Microbial Pathogenesis 77 (2014) 100e104
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Lineage variability in surface components expression within Porphyromonas gingivalis Silvia Regina Loureiro Teixeira a, Talyta Thereza Soares D'Epiro a, Ericka Tavares Pinheiro b, Kisielius c, Maria Regina L. Simionato a, Noemi Nosomi Taniwaki c, Jonas Jose a, * Marcia Pinto Alves Mayer a b c
~o Paulo, Av. Prof. Lineu Prestes, 1374, Sa ~o Paulo, SP 05508-900, Brazil Department of Microbiology, Institute of Biomedical Sciences, University of Sa ~o Paulo, Av. Prof. Lineu Prestes, 2227, Sa ~o Paulo, SP 05508-900, Brazil Department of Endodontics, School of Dentistry, University of Sa ~o Paulo, SP 01246-902, Brazil Department of Electron Microscopy, Adolfo Lutz Institute, Av Dr. Arnaldo, 355, Sa
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Article history: Received 5 June 2014 Received in revised form 9 October 2014 Accepted 3 November 2014 Available online 4 November 2014
The periodontopathogen Porphyromonas gingivalis is represented by a spectrum of phenotypes ranging from commensals to pathogenic lineages. Capsule and fimbriae are considered key virulence factors in this specie, involved in colonization and host defenses evasion. Since these virulence traits may not be expressed by certain strains, we aimed to test the hypothesis that certain clusters or genotypes of P. gingivalis correlate with the production of capsule and fimbriae. Sixteen P. gingivalis isolates were evaluated. Capsule (K) was detected by optical microscopy of negatively stained cells. The presence of fimbriae (F) was determined by TEM. Genotypes were determined by NotI macrorestriction fragments analysis through Pulsed-Field Gel Electrophoresis (PFGE) and Multi-locus sequence typing (MLST) based on seven house-keeping genes. The phenotypes included FþKþ (n ¼ 4), FKþ (n ¼ 5), FþK (n ¼ 5) and FK (n ¼ 2). The analysis of whole genome macrorestriction fragments revealed 14 different clusters. MLST data also revealed extensive genetic diversity; however, PFGE and MLST profiles showed evident differences. There was no association between P. gingivalis clusters and encapsulated and/or fimbriated phenotypes. Genotyping methods were not able to discriminate isolates according to the production of virulence factors such as capsule and major fimbriae, indicating that recombination played a key role in the expression of capsule and fimbriae in P. gingivalis. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Porphyromonas gingivalis MLST PFGE Capsule Fimbriae
1. Introduction Periodontal diseases result from complex interactions between pathogenic bacteria in subgingival biofilm and host tissues [38]. The disease does not only affect the oral tissues, but is associated with systemic conditions, such as cardiovascular diseases and increased risk of pre-term labor [30,34]. Porphyromonas gingivalis is a major pathogen involved in the etiology of periodontitis especially by manipulating host immune responses, driving a shift of
Abbreviations: MLST, Multi-locus sequence typing; PFGE, pulsed field gel electrophoresis. * Corresponding author. E-mail addresses: [email protected] (S.R.L. Teixeira), [email protected] (T.T.S. D'Epiro), [email protected] (E.T. Pinheiro), [email protected] (M.R.L. Simionato), [email protected] (N.N. Taniwaki), [email protected] (J.J. Kisielius), [email protected] (M.P.A. Mayer). http://dx.doi.org/10.1016/j.micpath.2014.11.001 0882-4010/© 2014 Elsevier Ltd. All rights reserved.
