Micrographs after reaction of glutathione&Sepharose 4B beads conjugated...
ContextPorphyromonas ( Por .) gingivalis is one of the major aetio- logical agents involved in advanced adult periodontitis and its virulence factors, such as lipopolysaccharide, fimbriae, haemagglutinins, vesicles and proteases, have been char- acterized (Slots, 1982; Slots & Genco, 1984). Among these virulence factors, arginine-specific cysteine proteinase (Arg- gingipain, Rgp) and lysine-specific cysteine proteinase (Lys- gingipain, Kgp), which specifically cleave synthetic and natural substrates at the carboxyl sides of arginine and lysine residues, respectively, have received considerable attention due to their strong ability to degrade a broad range of host proteins. Rgp and Kgp activities cause not only destruction of periodontal tissue but also disruption of host-defence mechanisms (Wingrove et al ., 1992; Kadowaki
et The al ., initial 1994; Imamura step of Por. et al ., gingivalis ; attachment Okamoto to et al the ., 1996; oral Abe tissue et has al ., 1998; been shown Calkins to et be al ., fimbriae-mediated 1998; Scragg et al ., (Hamada 1999). In addition, et al ., 1994; Rgp Sojar is involved et al ., 1999, in fimbriation, 2002). In by addition, processing the the attachment fimbrial can subunit be achieved protein by (FimA) Por. gingivalis to a mature adherence form (Nakayama to micro-organisms et al ., 1996; that Kadowaki have already et al colonized ., 1998). the periodontal regions (Slots & Gibbons, 1978). Adhesive interactions among bacterial cells can be observed as co-aggregation in vitro (Kolenbrander, 1988). We have previously reported that Por. gingivalis can co-aggregate with Prevotella ( Pre .) intermedia (Kamaguchi et al ., 2001). Pre. intermedia is detected not only in infected periodontal regions but also in the normal gingival crevice, indicating that Pre. intermedia is one of the early colonizers in periodontal niches (Loesche et al ., 1982; Slots & Listgarten, 1988; The initial step of Por. gingivalis attachment to the oral tissue has been shown to be fimbriae-mediated (Hamada et al ., 1994; Sojar et al ., ). In addition, the attachment can be achieved by Por. gingivalis adherence to micro-organisms that have already colonized the periodontal regions (Slots & Gibbons, 1978). Adhesive interactions among bacterial cells can be observed as co-aggregation in vitro (Kolenbrander, 1988). We have previously reported that Por. gingivalis can co-aggregate with Prevotella ( Pre .) intermedia (Kamaguchi et al ., 2001). Pre. intermedia is detected not only in infected periodontal regions but also in the normal gingival crevice, indicating that Pre. intermedia is one of the early colonizers in periodontal niches (Loesche et al ., 1982; Slots & Listgarten, 1988; Kononen, Previously, 1993; we found Raber-Durlacher that co-aggregation et al ., 1994; between Ashimoto Por. et gingivalis al ., 1996). and Therefore, Pre. intermedia it is possible was inhibited that Por. by L -arginine gingivalis participates and L -lysine, in and the by periodontal the potent biofilm Rgp/Kgp by inhibitors adhering leupep- to pre- tin existing and Pre. N a - intermedia p -tosyl- L -lysine . chloromethyl ketone hydrochloride (Kamaguchi et al ., 2001). Also, analysis with Por. gingivalis mutant strains revealed that the Rgp-/Kgp-related genes might be responsible for co-aggregation. In this study, we investigated aggregation factors of Por. gingivalis causing co-aggregation between Por. gingivalis and Pre. intermedia . We cloned a DNA region encoding one of the putative aggregation factors and found that this region encoded one of the adhesin domains (HGP17) within rgpA and kgp . In addition, recombinant HGP17-conjugated Sepharose 4B beads bound to Pre. intermedia . Previously, we found that co-aggregation between Por. gingivalis and Pre. intermedia was inhibited by L -arginine and L -lysine, and by the potent Rgp/Kgp inhibitors leupeptin and N a - p -tosyl- L -lysine chloromethyl ketone hydrochloride (Kamaguchi et al ., 2001). Also, analysis with Por. gingivalis mutant strains revealed that the Rgp-/Kgp-related genes might be responsible for co-aggregation. In