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. 2018 Feb 26:9:302.
doi: 10.3389/fmicb.2018.00302. eCollection 2018.

Conceptual Perspectives: Bacterial Antimicrobial Peptide Induction as a Novel Strategy for Symbiosis with the Human Host

Santosh K Ghosh  1 , Zhimin Feng  1 , Hisashi Fujioka  2 , Renate Lux  3 , Thomas S McCormick  1   4 , Aaron Weinberg  1
Affiliations

Conceptual Perspectives: Bacterial Antimicrobial Peptide Induction as a Novel Strategy for Symbiosis with the Human Host

Santosh K Ghosh et al. Front Microbiol. .

Abstract

Human beta defensins (hBDs) are small cationic peptides, expressed in mucosal epithelia and important agents of innate immunity, act as antimicrobial and chemotactic agents at mucosal barriers. In this perspective, we present evidence supporting a novel strategy by which the oral bacterium Fusobacterium nucleatum induces hBDs and other antimicrobial peptides (AMPs) in normal human oral epithelial cells (HOECs) and thereby protects them from other microbial pathogens. The findings stress (1) the physiological importance of hBDs, (2) that this strategy may be a mechanism that contributes to homeostasis and health in body sites constantly challenged with bacteria and (3) that novel properties identified in commensal bacteria could, one day, be harnessed as new probiotic strategies to combat colonization of opportunistic pathogens. With that in mind, we highlight and review the discovery and characterization of a novel lipo-protein, FAD-I (Fusobacterium Associated Defensin Inducer) associated with the outer membrane of F. nucleatum that may act as a homeostatic agent by activating endogenous AMPs to re-equilibrate a dysregulated microenvironment. FAD-I has the potential to reduce dysbiosis-driven diseases at a time when resistance to antibiotics is increasing. We therefore postulate that FAD-I may offer a new paradigm in immunoregulatory therapeutics to bolster host innate defense of vulnerable mucosae, while maintaining physiologically responsive states of inflammation.

Keywords: F. nucleatum; FAD-I; P. gingivalis; beta-defensin; symbiosis.

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Figures

Figure 1
Figure 1
(A–D) Representative F. nucleatum (A,C) and P. gingivalis (B,D) susceptibility to hBD-2 (A,B) and−3 (C,D), 2 × 105 bacteria were incubated with recombinant hBD-2 and 3 (indicated micro-molar concentrations) anaerobically, for 3 h, followed by serial dilutions and plating on sheep red blood agar plates.
Figure 2
Figure 2
(A) F. nucleatum stimulation of normal human oral epithelial cells (HOECs) confers protection against P. gingivalis invasion. Semi-confluent (80%) monolayers of HOECs were either unchallenged or challenged with F. nucleatum cell wall fraction (FnCW) (10 μg/ml) for approximately 18 hrs. P. gingivalis was then added at a multiplicity of infection (MOI) of 10:1 or 100:1, 90 min, 37°C, 5% CO2. After 1 h incubation with gentamycin and metronidazole, cells were harvested and subjected to flow cytometric analysis. Results revealed a 54.3 and 67.2% reduction in P. gingivalis invasion for the 100:1 and 10:1 MOI's respectively, when compared to non F. nucleatum challenged HOECs. (B–D) Immunogold transmission electron microscopy (TEM) of F. nucleatum and P. gingivalis incubated with rhBD-2. Overnight cultures of F. nucleatum (ATCC strain 25586) and P. gingivalis (ATCC strain 33277) (1.6 × 109 cells/ml) were incubated with recombinant hBD-2 (rhBD-2) (10 μg/ml), 3 h, 37°C anaerobically, and embedded in 1.5% low gel temperature agarose (Bio-Rad), respectively. Samples were fixed, 10 min at room temperature with 1% formaldehyde and 0.1% glutaraldehyde in 1x HEPES-buffered saline (pH 7.4), followed by washing 3X with 1X phosphate buffered saline (PBS) containing 0.05M glycine to block glutaraldehyde groups remaining on the cell surface. Samples were blocked in PBS with 1% BSA (bovine serum albumin; PBS-BSA), followed by incubation with goat anti-hBD-2 antibody (Cell Sciences, Canton, MA) (1:100) in PBS-BSA, 2 h, room temperature. After washing, samples were incubated, 2 h, in 5 nm gold-conjugated rabbit anti-goat IgG (BB International) (1:30) in PBS-BSA. To stabilize the gold particles, the samples were fixed with glutaraldehyde and post-fixed in 1% osmium tetroxide for 1 h. Samples were then block-stained in 0.5% of aqueous uranyl acetate, dehydrated in ascending concentrations of ethanol and embedded in Epon 812. Ultrathin sections were then stained with 2% uranyl acetate in 50% methanol and lead citrate, and examined in an electron microscope (Model Zeiss CEM902, Oberkochen, Germany). Black arrow points to F. nucleatum amorphous-like structures emanating from the organism's outer membrane to which immunogold labeled rhBD-2 (green circles) is sequestered, keeping it from interacting with the bacterium's outer membrane (red arrows) (C). Yellow arrow points to intact F. nucleatum cytoplasmic membrane. Extensive P. gingivalis cellular debris of outer (red arrow) and cytoplasmic membrane (yellow arrow) with rhBD-2 sequestered to these structures (green circles) are also shown (D). (E) Semi-confluent HOECs were treated with 10 μg/ml of either FnCW or recombinant FAD-I (rFAD-I) for 18 h. Levels of IL-8 and TNF-α in the supernatants were measured by ELISA (R&D systems, MN, US). Fold change in IL-8 and TNF- α released by each of the treatment compared to untreated cells were calculated.
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