the resident microbiota toward a periodontitis associated microbial community [12]. P. gingivalis isolates differ in their ability to induce periodontal destruction in humans and animal models [4,22], and in virulence factors such as the production of capsular polysaccharide, fimbriae and proteases [46]. Experimental animal studies revealed that encapsulated strains induce a more pronounced alveolar bone loss after oral challenge [20] and are more invasive after subcutaneous inoculation [21]. Furthermore, capsule production by certain P. gingivalis strains was associated with increased virulence [21,27], properties such as resistance against desiccation, osmotic stress, oxygen toxicity and phagocytic engulfment [11], induction of a lower host response [9,37], and biofilm formation [13]. The capsular polysaccharide locus shows evidence of being acquired by horizontal transfer [1,10], and is very polymorphic, leading to seven capsule known antigens [8] and non typable strains [26]. Although a gene encoding a glycosiltransferase (pg0106) was shown to be essential for capsule
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production [13], it is present in most P. gingivalis isolates independently on the capsule expression [10]. Fimbriae play a critical role in colonization of the oral cavity by mediating the interaction of bacteria with host cells [14,28], hard surfaces and host proteins [5,28]. It allows P. gingivalis to explore the Toll-like receptor signaling pathway and is associated with internalization in non phagocythic eukaryotic cells, inhibition of IL12p70 and persistence in macrophages [43]. The P. gingivalis population displays a spectrum of phenotypes from commensal to pathogenic [40] but there are no currently known markers for more virulent strains. The major fimbria of P. gingivalis is encoded by fimA and the specie is classified in 6 distinct fimA genotypes [2]. FimA type II is the most prevalent in periodontitis patients [3,31], although FimA IV correlated with disease severity in smokers [39]. FimA type II is dispersed among distinct genetic lineages of P. gingivalis [36] but whether all these clusters express the fimbriae is currently not known. Thus, we aimed to test the hypothesis that genotyping methods with high discriminatory power and reproducibility would highlight associations between certain genotypes or clusters and the expression of virulence factors in P. gingivalis. Profiles generated by whole genome macrorestriction fragments through Pulsed-Field Gel Electrophoresis (PFGE) vary in consequence of genome rearrangements [17,35], whereas Multilocus Sequence Typing (MLST), based on sequencing house-keeping genes, provides a much more stable genetic signature and enables clustering on the basis of evolutionary proximity [19]. Since both fimbriae and capsule may provide a selective advantage in the colonization of the host, and are considered key virulence factors in P. gingivalis, we evaluated the macrorestriction profiles and evolutionary patterns of P. gingivalis and determined their correlation with the production of capsule and fimbriae.
2. Methods 2.1. Bacterial strains
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2.3. Detection of fimbriae P. gingivalis cells grown anaerobically in TSBHK agar plates for 72 h were collected and resuspended in PBS. The bacterial suspensions were added to formvar-coated copper grid previously treated with 2% Alcian Blue solution, to improve adhesion of the bacterial cells to the grid. The bacterial cells were then negatively stained with 2% potassium phosphotungstate for 30 s [29], and the samples were observed with a JEOL electron microscope, model JEM1011. 2.4. MLST PCR primers and amplification conditions for MLST have been described earlier [16,24]. Seven genes (ftsQ, gdpxJ, hagB, mcmA, pepO, pga and recA) were evaluated. PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). Both strands of purified PCR products were sequenced at the Human Genome Center, Institute of Biology, University of S~ ao Paulo, Brazil. Bionumerics software version 6.1 (Applied Maths, SaintMartens-Latem, Belgium) was used for data entry, sequence alignment and the phylogenetic tree was constructed by the unweight method with arithmetic averages (UPGMA). Sequence data were submitted to the P. gingivalis MLST website (http://pubmlst. org/pgingivalis) and to Genbank (http://www.ncbi.nlm.nih.gov/ genbank). 2.5. PFGE P. gingivalis suspensions, at an OD660nme0.5 (3 109 CFU/ml), were treated with lyses solution [50 mM TriseHCl (pH 8.0), 50 mM EDTA, lysozyme (2.5 mg/mL), and proteinase K (1.5 mg/mL)], added to an equal volume of 1.2% low melting agarose (SeaKem Gold agarose, Cambrex Bio Science, Workingham, UK) and poured into CHEF Disposable Plug Mold (Bio-Rad Laboratories, Hercules, CA, USA). The proteolysis was performed with 0.5 M EDTA, 1% sarcosyl and 1 mg/mL of proteinase K; overnight at 50 C. After washings,
P. gingivalis isolates obtained from periodontitis patients of diverse geographical locations (Japan n ¼ 3; Sweden, n ¼ 4 and Brazil, n ¼ 7) and the reference strains ATCC 33277 and W83 were evaluated. Since human isolates were evaluated, this study was approved by the institutional ethical committee on research in humans (846-CEP-10.07.8). All strains were previously identified as P. gingivalis by polymerase chain reaction (PCR) using speciesspecific 16SrRNA primers [3], and genotyped by amplification using fimA specific primers followed by restriction analysis of amplicons for differentiation between fimA I and fimA Ib genotypes [2,32,42]. Frozen stocks of P. gingivalis strains were cultured anaerobically in TSAHK agar plates (Tryptic Soy Agar supplemented with 5% defibrinated sheep blood, 0.5 mg/mL hemin and 1 mg/mL menadione) for 7e10 days at 37 C in 85% N2, 5% CO2 e 10% H2 in an anaerobic chamber (Plas Labs, Lansing, MI, USA).