this study, we investigated aggregation factors of Por. gingivalis causing co-aggregation between Por. gingivalis and Pre. intermedia . We cloned a DNA region encoding one of the putative aggregation factors and found that this region encoded one of the adhesin domains (HGP17) within rgpA and kgp . In addition, recombinant HGP17-conjugated Sepharose 4B beads bound to Pre. intermedia . The n-heptyl- b - D -thioglucoside-solubilized Por. gingivalis vesicle proteins were gel-filtered (Fig. 1a). Aggregation activity of each fraction with Pre. intermedia cells was determined and aggregation-active fractions were pooled and applied to arginine–Sepharose 4B columns. Eluate with 1 M NaCl showed no aggregation with Pre. intermedia cells, while the eluate with 0 ? 5 M L -arginine aggregated Pre. intermedia cells (Fig. 1b). The L -arginine eluate showed about 240 times the aggregation activity in comparison with that of Por. gingivalis culture supernatants (data not shown). SDS-PAGE of the eluates gave three major bands, with molecular masses of 44, 41 and 18 kDa, and several minor protein bands (Fig. 1c). To further purify the 44, 41 and 18 kDa proteins, the respective bands were cut out of the SDS-PAGE gel and the proteins were extracted from the gel pieces. To examine whether the purified proteins were responsible for Por. gingivalis vesicle-mediated aggregation of Pre. intermedia , we determined the effect of antisera against the purified proteins on vesicle-mediated aggregation. Vesicle solution (20 m g ml 2 1 ) and buffer, normal serum or each antiserum (10 6 dilution, 50 m l) were added to each test tube. Anti-44 kDa protein antiserum and anti-18 kDa protein antiserum inhibited the vesicle-mediated aggregation of Pre. intermedia (mean aggregation values of 21 ? 4 ± 3 ? 7 % and 9 ? 6 ± 8 ? 7 %, n = 3, results shown ± SE ), whereas anti-41 kDa protein antiserum failed to inhibit this aggregation (mean aggregation = 47 ? 1 ± 1 ? 7 %, compared to 84 ? 7 ± 1 ? 2 % for Pre. intermedia with vesicle + buffer and 44 ? 3 ± 3 ? 3 % for Pre. intermedia with vesicle + normal serum). These results indicated that the 18 and 44 kDa proteins might contribute to Por. gingivalis vesicle-mediated aggregation of Pre. intermedia cells. When BSA was added to the mixture of Por. gingivalis and Pre. intermedia , co-aggregation of Por. gingivalis with Pre. intermedia was suppressed. Apparent inhibition of aggregation in the presence of normal serum would be caused by a non-specific interaction between proteins and bacterial cells. Molecular cloning of a gene encoding the Por. gingivalis 18 kDa protein was performed using l gt11 and anti- 18 kDa protein antiserum. About 2000 plaques were screened, with one positive plaque obtained. Phage DNA was purified from the positive plaque. DNA sequencing revealed that the phage contained a 726 bp insert (data not shown). Interestingly, the insert DNA lacked Eco RI sites at both ends, although Eco RI digests of Por. gingivalis genomic DNA had been ligated into the l gt11 Eco RI site, suggesting that illegitimate ligation might have taken place, resulting in a positive clone. From similarity searches against the GenBank database ( BLAST ), the cloned DNA sequence was found to correspond to an intragenic region of the Por. gingivalis H66 rgpA gene encoding Rgp (GenBank accession no. U15282). The rgpA gene encodes a proteolytic domain and four adhesin domains (HGP44, HGP15, HGP17 and HGP27). As shown in Fig. 2, the cloned DNA encoded the HGP15 C-terminal region, the entire HGP17 region and the HGP27 N-terminal region. This DNA also had homology to kgp (GenBank accession no. U54691). In addition, the 5 9 end (nucleotides 1–348) of the cloned DNA sequence had homology to hagA (GenBank accession no. U41807). These results indicated that the 18 kDa protein is closely related to the HGP17 domain protein encoded by rgpA and kgp . The DNA encoding the HGP17 domain (474 bp) was amplified by PCR from Por. gingivalis genomic DNA and ligated into a GST fusion protein plasmid (pGEX-6P-3). The GST–HGP17 fusion protein overproduced in E. coli was purified using glutathione-conjugated Sepharose 4B beads. We determined whether anti-18 