzola, & Rizzo, 2009;Vora et al, 2004), human vaginal epithelial cells (Rizzo et al, 2013), human oral epithelial cells (Bhattacharyya et al, 2016;Gupta et al, 2010;Ji et al, 2009), human ear epithelial cells (Lee et al, 2008), human corneal epithelial cells (Kumar, Zhang, & Yu, 2006), human epidermal keratinocytes (Li et al, 2013), and ovine ruminal epithelial cells (Jin et al, 2018). Herein, we show for the first time that TLR2 mediates induction of AvBD9 by probiotic L. rhamnosus and its cell wall component WPG in chicken intestinal epithelial cells.…”