2.2. Detection of capsule The isolates were also grown to the exponential growth phase in TSBHK (Tryptic Soy Broth supplemented with 0.5 mg/mL hemin and 1 mg/mL menadione) to an OD660nme0.5e0.8. Cells were negatively stained by crystal violet according to Hiss method. The presence of capsule was evident as clear areas around the bacterial cell under light optical microscopy at 1000 magnification.
Fig. 1. Microscopic examination of capsule in P. gingivalis strain W83 at 1000 magnification. The cells were negatively stained with crystal violet, the clear zone around the cells indicates the presence of capsule.
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DNA was restricted with NotI (New England Biolabs, Hitchin, UK) [6]. Electrophoresis was performed in 1.2% agarose gels (SeaKem Gold agarose), by using the contour homogenous electric fields device (CHEF-DR III, Bio-Rad, Hercules, CA, USA); the pulse time was increased from 1 to 30 s over 20 h at 6 V/cm (200 V). Ethidium bromide stained gels were photographed under UV light. The macrorestriction fingerprints were analyzed by Bionumerics Software. A tolerance in the band position of 1% was applied for comparison of PFGE patterns. The similarities of isolates were determined by using the Dice coefficient, and a dendrogram was constructed by UPGMA. Clusters were defined above the 70% similarity level.
3. Results The presence of capsule was determined in 9 of the 16 studied P. gingivalis strains, including the reference encapsulated strain W83. As shown in Fig. 1, the encapsulated strains demonstrated a clear zone around the cell, indicative of the capsular polysaccharide. The presence of fimbriae was determined by TEM in negatively stained cells, as shown in Fig. 2 for the reference strain ATCC 33277. 9 out of 16 strains presented hair-like appendages on their cell surfaces resembling the major fimbriae (including ATCC 33277). The phenotypes included four strains presenting both fimbriae and capsule at the studied conditions, 2 strains exhibiting none of these surface structures, and 5 strains were fimbriated but not encapsulated and 5 were encapsulated but not fimbriated. 14 macrorestriction profiles were determined by PFGE (above 70% similarity), whereas MLST revealed 15 Sequence Types (STs) distributed in 7 clusters with at least 70% of similarity. The dendrograms constructed from the MLST and PFGE data were largely divergent, as shown in Fig. 3. Data on capsule and fimbriae and FimA genotypes of the P. gingivalis isolates are also shown in Fig. 3. No specific cluster was associated with capsule and/or fimbriae expression, since fimbriated and encapsulated strains were distributed along both dendrograms.
Fig. 2. TEM examination of fimbriae in P. gingivalis strain 33277. The hair-like appendages on the cell surfaces indicate the presence of fimbriae.