kDa protein antiserum reacted to the GST–HGP17 fusion protein (Fig. 3). Anti- 18 kDa protein antiserum strongly reacted to the GST– HGP17 fusion protein, indicating that the 18 kDa protein was HGP17. Since HGP17 and HGP44 contain a common amino acid sequence region (Pavloff et al ., 1995), we also constructed and obtained a GST–HGP44 fusion protein. The anti-18 kDa protein antiserum also reacted to the GST–HGP44 protein (Fig. 3b), suggesting that the antiserum may recognize the HGP17 and HGP44 common region. To determine whether HGP17 and HGP44 domain proteins have the ability to bind Pre. intermedia cells, the recombinant HGP17 and HGP44 proteins were mixed with Pre. intermedia cells. These proteins failed to aggregate Pre. intermedia cells (data not shown). Then, glutathione– Sepharose 4B beads conjugated with GST–HGP17, GST– HGP44 or GST alone were mixed with Pre. intermedia cells. Pre. intermedia cells adhered to the GST–HGP17- and GST–HGP44-conjugated beads, whereas Pre. intermedia cells could not adhere to the GST-conjugated beads (Fig. 4). Interestingly, when the recombinant GST–HGP17 proteins were simultaneously added to the mixture of the GST– HGP17-conjugated beads and Pre. intermedia cells, the binding of Pre. intermedia cells to the beads took place as well as without the addition of the recombinant GST– HGP17 proteins (data not shown). Moreover, Pre. intermedia cells were mixed with the recombinant GST–HGP17 proteins, washed with PBS and mixed with glutathione– Sepharose 4B beads, resulting in no binding of Pre. intermedia cells to the beads (data not shown). These results suggested that HGP17 proteins fixed on the surface of the beads might have the ability to bind Pre. intermedia cells, whereas free HGP17 proteins might lose the ability to bind. We Por. gingivalis obtained produces aggregation-active vesicles from protein its outer fractions membrane. from These vesicles cause the aggregation of Pre. intermedia cells, and this aggregation is inhibited by the same compounds that inhibit co-aggregation of Por. gingivalis and Pre. intermedia (Kamaguchi et al ., 2001). The composition of the vesicles is the almost same as that of the outer membrane of Por. gingivalis cells (Grenier & Mayrand, 1987). These results imply that the aggregation factor of Por. gingivalis vesicles to Pre. intermedia cells may be identical to the components of the outer membrane of Por. gingivalis that are involved in the co-aggregation of Por. gingivalis and Pre. intermedia . Vesicles can be prepared easily from culture supernatants of Por. gingivalis . Hence, we purified the aggregation factor from Por. gingivalis vesicles. We obtained aggregation-active protein fractions from Several lines of evidence show that HGP17 is responsible for co-aggregation between Por. gingivalis and Pre. intermedia as a Por. gingivalis aggregation factor. First, partially purified protein fractions from Por. gingivalis vesicles that cause Pre. intermedia aggregation contain a protein with a molecular mass of 18 kDa. Second, antiserum against the 18 kDa protein markedly inhibited Por. gingivalis vesicle- mediated aggregation of Pre. intermedia . Third, one recombinant clone from the Por. gingivalis genomic library that reacted to antiserum against the 18 kDa protein contained a DNA region encoding HGP17. Fourth, the GST–HGP17-conjugated beads had the ability to bind Pre. intermedia . Finally, we found in a previous study that Por. gingivalis rgpA rgpB , rgpA kgp , rgpA rgpB kgp and rgpA kgp hagA mutants, which were producing reduced or negligible amounts of HGP17, failed to co-aggregate with Pre. intermedia (Kamaguchi et al ., 2001). We also found that the aggregation-active protein fractions contained the 44 kDa protein and that antiserum against the 44 kDa protein inhibited Por. gingivalis vesicle-mediated aggregation of Pre. intermedia . In addition, Pre. intermedia could bind to the GST–HGP44-conjugated beads. Since HGP17 and HGP44 have a common amino acid sequence region, the common region may contribute to the aggregation activity of these two proteins. The cross-reactivity of anti-18 kDa protein antiserum and anti-44 kDa protein antiserum to HGP17 and