Cu(II) complexes with peptides from FomA protein containing -His-Xaa-Yaa-Zaa-His and -His-His-motifs. ROS generation and DNA degradation.

Mono- and dinuclear Cu(II) complexes with Ac-PTVHNEYH-NH2 (L1) and Ac-NHHTLND-NH2 (L2) peptides from FomA protein of Fusobacterium nucleatum were studied by potentiometry, spectroscopic methods (UV-Vis, CD, EPR) and MS technique. The dominant mononuclear complexes for L1 ligand are: CuHL (pH range 5.0-6.0) with 2N {2Nim}, CuH-2L (pH range 8.0-8.5) and CuH-3L species (above pH 9.0) with 4N {Nim, 3N-} coordination modes. The complexes: CuH-1L with 3N {2Nim, N-}, CuH-2L with 3N {Nim, 2N-} and CuH-3L with 4N {Nim, 3N-} binding sites are proposed for the L2 ligand. Probably in the CuH-2L complex for CuL2 system the second His residue in His-His sequence is bound to Cu(II) ion, while the first His residue may stabilize this complex by His-His and/or His-Cu(II) interactions. The dominant dinuclear Cu2L1 complexes in the pH range 6.5-10.5 are: the Cu2H-4L and Cu2H-6L species with 3N{Nim, 2N-}4N{Nim, 3N-} and 4N{Nim, 3N-}4N{Nim, 3N-} binding sites, respectively. In the case of the Cu2L2 complex in the pH range 7.2-10.5, the Cu2H-4L and Cu2H-7L species dominate with 2N{Nim, N-}4N{Nim, 3N-} and (Cu(OH)42-4N{Nim, 3N-}) coordination modes, respectively. The ability to generate reactive oxygen species (ROS) by uncomplexed Cu(II) ions, ligands and their complexes at pH 7.4 in the presence of hydrogen peroxide or ascorbic acid was studied. UV-Vis, luminescence, EPR spin trapping and gel electrophoresis methods were used. Both complexes produce higher level of ROS compared to those of their ligands. ROS produced by Cu(II) complexes are hydroxyl radical and singlet oxygen, which contribute to oxidative DNA cleavage.

[Acquisition and the immunogenicity of the outer membrane FomA protein of Fusobacterium nucleatum].

Objective: To express and purify outer membrane protein FomA of Fusobacterium nucleatum (Fn) through gene recombination technique with Escherichia coli (Ec) expression system, and to detect the immunogenicity and the immune effects of the recombinant protein on gingival tissues. Methods: The gene recombination technique and Ec expression system were used to express and purified the FomA protein. Totally 20 C57 mice were immuned with the protein or the phosphate buffer solution (PBS) buffer by subcutaneous injection (each 10 mice), and the specific FomA antibody was detection in mice serum. The immunogenicity of FomA protein was assessed by comparing the differences between groups. Furthermore, the model of mice gum abscess was constructed with Fn or Fn and Porphyromonas gingivalis (Pg) mixed suspension used the above mice. The score of the gingival abscess was recorded and the interleukin (IL)-1ß in gum tissue and mice serum was determined by enzyme-linked immunosorbent assay method, and the differences of the indexes between groups were compared to evaluate the effect of the FomA protein immunization. Results: Totally 1.0-1.5 g FomA protein were successfully obtained and the protein purity was over 90%. The FomA specific antibody was detected in the serum of mice by subcutaneous injection of the protein, and the antibody titer reached the highest level in 2 weeks after secondary immunization. The model of submaxillary gingival abscess was successfully constructed. In the Fn model, the score of the FomA protein immune group was (1.82±0.35), and the PBS control group was (2.62±0.71), with statistically significant difference between the two groups (P=0.049). In the Fn+Pg mixture model, the score of gingival abscess in the FomA immune group (2.31±0.55) was lower than that in PBS group (3.63±0.45), and the difference was statistically significant (P=0.003). Both in Fn and Fn+Pg injection group, the concentration of IL-1ß in the serum of FomA immune mice and gingival tissues was lower than that of PBS control mice (P<0.001). Conclusions: The recombinant FomA protein can be acquired by Ec expression system, and it can produce a certain level antibodies in the mice serum. The way of mice subcutaneously injected with the recombinant FomA protein can reduce the severity of periodontal infections caused by Fn and Pg.