4. Discussion Most studies on P. gingivalis virulence factors use reference strains which possess either capsule or fimbriae, but not both or none of these surface components [23,37]. Fimbriae are considered an important virulence factor and it is involved in adhesion to epithelial cells, invasion and biofilm formation [25]. Furthermore, the loss of capsule also leads to reduced virulence [23,37,44] although its absence results in increased biofilm formation [13], suggesting that these two surface components may have complementary roles in the colonization of host tissues. In the present study nine strains were shown to be fimbriated, and nine were encapsulated. Four of the studied isolates possessed both surface components, while the major fimbriae and capsule were not detected in two isolates. As far as we know, this is the first report showing P. gingivalis isolates which express both the fimbriae and the capsule whereas others may not produce any of these structures, at least under the studied conditions. The ability to produce fimbriae or capsule was not associated to any particular fimA genotype, and even strains classified in the highly prevalent fimA genotype II were a fimbriated. Variation within a species is often introduced by random mutation and recombination. In this context, PFGE restriction profiles are mostly affected by recombination events, including horizontal gene transfer, whereas MLST allows a phylogenetic analysis based on variations of house-keeping genes [17]. P. gingivalis exhibits a non-clonal structure population [15], and a mechanism for chromosomal DNA transfer via transformation seems to be responsible for DNA exchange within this specie [41]. The periodontal pocket may be a favorable environment for horizontal gene transfer and recombination [24] and previous data suggested that the high rates of recombination of P. gingivalis resulted in plasticity in the genome [35]. The strains W83 (encapsulated, non-fimbriated) and 33277 (non-encapsulated, fimbriated) were classified in the same phylogenetic group by MLST, with a genetic distance of about 50%, as previously shown [15]. Also the analysis of macrorestriction fragments revealed about 50% similarity between 33277 and W83, indicating recombination events in the evolutionary process differentiating these two strains. In fact, chromosome sequencing analysis of these two strains revealed differences in the operon encoding the biosynthesis of capsular polysaccharide and in another operon with low G þ C content [10]. Furthermore, an insertion in FimS, which is part of the FIMS-FIMR two components regulatory system, regulating fimbriae expression, was reported in W83, leading to the malfunction of FIMS [33]. However, the allocation of W83 and 33277 in the dendrograms highlights that a much larger divergence has occurred in P. gingivalis than represented by these two strains. Previous data on MLST revealed that fimA genotypes tended to cluster [15]. In addition, some studies related the diversity by PFGE to the fimA genotype [6]; [35]. Our data confirm the panmitic structure of P. gingivalis populations [18], and indicated that the production of capsule and fimbriae is widely spread among P. gingivalis phylogenetic lineages, although no clustering according to the fimA type was observed, possibly due to the high diverse origin of the tested strains. Some limitations of our study should be mentioned. The number of isolates was low, although they were obtained from very diverse regions, and all fimA genotypes were represented. In addition, the capsule and fimbriae were detected by visualization after in vitro cultivation, which may not reflect in vivo conditions for the expression of these virulence factors. It is known that certain conditions may affect gene expression in P. gingivalis [4,7,29,45]. In fact, the proportion of strains with detectable capsule after growth
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Fig. 3. Dendrograms representing the similarity of 16 strains of Porphyromonas gingivalis strains constructed with the UPGMA clustering method. MLST tree shows similarity based in sequence analysis of 7 housekeeping genes, whereas PFGE tree shows the NotI pattern similarity among the strains. Encapsulated strains are highlighted in boxes. * fimbriated strains.
in blood agar plates was much lower than that observed after cultivation in broth (data not shown). Furthermore, the cells grown in liquid media did not preserve intact major fimbriae as observed for cells grown in blood agar plates. It is well known that differences in P. gingivalis capsular (K) and FIMA antigens may influence virulence [8,9,27,32,42]. However, our choice for microscopic detection of capsule and fimbriae instead of immune detection of their antigens was due to the large variability of the capsular and major fimbriae antigens within P. gingivalis, requiring the use of several non commercially available sera, plus the existence of still not typable strains based on the K or FIMA antigens. The lack of correlation of production of capsule and fimbriae with evolutionary lineages or macrorestriction profiles suggests that comparative genomes aiming to map sequencing reads against reference sequences in P. gingivalis may not be able to identify genetic markers for virulence due to the significant genomic rearrangement and divergence found in this species. Our data indicating that the encapsulated/fimbriated phenotypes were spread among different evolutionary lineages of P. gingivalis suggested that recombination played a key role in the expression of capsule and fimbriae. Thus, no genetic markers to be used in larger studies are already available for discriminating P. gingivalis strains according to the expression of these relevant virulence factors.
Acknowledgments This study was supported by FAPESP, grants 09/53958-1, 09/ 50191-1 and 09/13029-1.