HGP44 may support this hypothesis (Fig. 3). Por. Although We Several also gingivalis lines found Pre. of intermedia that vesicles evidence the aggregation-active by show cells gel markedly that filtration HGP17 adhered protein and is responsible arginine– fractions to GST– Sepharose HGP17-conjugated contained for co-aggregation the 4B 44 column kDa between beads, protein chromatography, the Por. and recombinant gingivalis that antiserum and purified GST–HGP17 Pre. against inter- three major proteins the media 44 as kDa proteins failed a Por. protein to gingivalis with aggregate inhibited molecular aggregation Pre. Por. intermedia gingivalis masses factor. vesicle-mediated of cells First, 44, when partially 41 and the 18 proteins aggregation purified kDa from protein and of the the Pre. fractions protein bacterial intermedia fractions, from cells . In Por. were addition, and gingivalis mixed. generated Pre. In vesicles intermedia addition, antisera that against the could cause recombinant Pre. bind these intermedia to proteins. the GST–HGP17 GST–HGP44-conjugated aggregation The antisera proteins contain against a could protein beads. the not 18 with Since sup- and a 44 press HGP17 molecular kDa the and proteins binding mass HGP44 of of showed 18 have Pre. kDa. intermedia a common Second, inhibition antiserum cells amino of to Por. GST–HGP17- acid against gingivalis sequence the vesicle-mediated conjugated region, 18 kDa the protein beads. common markedly aggregation Moreover, region inhibited may Pre. of Pre. intermedia contribute Por. intermedia gingivalis cells to , the while that vesicle- aggre- were the antiserum pre-treated gation mediated activity against aggregation with of the these the recombinant 41 two of kDa Pre. proteins. protein intermedia GST–HGP17 failed The cross-reactivity . to Third, inhibit proteins one this aggregation. of recombinant anti-18 kDa Molecular clone protein from cloning the antiserum Por. of gingivalis the and gene anti-44 genomic encoding kDa library pro- the Por. tein that antiserum gingivalis reacted to 18 to antiserum kDa HGP17 protein and against by HGP44 using the may the 18 kDa support anti-18 protein kDa this antiserum hypothesis contained revealed a (Fig. 3). DNA region that the encoding 18 kDa protein HGP17. was Fourth, encoded the by GST–HGP17-conjugated rgpA and kgp as a domain beads protein had the (HGP17). ability The to rgpA bind and Pre. kgp intermedia genes encode . Finally, polyproteins: we found in proteolytic a previous and study adhesin that Por. domain gingivalis proteins rgpA (Kadowaki rgpB , rgpA et kgp al , ., rgpA 1994; rgpB Okamoto kgp and et rgpA al ., kgp 1996). hagA The mutants, adhesin domains which were consist producing of HGP44, reduced HGP15, or negligible HGP17 and amounts HGP27. of HGP44 HGP17, and failed HGP17 to co-aggregate are believed with to Pre. be involved intermedia in (Kamaguchi haemagglutination, et al ., 2001). while HGP15 has the ability to bind haemoglobin (Curtis et al ., 1996; Booth & Lehner, 1997; Kelly et al ., 1997; Nakayama et al ., 1998; Shi et al ., 1999; Shibata et al ., 1999). Although Pre. intermedia cells markedly adhered to GST– HGP17-conjugated beads, the recombinant GST–HGP17 proteins failed to aggregate Pre. intermedia cells when the proteins and the bacterial cells were mixed. In addition, the recombinant GST–HGP17 proteins could not suppress the binding of Pre. intermedia cells to GST–HGP17- conjugated beads. Moreover, Pre. intermedia cells that were pre-treated with the recombinant GST–HGP17 proteins L -Arginine and L -lysine, and leupeptin and N a - p -tosyl- L lysine chloromethyl ketone hydrochloride (TLCK), which are potent inhibitors of Rgp and Kgp, respectively, were found to suppress co-aggregation between Por. gingivalis and Pre. intermedia , suggesting that Rgp and Kgp activities may be involved in co-aggregation. However, Pre. intermedia adherence to the GST–HGP17-conjugated beads was not inhibited by the addition of L -arginine, L -lysine, leupeptin or TLCK (data not shown). HGP17, as well as other adhesin domain proteins, seems to be associated with the catalytic domain proteins on the cell surface. The conformation