SummaryPlant exosomes protect plants against infection; however, whether edible plant exosomes can protect mammalian hosts against infection is not known. In this study, we show that ginger exosome-like nanoparticles (GELNs) are selectively taken up by the periodontal pathogen Porphyromonas gingivalis in a GELN phosphatidic acid (PA) dependent manner via interactions with hemin-binding protein 35 (HBP35) on the surface of P. gingivalis. Compared with PA (34:2), PA (34:1) did not interact with HBP35, indicating that the degree of unsaturation of PA plays a critical role in GELN-mediated interaction with HBP35. On binding to HBP35, pathogenic mechanisms of P. gingivalis were significantly reduced following interaction with GELN cargo molecules, including PA and miRs. These cargo molecules interacted with multiple pathogenic factors in the recipient bacteria simultaneously. Using edible plant exosome-like nanoparticles as a potential therapeutic agent to prevent/treat chronic periodontitis was further demonstrated in a mouse model.

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Background:The Treponema denticola FhbB protein binds FH, a complement regulator. Results: The structure of FhbB was solved, and its interaction with FH was further defined. Conclusion: The structurally unique FhbB protein interacts with CCP7 of FH through electrostatic interactions. Significance: The T. denticola/FH interaction may perturb complement regulation resulting in conditions that favor the development of periodontal disease.

Anaplasma phagocytophilum causes granulocytic anaplasmosis, an emerging disease of humans and domestic animals. The obligate intracellular bacterium uses its invasins OmpA, Asp14, and AipA to infect myeloid and non-phagocytic cells. Identifying the domains of these proteins that mediate binding and entry, and determining the molecular basis of their interactions with host cell receptors would significantly advance understanding of A. phagocytophilum infection. Here, we identified the OmpA binding domain as residues 59 to 74. Polyclonal antibody generated against a peptide spanning OmpA residues 59 to 74 inhibited A. phagocytophilum infection of host cells and binding to its receptor, sialyl Lewis x (sLex-capped P-selectin glycoprotein ligand 1. Molecular docking analyses predicted that OmpA residues G61 and K64 interact with the two sLex sugars that are important for infection, α2,3-sialic acid and α1,3-fucose. Amino acid substitution analyses demonstrated that K64 was necessary, and G61 was contributory, for recombinant OmpA to bind to host cells and competitively inhibit A. phagocytophilum infection. Adherence of OmpA to RF/6A endothelial cells, which express little to no sLex but express the structurally similar glycan, 6-sulfo-sLex, required α2,3-sialic acid and α1,3-fucose and was antagonized by 6-sulfo-sLex antibody. Binding and uptake of OmpA-coated latex beads by myeloid cells was sensitive to sialidase, fucosidase, and sLex antibody. The Asp14 binding domain was also defined, as antibody specific for residues 113 to 124 inhibited infection. Because OmpA, Asp14, and AipA each contribute to the infection process, it was rationalized that the most effective blocking approach would target all three. An antibody cocktail targeting the OmpA, Asp14, and AipA binding domains neutralized A. phagocytophilum binding and infection of host cells. This study dissects OmpA-receptor interactions and demonstrates the effectiveness of binding domain-specific antibodies for blocking A. phagocytophilum infection.

The c-di-GMP network of Borrelia burgdorferi, a causative agent of Lyme disease, consists of Rrp1, a diguanylate cyclase/response regulator; Hpk1, a histidine kinase; PdeA and PdeB, c-di-GMP phosphodiesterases; and PlzA, a PilZ domain c-di-GMP receptor. Borrelia hermsii, a causative agent of tick-borne relapsing fever, possesses a putative c-di-GMP regulatory network that is uncharacterized. While B. burgdorferi requires c-di-GMP to survive within ticks, the associated effector mechanisms are poorly defined. Using site-directed mutagenesis, size exclusion chromatography, isothermal titration calorimetry and fluorescence resonance energy transfer, we investigate the interaction of c-di-GMP with the Borrelia PilZ domain-containing Plz proteins: B. burgdorferi PlzA and B. hermsii PlzC. The Plz proteins were determined to be monomeric in their apo and holo forms and to bind c-di-GMP with high affinity with a 1:1 stoichiometry. C-di-GMP binding induced structural rearrangements in PlzA and PlzC. C-di-GMP binding proved to be dependent on positive charge at R of the PilZ domain motif, RxxxR. Comparative sequence analyses led to the identification of Borrelia consensus sequences for the PilZ domain signature motifs. This study provides insight into c-di-GMP:Plz receptor interaction and identifies a possible switch mechanism that may regulate Plz protein effector functions.

The polymicrobial microbiome of the oral cavity is a direct precursor of periodontal diseases, and changes in microhabitat or shifts in microbial composition may also be linked to oral squamous cell carcinoma. Dysbiotic oral epithelial responses provoked by individual organisms, and which underlie these diseases, are widely studied. However, organisms may influence community partner species through manipulation of epithelial cell responses, an aspect of the host microbiome interaction that is poorly understood. We report here thatPorphyromonas gingivalis, a keystone periodontal pathogen, can up-regulate expression of ZEB2, a transcription factor which controls epithelial–mesenchymal transition and inflammatory responses. ZEB2 regulation byP. gingivaliswas mediated through pathways involving β-catenin and FOXO1. Among the community partners ofP. gingivalis,Streptococcus gordoniiwas capable of antagonizing ZEB2 expression. Mechanistically,S. gordoniisuppressed FOXO1 by activating the TAK1-NLK negative regulatory pathway, even in the presence ofP. gingivalis. Collectively, these results establishS. gordoniias homeostatic commensal, capable of mitigating the activity of a more pathogenic organism through modulation of host signaling.

SUMMARYTreponema denticola, a periodontal pathogen, binds the complement regulatory protein, Factor H (FH). FhbB (FH binding protein B) is the sole FH binding protein produced by T.denticola. The interaction of FhbB with FH is unique in that FH is bound to the cell and then cleaved by the T.denticola protease, dentilisin. A ~50kDa product generated by dentilisin cleavage is retained at the cell surface. Until this study, a direct role for the FhbB-FH interaction in complement evasion and serum sensitivity has not been demonstrated. Here we assess the serum resistance of T.denticola strain 35405 (Td35405wt) and isogeneic mutants deficient in dentilisin (Td35405-CCE) and FhbB production (Td35405)fhbB), respectively. Both dentilisin and FhbB have been postulated to be key virulence factors that mediate complement evasion. Consistent with conditions in the sub-gingival crevice, an environment with a significant concentration of complement, Td35405wt was resistant to serum concentrations as high as 25%. Deletion of fhbB (Td35405)fhbB), which resulted in the complete loss of FH binding ability, but not inactivation of dentilisin activity (Td35405-CCE), rendered T.denticola highly sensitive to 25% human serum with 80% of the cells being disrupted after 4 hours of incubation. Heat treatment of the serum to inactivate complement confirmed that killing was mediated by complement. These results indicate that the FH-FhbB interaction is required for serum resistance while dentilisin is not. This report provides new insight into the novel complement evasion mechanisms of T. denticola.

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