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Microbial Pathogenesis journal homepage: www.elsevier.com/locate/micpath
Lineage variability in surface components expression within Porphyromonas gingivalis Silvia Regina Loureiro Teixeira a, Talyta Thereza Soares D'Epiro a, Ericka Tavares Pinheiro b, Kisielius c, Maria Regina L. Simionato a, Noemi Nosomi Taniwaki c, Jonas Jose a, * Marcia Pinto Alves Mayer a b c
~o Paulo, Av. Prof. Lineu Prestes, 1374, Sa ~o Paulo, SP 05508-900, Brazil Department of Microbiology, Institute of Biomedical Sciences, University of Sa ~o Paulo, Av. Prof. Lineu Prestes, 2227, Sa ~o Paulo, SP 05508-900, Brazil Department of Endodontics, School of Dentistry, University of Sa ~o Paulo, SP 01246-902, Brazil Department of Electron Microscopy, Adolfo Lutz Institute, Av Dr. Arnaldo, 355, Sa
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a b s t r a c t
Article history: Received 5 June 2014 Received in revised form 9 October 2014 Accepted 3 November 2014 Available online 4 November 2014
The periodontopathogen Porphyromonas gingivalis is represented by a spectrum of phenotypes ranging from commensals to pathogenic lineages. Capsule and fimbriae are considered key virulence factors in this specie, involved in colonization and host defenses evasion. Since these virulence traits may not be expressed by certain strains, we aimed to test the hypothesis that certain clusters or genotypes of P. gingivalis correlate with the production of capsule and fimbriae. Sixteen P. gingivalis isolates were evaluated. Capsule (K) was detected by optical microscopy of negatively stained cells. The presence of fimbriae (F) was determined by TEM. Genotypes were determined by NotI macrorestriction fragments analysis through Pulsed-Field Gel Electrophoresis (PFGE) and Multi-locus sequence typing (MLST) based on seven house-keeping genes. The phenotypes included FþKþ (n ¼ 4), FKþ (n ¼ 5), FþK (n ¼ 5) and FK (n ¼ 2). The analysis of whole genome macrorestriction fragments revealed 14 different clusters. MLST data also revealed extensive genetic diversity; however, PFGE and MLST profiles showed evident differences. There was no association between P. gingivalis clusters and encapsulated and/or fimbriated phenotypes. Genotyping methods were not able to discriminate isolates according to the production of virulence factors such as capsule and major fimbriae, indicating that recombination played a key role in the expression of capsule and fimbriae in P. gingivalis. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Porphyromonas gingivalis MLST PFGE Capsule Fimbriae
1. Introduction Periodontal diseases result from complex interactions between pathogenic bacteria in subgingival biofilm and host tissues [38]. The disease does not only affect the oral tissues, but is associated with systemic conditions, such as cardiovascular diseases and increased risk of pre-term labor [30,34]. Porphyromonas gingivalis is a major pathogen involved in the etiology of periodontitis especially by manipulating host immune responses, driving a shift of
Abbreviations: MLST, Multi-locus sequence typing; PFGE, pulsed field gel electrophoresis. * Corresponding author. E-mail addresses: [email protected] (S.R.L. Teixeira), [email protected] (T.T.S. D'Epiro), [email protected] (E.T. Pinheiro), [email protected] (M.R.L. Simionato), [email protected] (N.N. Taniwaki), [email protected] (J.J. Kisielius), [email protected] (M.P.A. Mayer). http://dx.doi.org/10.1016/j.micpath.2014.11.001 0882-4010/© 2014 Elsevier Ltd. All rights reserved.