or location of HGP17 on the cell surface might be affected by a conformational change of the catalytic domain proteins caused by the inhibitory chemicals, resulting in loss of co-aggregation activity. L Proteinase–adhesin failed -Arginine to adhere and L -lysine, to complexes, glutathione–Sepharose and leupeptin encoded and by rgpA N a 4B - p and -tosyl- beads, kgp L - , appear lysine suggesting chloromethyl to bind that several HGP17 ketone human proteins hydrochloride proteins fixed on such the (TLCK), as solid fibrinogen, surfaces which fibronectin are may potent have the inhibitors and ability laminin to of bind Rgp (Pike Pre. and et intermedia Kgp, al ., 1996). respectively, cells, Since whereas these were complexes found free HGP17 to suppress are proteins on co-aggregation the might cell lose surface the between ability (Bhogal to Por. bind. et al gingivalis ., A 1997; simi- DeCarlo and lar phenomenon Pre. intermedia & Harber, has , 1997), suggesting been they observed that may Rgp in play the and important attachment Kgp activities roles of in may Actinomyces the be attachment involved viscosus in of co-aggregation. cells Por. to gingivalis apatitic to However, surfaces host-cell fixed Pre. surfaces. inter- with HRgp, media salivary adherence which acidic consists proline-rich to the of GST–HGP17-conjugated an Rgp proteins domain (PRPs) and HGP44 (Gibbons beads (Pike was & et not Hay, al inhibited ., 1988). 1994; Gibbons Curtis by the & et addition Hay al ., (), of found L actually -arginine, that adheres although L -lysine, to leupeptin erythrocytes PRP molecules or and TLCK adsorbed platelets, (data not resulting on shown). apatitic in HGP17, haemagglutination surfaces as interact well as other and strongly platelet adhesin with aggregation, A. domain viscosus cells, proteins, respectively the same seems proteins (Pike to be et in associated al solution ., 1994; with Shibata do not the appear et catalytic al ., to 1999; bind domain to Lourbakos cells proteins of the on et organism, the al ., cell 2001). surface. nor do In they The this conformation affect study, its we attachment demonstrated or location to pellicles. that of HGP17 HGP17 Their and on explanation the HGP44 cell surface of of this the might unexpected proteinase–adhesin be affected behaviour by complexes a conformational was that hidden play an change molecular important of the segments role cataly- in tic of Por. PRPs domain gingivalis became proteins vesicle-mediated exposed caused as by a aggregation the result inhibitory of conformational of Pre. chemicals, inter- resulting changes media . This in in the loss finding protein of co-aggregation provides when it a adsorbed activity. novel function to apatitic of sur- the faces, adhesin which domains could with react regard with to the Por. adhesins gingivalis of A. adherence. viscosus cells. Interactions Further of work various is needed micro-organisms to clarify this in the issue. periodontal region result in a complex bacterial network in the gingival biofilm, which is believed to cause periodontal diseases. The adhesin domain proteins, such as HGP17 and HGP44, may make a significant contribution to the formation of the complex bacterial network as well as to the adhesion of Por. gingivalis to the cells of several different species of bacteria. Proteinase–adhesin complexes, encoded by rgpA and kgp , appear to bind several human proteins such as fibrinogen, fibronectin and laminin (Pike et al ., 1996). Since these complexes are on the cell surface (Bhogal et al ., 1997; DeCarlo & Harber, 1997), they may play important roles in the attachment of Por. gingivalis to host-cell surfaces. HRgp, which consists of an Rgp domain and HGP44 (Pike et al ., 1994; Curtis et al ., 1999), actually adheres to erythrocytes and platelets, resulting in haemagglutination and platelet aggregation, respectively (Pike et al ., 1994; Shibata et al ., 1999; Lourbakos et al ., 2001). In this study, we demonstrated that HGP17 and HGP44 of the proteinase–adhesin complexes play an important role in Por. gingivalis vesicle-mediated aggregation of Pre. intermedia . This finding provides a novel function of the adhesin domains with regard to Por. gingivalis adherence. Interactions of various micro-organisms in the periodontal region result in a complex bacterial network in the gingival biofilm, which is believed to cause periodontal diseases. The adhesin domain proteins, such as HGP17 and HGP44, may make a significant contribution to the formation of the complex bacterial network as well as to the adhesion of Por. gingivalis to the cells of several different species of bacteria.Join ResearchGate to access over 30 million figures and 100+ million publications – all in one place.Copy referenceCopy captionEmbed figurePublished in