Biochemical characterization of a recombinant Lactobacillus acidophilus strain expressing exogenous FomA protein.

In previous research, to combine the immunogenicity of Fusobacterium nucleatum (F. nucleatum) and the probiotic properties of Lactobacillus acidophilus (L. acidophilus), we constructed a FomA-expressing L. acidophilus strain and assessed its immunogenicity. Our findings indicated that oral administration of the recombinant L. acidophilus strain reduced the risk of periodontal infection by Porphyromonas gingivalis (P. gingivalis) and F. nucleatum. However, because the exogenous FomA is an heterologous protein for the original bacterium, in this study, we assessed whether the biochemical characteristics of the recombinant L. acidophilus strain change due to the expression of the exogenous FomA protein. OBJECTIVES: To test the biochemical characteristics of a recombinant L. acidophilus strain expressing exogenous FomA and assess its antibiotic sensitivity. DESIGNS: We assessed the colony morphology, growth, acid production, and carbohydrate fermentation abilities of the recombinant L. acidophilus strain. In addition, we tested the adhesive ability and antimicrobial activity of the recombinant and assessed its antibiotic sensitivity through a drug susceptibility test. RESULTS: The experimental results showed that the colony and microscopic morphology of the recombinant L. acidophilus strain was consistent with the original strain, and the recombinant strain grew well when cultured under aerobic or anaerobic conditions, exhibiting a growth rate that was identical to that of the standard strain. Similarly, the supernatants of the recombinant L. acidophilus can inhibit the growth of E. coli and P. gingivalis at different concentrations, and the recombinant strain displayed essentially the same drug sensitivity profile as the original L. acidophilus. However, to our surprise, the recombinant strains exhibited a greater adhesion ability than the reference strain. CONCLUSIONS: Our study demonstrated that, in addition to an increased adhesion ability, the recombinant L. acidophilus strain maintained the basic characteristics of the standard strain ATCC 4356, including antibiotic sensitivity. Thus, the recombinant strains have great potential to be utilized as a safe and effective periodontitis vaccine in the future.

Acquisition and the immunogenicity of the outer membrane FomA protein of Fusobacterium nucleatum / 中华口腔医学杂志

Objective@#To express and purify outer membrane protein FomA of Fusobacterium nucleatum (Fn) through gene recombination technique with Escherichia coli (Ec) expression system, and to detect the immunogenicity and the immune effects of the recombinant protein on gingival tissues.@*Methods@#The gene recombination technique and Ec expression system were used to express and purified the FomA protein. Totally 20 C57 mice were immuned with the protein or the phosphate buffer solution (PBS) buffer by subcutaneous injection (each 10 mice), and the specific FomA antibody was detection in mice serum. The immunogenicity of FomA protein was assessed by comparing the differences between groups. Furthermore, the model of mice gum abscess was constructed with Fn or Fn and Porphyromonas gingivalis (Pg) mixed suspension used the above mice. The score of the gingival abscess was recorded and the interleukin (IL)-1β in gum tissue and mice serum was determined by enzyme-linked immunosorbent assay method, and the differences of the indexes between groups were compared to evaluate the effect of the FomA protein immunization.@*Results@#Totally 1.0-1.5 g FomA protein were successfully obtained and the protein purity was over 90%. The FomA specific antibody was detected in the serum of mice by subcutaneous injection of the protein, and the antibody titer reached the highest level in 2 weeks after secondary immunization. The model of submaxillary gingival abscess was successfully constructed. In the Fn model, the score of the FomA protein immune group was (1.82±0.35), and the PBS control group was (2.62±0.71), with statistically significant difference between the two groups (P=0.049). In the Fn+Pg mixture model, the score of gingival abscess in the FomA immune group (2.31±0.55) was lower than that in PBS group (3.63±0.45), and the difference was statistically significant (P=0.003). Both in Fn and Fn+Pg injection group, the concentration of IL-1β in the serum of FomA immune mice and gingival tissues was lower than that of PBS control mice (P<0.001).@*Conclusions@#The recombinant FomA protein can be acquired by Ec expression system, and it can produce a certain level antibodies in the mice serum. The way of mice subcutaneously injected with the recombinant FomA protein can reduce the severity of periodontal infections caused by Fn and Pg.