the resident microbiota toward a periodontitis associated microbial community [12]. P. gingivalis isolates differ in their ability to induce periodontal destruction in humans and animal models [4,22], and in virulence factors such as the production of capsular polysaccharide, fimbriae and proteases [46]. Experimental animal studies revealed that encapsulated strains induce a more pronounced alveolar bone loss after oral challenge [20] and are more invasive after subcutaneous inoculation [21]. Furthermore, capsule production by certain P. gingivalis strains was associated with increased virulence [21,27], properties such as resistance against desiccation, osmotic stress, oxygen toxicity and phagocytic engulfment [11], induction of a lower host response [9,37], and biofilm formation [13]. The capsular polysaccharide locus shows evidence of being acquired by horizontal transfer [1,10], and is very polymorphic, leading to seven capsule known antigens [8] and non typable strains [26]. Although a gene encoding a glycosiltransferase (pg0106) was shown to be essential for capsule
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production [13], it is present in most P. gingivalis isolates independently on the capsule expression [10]. Fimbriae play a critical role in colonization of the oral cavity by mediating the interaction of bacteria with host cells [14,28], hard surfaces and host proteins [5,28]. It allows P. gingivalis to explore the Toll-like receptor signaling pathway and is associated with internalization in non phagocythic eukaryotic cells, inhibition of IL12p70 and persistence in macrophages [43]. The P. gingivalis population displays a spectrum of phenotypes from commensal to pathogenic [40] but there are no currently known markers for more virulent strains. The major fimbria of P. gingivalis is encoded by fimA and the specie is classified in 6 distinct fimA genotypes [2]. FimA type II is the most prevalent in periodontitis patients [3,31], although FimA IV correlated with disease severity in smokers [39]. FimA type II is dispersed among distinct genetic lineages of P. gingivalis [36] but whether all these clusters express the fimbriae is currently not known. Thus, we aimed to test the hypothesis that genotyping methods with high discriminatory power and reproducibility would highlight associations between certain genotypes or clusters and the expression of virulence factors in P. gingivalis. Profiles generated by whole genome macrorestriction fragments through Pulsed-Field Gel Electrophoresis (PFGE) vary in consequence of genome rearrangements [17,35], whereas Multilocus Sequence Typing (MLST), based on sequencing house-keeping genes, provides a much more stable genetic signature and enables clustering on the basis of evolutionary proximity [19]. Since both fimbriae and capsule may provide a selective advantage in the colonization of the host, and are considered key virulence factors in P. gingivalis, we evaluated the macrorestriction profiles and evolutionary patterns of P. gingivalis and determined their correlation with the production of capsule and fimbriae.
2. Methods 2.1. Bacterial strains
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2.3. Detection of fimbriae P. gingivalis cells grown anaerobically in TSBHK agar plates for 72 h were collected and resuspended in PBS. The bacterial suspensions were added to formvar-coated copper grid previously treated with 2% Alcian Blue solution, to improve adhesion of the bacterial cells to the grid. The bacterial cells were then negatively stained with 2% potassium phosphotungstate for 30 s [29], and the samples were observed with a JEOL electron microscope, model JEM1011. 2.4. MLST PCR primers and amplification conditions for MLST have been described earlier [16,24]. Seven genes (ftsQ, gdpxJ, hagB, mcmA, pepO, pga and recA) were evaluated. PCR products were purified using QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA). Both strands of purified PCR products were sequenced at the Human Genome Center, Institute of Biology, University of S~ ao Paulo, Brazil. Bionumerics software version 6.1 (Applied Maths, SaintMartens-Latem, Belgium) was used for data entry, sequence alignment and the phylogenetic tree was constructed by the unweight method with arithmetic averages (UPGMA). Sequence data were submitted to the P. gingivalis MLST website (http://pubmlst. org/pgingivalis) and to Genbank (http://www.ncbi.nlm.nih.gov/ genbank). 2.5. PFGE P. gingivalis suspensions, at an OD660nme0.5 (3 109 CFU/ml), were treated with lyses solution [50 mM TriseHCl (pH 8.0), 50 mM EDTA, lysozyme (2.5 mg/mL), and proteinase K (1.5 mg/mL)], added to an equal volume of 1.2% low melting agarose (SeaKem Gold agarose, Cambrex Bio Science, Workingham, UK) and poured into CHEF Disposable Plug Mold (Bio-Rad Laboratories, Hercules, CA, USA). The proteolysis was performed with 0.5 M EDTA, 1% sarcosyl and 1 mg/mL of proteinase K; overnight at 50 C. After washings,
P. gingivalis isolates obtained from periodontitis patients of diverse geographical locations (Japan n ¼ 3; Sweden, n ¼ 4 and Brazil, n ¼ 7) and the reference strains ATCC 33277 and W83 were evaluated. Since human isolates were evaluated, this study was approved by the institutional ethical committee on research in humans (846-CEP-10.07.8). All strains were previously identified as P. gingivalis by polymerase chain reaction (PCR) using speciesspecific 16SrRNA primers [3], and genotyped by amplification using fimA specific primers followed by restriction analysis of amplicons for differentiation between fimA I and fimA Ib genotypes [2,32,42]. Frozen stocks of P. gingivalis strains were cultured anaerobically in TSAHK agar plates (Tryptic Soy Agar supplemented with 5% defibrinated sheep blood, 0.5 mg/mL hemin and 1 mg/mL menadione) for 7e10 days at 37 C in 85% N2, 5% CO2 e 10% H2 in an anaerobic chamber (Plas Labs, Lansing, MI, USA).
