These data confirm that HmuY protein may be among the proteins important for biofilm accumulation by P. gingivalis. Figure 6 Production of anti-HmuY antibodies in rabbits. The reactivity of serial dilutions of rabbit pre-immune and immune anti-HmuY (test I, test II, and immune-serum) sera with 100 ng per well HmuY immobilized on the microtiter plate SHP099 (A) and the reactivity of pre-immune and immune anti-HmuY (test I, test II, and immune-serum) sera diluted 1:10,000 with varying amounts of HmuY immobilized in the

wells of a microtiter plate (B) are shown. Data from three sera analyzed in triplicate are shown as the mean ± SD. Figure 7 Inhibition of P. gingivalis growth by anti-HmuY IgG antibodies. The P. gingivalis wild-type A7436 and ATCC 33277 strains and the hmuY deletion mutant (TO4) strain were grown in basal medium PD0325901 supplemented with dipyridyl. The cells were then washed

with PBS, incubated without IgGs (-), with purified pre-immune (pre), or immune (im) anti-HmuY IgGs and inoculated into fresh BM supplemented with hemin (Hm). Figure 8 Inhibition of P. gingivalis biofilm formation by anti-HmuY IgG antibodies. P. gingivalis wild-type (A7436, W83, and ATCC 33277) strains and the hmuY deletion mutant strain constructed in A7436 (TO4) were grown in basal medium supplemented with hemin (Hm) or dipyridyl (DIP). The cells were washed with PBS, incubated with purified pre-immune or immune anti-HmuY IgGs, and inoculated into fresh media. The microtiter plate biofilms were Doramapimod mouse stained with crystal violet. Data are shown as the mean ± SD of three independent experiments (n =

6). Differences between the cells incubated with pre-immune IgGs and cells incubating with immune anti-HmuY IgGs expressed as p values are given above the respective bars. Conclusions As the prevalence of antibiotic-resistant strains of bacteria increases, novel ways of treating infections Mannose-binding protein-associated serine protease need to be developed. This is particularly important with respect to periodontal diseases, which are the most common chronic bacterial infections of man. First of all, HmuY may be important for a better understanding of the pathology caused by P. gingivalis. The surface exposure, high abundance, and immunogenicity of P. gingivalis HmuY protein suggest that its detailed examination may yield novel diagnostic methods. Knowledge of the molecular bases of the host immune response against P. gingivalis HmuY may be further essential for developing approaches to control and treat chronic periodontitis. To confirm these hypotheses, studies of anti-HmuY antibodies produced in patients with various forms of periodontal diseases and the influence of HmuY and anti-HmuY antibodies on the experimental periodontitis in a mouse model are now underway. Methods Amino-acid sequence analyses HmuY homologues were identified using the Basic Local Alignment Search Tool (BLAST; http://​blast.​ncbi.​nlm.​nih.​gov/​Blast.​cgi) [44]. Prediction of signal peptides was performed with the LipoP 1.