Species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of Porphyromonas gingivalis HmuY

Abstract

BackgroundPorphyromonas gingivalis is a major etiological agent of chronic periodontitis. The aim of this study was to examine the species specificity, surface exposure, protein expression, immunogenicity, and participation in biofilm formation of the P. gingivalis heme-binding protein HmuY.ResultsHmuY is a unique protein of P. gingivalis since only low amino-acid sequence homology has been found to proteins encoded in other species. It is exposed on the cell surface and highly abundant in the outer membrane of the cell, in outer-membrane vesicles, and is released into culture medium in a soluble form. The protein is produced constitutively at low levels in bacteria grown under high-iron/heme conditions and at higher levels in bacteria growing under the low-iron/heme conditions typical of dental plaque. HmuY is immunogenic and elicits high IgG antibody titers in rabbits. It is also engaged in homotypic biofilm formation by P. gingivalis. Anti-HmuY antibodies exhibit inhibitory activity against P. gingivalis growth and biofilm formation.ConclusionsHere it is demonstrated that HmuY may play a significant role not only in heme acquisition, but also in biofilm accumulation on abiotic surfaces. The data also suggest that HmuY, as a surface-exposed protein, would be available for recognition by the immune response during chronic periodontitis and the production of anti-HmuY antibodies may inhibit biofilm formation.

Gallium(III), cobalt(III) and copper(II) protoporphyrin IX exhibit antimicrobial activity against Porphyromonas gingivalis by reducing planktonic and biofilm growth and invasion of host epithelial cells

“…It has been shown that Bacteroides spp. can grow in and subsequently incorporate deuteroheme and mesoheme into a functional b-type cytochrome (McCall and Caldwell 1977;Fuller and Caldwell 1982), and that versatility in tetrapyrrole utilization may be a common feature within the phylum Bacteroidetes (Olczak et al 2010). Also, PPIX supports planktonic growth of P. gingivalis in basal medium (data not shown), similar to some non-iron metalloporphyrins examined (Fig.…”

“…The best characterized heme acquisition system comprises the HmuR and HmuY proteins. HmuR is an outer-membrane TonB-dependent receptor engaged in heme transport through the outer membrane (Olczak et al 2001), whereas HmuY is a membrane-associated heme-binding lipoprotein (Olczak et al 2008(Olczak et al , 2010, which also binds iron(III) mesoporphyrin IX (mesoheme) and iron(III) deuteroporphyrin IX (deuteroheme) (Wojaczynski et al 2011). Detailed characterization of the HmuY-heme complex has revealed a unique protein all-beta fold structure in which histidines 134 and 166 bind heme in a low-spin Fe(III) hexa-coordinate arrangement (Wojtowicz et al 2009a, b).…”

“…Therefore, we examined the eVect of metalloporphyrins on P. gingivalis homotypic bioWlm accumulation. Homotypic bioWlms formed by P. gingivalis were analyzed as previously described (Olczak et al 2010). Cells in BM supplemented with PPIX or metalloporphyrins were transferred (200 l) into sterile round-bottom 96-well microtiter plates (Sarstedt) and incubated under anaerobic conditions at 37°C for 48 h. The resulting bioWlms were stained with 1 % crystal violet and absorbance at 570 nm determined using a Multiskan Ascent microplate reader (Thermo Electron Corporation).…”

Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

“…The absorbance (OD 600 ) of cultures at given times was measured using a Genesys spectrometer (Thermo Scientific, Waltham, MA). Biofilm formation was measured as previously described (24,46,77) with slight modifications. Briefly, Pg83 and ⌬PG352 strains were grown to mid-log phase, and then the cell densities (OD 600 ) were measured to ensure that the same amounts of Pg83 and ⌬PG352 cells were used for biofilm measurements.…”

“…The assay was carried out in 96-well microtiter plates as previously described (24,46). The upper panel shows representative wells of Pg83 and the ⌬PG352 mutant, and the lower panel gives the average absorbances of Pg83 and the mutant.…”