2.2. Detection of capsule The isolates were also grown to the exponential growth phase in TSBHK (Tryptic Soy Broth supplemented with 0.5 mg/mL hemin and 1 mg/mL menadione) to an OD660nme0.5e0.8. Cells were negatively stained by crystal violet according to Hiss method. The presence of capsule was evident as clear areas around the bacterial cell under light optical microscopy at 1000 magnification.
Fig. 1. Microscopic examination of capsule in P. gingivalis strain W83 at 1000 magnification. The cells were negatively stained with crystal violet, the clear zone around the cells indicates the presence of capsule.
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DNA was restricted with NotI (New England Biolabs, Hitchin, UK) [6]. Electrophoresis was performed in 1.2% agarose gels (SeaKem Gold agarose), by using the contour homogenous electric fields device (CHEF-DR III, Bio-Rad, Hercules, CA, USA); the pulse time was increased from 1 to 30 s over 20 h at 6 V/cm (200 V). Ethidium bromide stained gels were photographed under UV light. The macrorestriction fingerprints were analyzed by Bionumerics Software. A tolerance in the band position of 1% was applied for comparison of PFGE patterns. The similarities of isolates were determined by using the Dice coefficient, and a dendrogram was constructed by UPGMA. Clusters were defined above the 70% similarity level.
3. Results The presence of capsule was determined in 9 of the 16 studied P. gingivalis strains, including the reference encapsulated strain W83. As shown in Fig. 1, the encapsulated strains demonstrated a clear zone around the cell, indicative of the capsular polysaccharide. The presence of fimbriae was determined by TEM in negatively stained cells, as shown in Fig. 2 for the reference strain ATCC 33277. 9 out of 16 strains presented hair-like appendages on their cell surfaces resembling the major fimbriae (including ATCC 33277). The phenotypes included four strains presenting both fimbriae and capsule at the studied conditions, 2 strains exhibiting none of these surface structures, and 5 strains were fimbriated but not encapsulated and 5 were encapsulated but not fimbriated. 14 macrorestriction profiles were determined by PFGE (above 70% similarity), whereas MLST revealed 15 Sequence Types (STs) distributed in 7 clusters with at least 70% of similarity. The dendrograms constructed from the MLST and PFGE data were largely divergent, as shown in Fig. 3. Data on capsule and fimbriae and FimA genotypes of the P. gingivalis isolates are also shown in Fig. 3. No specific cluster was associated with capsule and/or fimbriae expression, since fimbriated and encapsulated strains were distributed along both dendrograms.
Fig. 2. TEM examination of fimbriae in P. gingivalis strain 33277. The hair-like appendages on the cell surfaces indicate the presence of fimbriae.