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OMV production is induced by stresses associated with host colonization (McBroom and Kuehn, 2007), for example by exposure to host muscle tissue (Dutson et al., 1971). They are able to adhere to host cells (Inagaki et al., 2006), and promote biofilm formation in clinically important bacteria (Grenier and Mayrand, 1987; Kamaguchi et al., 2003; Yonezawa et al., 2009). The OMVs of many pathogens have been documented to contain toxins and other virulence factors (Elluri et al., 2014; Roier et al., 2014; Thay et al., 2014; Vanhove et al., 2015), and OMV-packaging has been shown to stabilize, activate and/or regulate toxin activity (Fahie et al., 2013; Bielaszewska et al., 2014; Elluri et al., 2014). ABSTRACT: Outer membrane vesicles (OMVs) shed from bacteria contribute to pathogenesis by promoting colonization of host tissues and trafficking virulence factors into host cells via fusion with the host cell plasma membrane. Glyeraldehyde-3-phosphate dehydrogenase (GAPDH) is also secreted by prokaryotes, but enhances pathogenesis by promoting adhesion of bacteria to host cell surfaces. However, GAPDH is also known to catalyze the fusion of membranes, and it has been shown to promote OMV activity in the non-pathogen Myxococcus xanthus. We suggest that during infection by Gram-negative bacteria, GAPDH and OMVs work synergistically to stimulate pathogenesis. Full-text · Article · Nov 2015 Analysis of bacterial species isolated from subgingival samples has revealed the presence and relative abundance of periodontal pathogens, including the & red complex & members (Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola), associated with the clinical features of chronic periodontitis4567. In addition, Prevotella intermedia, a member of the &orange complex&, serves as a bridging species through binding to members of the &red complex& [8,9] . Among periodontal pathogens, P. gingivalis is considered the main etiologic agent and a key pathogen responsible for initiation and progression of chronic periodon- titis [10,11]. ABSTRACT: Given the emerging evidence of an association between periodontal infections and systemic conditions, the search for specific methods to detect the presence of P. gingivalis, a principal etiologic agent in chronic periodontitis, is of high importance. The aim of this study was to characterize antibodies raised against purified P. gingivalis HmuY protein and selected epitopes of the HmuY molecule. Since other periodontopathogens produce homologs of HmuY, we also aimed to characterize responses of antibodies raised against the HmuY protein or its epitopes to the closest homologous proteins from Prevotella intermedia and Tannerella forsythia. Rabbits were immunized with purified HmuY protein or three synthetic, KLH-conjugated peptides, derived from the P. gingivalis HmuY protein. The reactivity of anti-HmuY antibodies with purified proteins or bacteria was determined using Western blotting and ELISA assay. First, we found homologs of P. gingivalis HmuY in P. intermedia (PinO and PinA proteins) and T. forsythia (Tfo protein) and identified corrected nucleotide and amino acid sequences of Tfo. All proteins were overexpressed in E. coli and purified using ion-exchange chromatography, hydrophobic chromatography and gel filtration. We demonstrated that antibodies raised against P. gingivalis HmuY are highly specific to purified HmuY protein and HmuY attached to P. gingivalis cells. No reactivity between P. intermedia and T. forsythia or between purified HmuY homologs