4. Discussion Most studies on P. gingivalis virulence factors use reference strains which possess either capsule or fimbriae, but not both or none of these surface components [23,37]. Fimbriae are considered an important virulence factor and it is involved in adhesion to epithelial cells, invasion and biofilm formation [25]. Furthermore, the loss of capsule also leads to reduced virulence [23,37,44] although its absence results in increased biofilm formation [13], suggesting that these two surface components may have complementary roles in the colonization of host tissues. In the present study nine strains were shown to be fimbriated, and nine were encapsulated. Four of the studied isolates possessed both surface components, while the major fimbriae and capsule were not detected in two isolates. As far as we know, this is the first report showing P. gingivalis isolates which express both the fimbriae and the capsule whereas others may not produce any of these structures, at least under the studied conditions. The ability to produce fimbriae or capsule was not associated to any particular fimA genotype, and even strains classified in the highly prevalent fimA genotype II were a fimbriated. Variation within a species is often introduced by random mutation and recombination. In this context, PFGE restriction profiles are mostly affected by recombination events, including horizontal gene transfer, whereas MLST allows a phylogenetic analysis based on variations of house-keeping genes [17]. P. gingivalis exhibits a non-clonal structure population [15], and a mechanism for chromosomal DNA transfer via transformation seems to be responsible for DNA exchange within this specie [41]. The periodontal pocket may be a favorable environment for horizontal gene transfer and recombination [24] and previous data suggested that the high rates of recombination of P. gingivalis resulted in plasticity in the genome [35]. The strains W83 (encapsulated, non-fimbriated) and 33277 (non-encapsulated, fimbriated) were classified in the same phylogenetic group by MLST, with a genetic distance of about 50%, as previously shown [15]. Also the analysis of macrorestriction fragments revealed about 50% similarity between 33277 and W83, indicating recombination events in the evolutionary process differentiating these two strains. In fact, chromosome sequencing analysis of these two strains revealed differences in the operon encoding the biosynthesis of capsular polysaccharide and in another operon with low G þ C content [10]. Furthermore, an insertion in FimS, which is part of the FIMS-FIMR two components regulatory system, regulating fimbriae expression, was reported in W83, leading to the malfunction of FIMS [33]. However, the allocation of W83 and 33277 in the dendrograms highlights that a much larger divergence has occurred in P. gingivalis than represented by these two strains. Previous data on MLST revealed that fimA genotypes tended to cluster [15]. In addition, some studies related the diversity by PFGE to the fimA genotype [6]; [35]. Our data confirm the panmitic structure of P. gingivalis populations [18], and indicated that the production of capsule and fimbriae is widely spread among P. gingivalis phylogenetic lineages, although no clustering according to the fimA type was observed, possibly due to the high diverse origin of the tested strains. Some limitations of our study should be mentioned. The number of isolates was low, although they were obtained from very diverse regions, and all fimA genotypes were represented. In addition, the capsule and fimbriae were detected by visualization after in vitro cultivation, which may not reflect in vivo conditions for the expression of these virulence factors. It is known that certain conditions may affect gene expression in P. gingivalis [4,7,29,45]. In fact, the proportion of strains with detectable capsule after growth
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Fig. 3. Dendrograms representing the similarity of 16 strains of Porphyromonas gingivalis strains constructed with the UPGMA clustering method. MLST tree shows similarity based in sequence analysis of 7 housekeeping genes, whereas PFGE tree shows the NotI pattern similarity among the strains. Encapsulated strains are highlighted in boxes. * fimbriated strains.
in blood agar plates was much lower than that observed after cultivation in broth (data not shown). Furthermore, the cells grown in liquid media did not preserve intact major fimbriae as observed for cells grown in blood agar plates. It is well known that differences in P. gingivalis capsular (K) and FIMA antigens may influence virulence [8,9,27,32,42]. However, our choice for microscopic detection of capsule and fimbriae instead of immune detection of their antigens was due to the large variability of the capsular and major fimbriae antigens within P. gingivalis, requiring the use of several non commercially available sera, plus the existence of still not typable strains based on the K or FIMA antigens. The lack of correlation of production of capsule and fimbriae with evolutionary lineages or macrorestriction profiles suggests that comparative genomes aiming to map sequencing reads against reference sequences in P. gingivalis may not be able to identify genetic markers for virulence due to the significant genomic rearrangement and divergence found in this species. Our data indicating that the encapsulated/fimbriated phenotypes were spread among different evolutionary lineages of P. gingivalis suggested that recombination played a key role in the expression of capsule and fimbriae. Thus, no genetic markers to be used in larger studies are already available for discriminating P. gingivalis strains according to the expression of these relevant virulence factors.
Acknowledgments This study was supported by FAPESP, grants 09/53958-1, 09/ 50191-1 and 09/13029-1.
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