from these bacteria and anti-HmuY antibodies was detected. The results obtained in this study demonstrate that P. gingivalis HmuY protein may serve as an antigen for specific determination of serum antibodies raised against this bacterium. Full-text · Article · Feb 2015 +1 more author...Biofilm formation is characterized by the expression of genes responsible for exopolysaccharide production and co-aggregation of cells. OMVs were found to be involved in co-aggregation of cells (Grenier & Mayrand, 1987; Whitchurch et al., 2002; Kamaguchi et al., 2003; Inagaki et al., 2006; Ellis et al., 2010). Also, it has been suggested that OMVs provide a platform for the interactions of exopolysaccharides, DNA, proteins and the attachment surface, along with the bacterial cells (Schooling & Beveridge, 2006; Schooling et al., 2009). ABSTRACT: Outer membrane vesicles (OMVs) released from Gram-negative bacteria are made up of lipids, proteins, lipopolysaccharides and other molecules. OMVs are associated with several biological functions such as horizontal gene transfer, intra and intercellular communication, transfer of contents to host cells, and eliciting an immune response in host cells. Though there are some hypotheses concerning the mechanism of biogenesis of these vesicles, research on OMV formation is far from complete. The roles of outer membrane components, bacterial quorum sensing molecules, and some specific proteins in OMV biogenesis have been studied. This review will discuss different models that have been proposed for OMV biogenesis, along with details of the biological functions of OMVs, and the likely scope of future research.Article · Jul 2014 It has been suggested that P. gingivalis selectively sorts outer membrane proteins into OMVs, resulting in an enrichment of gingipains in OMVs [49]. Besides the catalytic domain, gingipains also encode non-catalytic adhesin domains and these adhesins have been shown to be responsible for the interaction of P. gingivalis with other bacteria, including T. denticola [20,50,51]. It is possible that this enrichment of adhesins in OMVs contributed to the synergistic biofilm formation by P. gingivalis and T. denticola. ABSTRACT: Chronic periodontitis has a polymicrobial biofilm aetiology and interactions between key bacterial species are strongly implicated as contributing to disease progression. Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia have all been implicated as playing roles in disease progression. P. gingivalis cell-surface-located protease/adhesins, the gingipains, have been suggested to be involved in its interactions with several other bacterial species. The aims of this study were to determine polymicrobial biofilm formation by P. gingivalis, T. denticola and T. forsythia, as well as the role of P. gingivalis gingipains in biofilm formation by using a gingipain null triple mutant. To determine homotypic and polymicrobial biofilm formation a flow cell system was employed and the biofilms imaged and quantified by fluorescent in situ hybridization using DNA species-specific probes and confocal scanning laser microscopy imaging. Of the three species, only P. gingivalis and T. denticola formed mature, homotypic biofilms, and a strong synergy was observed between P. gingivalis and T. denticola in polymicrobial biofilm formation. This synergy was demonstrated by significant increases in biovolume, average biofilm thickness and maximum biofilm thickness of both species. In addition there was a morphological change of T. denticola in polymicrobial biofilms when compared with homotypic biofilms, suggesting reduced motility in homotypic biofilms. P. gingivalis gingipains were shown to play an essential role in synergistic polymicrobial biofilm formation with T. denticola. Full-text · Article · Aug 2013 +1 more author...InpA-pretreated oxyhaemoglobin (A); auto-oxidized haemoglobin (B). Experimental conditions were as described in Fig. 4. See text for details. that P. gingivalis gingipains mediate co-aggregation with Pr. intermedia (Kamaguchi et al., 2003). Such interactions are considered to provide growth substrates and reduce oxygen tension allowing anaerobic growth. ABSTRACT: Haem (iron protoporphyrin IX) is both an essential growth factor and a virulence regulator of the periodontal pathogens Porphyromonas gingivalis and Prevotella intermedia, which acquire it through the proteolytic degradation of haemoglobin and other haem-carrying plasma proteins. The haem-binding lipoprotein HmuY haemophore and the gingipain proteases of P. gingivalis form a unique synthrophic system responsible for capture of haem from haemoglobin and methaemalbumin. In this system, methaemoglobin is formed from oxyhaemoglobin by the activities of gingipain proteases and serves as a facile substrate from which HmuY can capture haem. This study examined the possibility of cooperation between HmuY and the cysteine protease interpain A (InpA) of Pr. intermedia in the haem acquisition process. Using UV-visible spectroscopy and polyacrylamide gel electrophoresis, HmuY was demonstrated to be resistant to proteolysis and so able to cooperate with InpA to extract haem from haemoglobin, which was proteolytically converted to methaemoglobin by the protease. Spectroscopic pH titrations showed that both the iron(II) and iron(III) protoporphyrin IX-HmuY complexes were stable over the pH range 4-10, demonstrating that the haemophore could function over a range of pH that may be encountered in the dental plaque biofilm. This is the first demonstration of a bacterial haemophore working in conjunction with a protease from another bacterial species to acquire haem from haemoglobin and may represent mutualism between P. gingivalis and Pr. intermedia co-inhabiting the periodontal pocket.Article · Dec 2012 In contrast to T. forsythia and F. nucleatum/periodonticum, however, reports about a possible mutualistic relationship of these organisms have been controversial. Based on in vitro co-aggregation experiments of P. gingivalis vesicles with P. intermedia cells, Kamaguchi et al. [42] concluded that P. gingivalis and P. intermedia physically interact via a HPG17 domain protein. In contrast, Kolenbrander and coworkers [23,43] did not observe such interaction between these two species. ABSTRACT: The polymicrobial nature of periodontal diseases is reflected by the diversity of phylotypes detected in subgingival plaque and the finding that consortia of suspected pathogens rather than single species are associated with disease development. A number of these microorganisms have been demonstrated in vitro to interact and enhance biofilm integration, survival or even pathogenic features. To examine the in vivo relevance of these proposed interactions, we extended the spatial arrangement analysis tool of the software daime (digital image analysis in microbial ecology). This modification enabled the quantitative analysis of microbial co-localization in images of subgingival biofilm species, where the biomass was confined to fractions of the whole-image area, a situation common for medical samples. Selected representatives of the disease-associated red and orange complexes that were previously suggested to interact with each other in vitro (Tannerella forsythia with Fusobacterium nucleatum and Porphyromonas gingivalis with Prevotella intermedia) were chosen for analysis and labeled with specific fluorescent probes via fluorescence in situ hybridization. Pair cross-correlation analysis of in vivo grown biofilms revealed tight clustering of F. nucleatum/periodonticum and T. forsythia at short distances (up to 6 um) with a pronounced peak at 1.5 um. While these results confirmed previous in vitro observations for F. nucleatum and T. forsythia, random spatial distribution was detected between P. gingivalis and P. intermedia in the in vivo samples. In conclusion, we successfully employed spatial arrangement analysis on the single cell level in clinically relevant medical samples and demonstrated the utility of this approach for the in vivo validation of in vitro observations by analyzing statistically relevant numbers of different patients. More importantly, the culture-independent nature of this approach enables similar quantitative analyses for &as-yet-uncultured& phylotypes which cannot be characterized in vitro. Full-text · Article · May 2012 +1 more author...}