Advertisement
Browse Subject Areas
?

Click through the PLOS taxonomy to find articles in your field.

For more information about PLOS Subject Areas, click here.

  • Loading metrics

Comparative gene expression analysis of Porphyromonas gingivalis ATCC 33277 in planktonic and biofilms states

  • P. Romero-Lastra,

    Affiliation Laboratory of Dental Research, University Complutense, Madrid, Spain

    ORCID http://orcid.org/0000-0003-4421-7845

  • MC. Sánchez,

    Affiliation Laboratory of Dental Research, University Complutense, Madrid, Spain

  • H. Ribeiro-Vidal,

    Affiliation Laboratory of Dental Research, University Complutense, Madrid, Spain

  • A. Llama-Palacios,

    Affiliation Laboratory of Dental Research, University Complutense, Madrid, Spain

  • E. Figuero,

    Affiliations Laboratory of Dental Research, University Complutense, Madrid, Spain, ETEP (Etiology and Therapy of Periodontal Diseases) Research Group, University Complutense, Madrid, Spain

  • D. Herrera,

    Affiliation ETEP (Etiology and Therapy of Periodontal Diseases) Research Group, University Complutense, Madrid, Spain

  • M. Sanz

    marsan@ucm.es

    Affiliation ETEP (Etiology and Therapy of Periodontal Diseases) Research Group, University Complutense, Madrid, Spain

Comparative gene expression analysis of Porphyromonas gingivalis ATCC 33277 in planktonic and biofilms states

  • P. Romero-Lastra, 
  • MC. Sánchez, 
  • H. Ribeiro-Vidal, 
  • A. Llama-Palacios, 
  • E. Figuero, 
  • D. Herrera, 
  • M. Sanz
PLOS
x

Abstract

Background and objective

Porphyromonas gingivalis is a keystone pathogen in the onset and progression of periodontitis. Its pathogenicity has been related to its presence and survival within the subgingival biofilm. The aim of the present study was to compare the genome-wide transcription activities of P. gingivalis in biofilm and in planktonic growth, using microarray technology.

Material and methods

P. gingivalis ATCC 33277 was incubated in multi-well culture plates at 37°C for 96 hours under anaerobic conditions using an in vitro static model to develop both the planktonic and biofilm states (the latter over sterile ceramic calcium hydroxyapatite discs). The biofilm development was monitored by Confocal Laser Scanning Microscopy (CLSM) and Scanning Electron Microscopy (SEM). After incubation, the bacterial cells were harvested and total RNA was extracted and purified. Three biological replicates for each cell state were independently hybridized for transcriptomic comparisons. A linear model was used for determining differentially expressed genes and reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to confirm differential expression. The filtering criteria of ±2 change in gene expression and significance p-values of <0.05 were selected.

Results

A total of 92 out of 1,909 genes (4.8%) were differentially expressed by P. gingivalis growing in biofilm compared to planktonic. The 54 up-regulated genes in biofilm growth were mainly related to cell envelope, transport, and binding or outer membranes proteins. Thirty-eight showed decreased expression, mainly genes related to transposases or oxidative stress.

Conclusion

The adaptive response of P. gingivalis in biofilm growth demonstrated a differential gene expression.

Introduction

Human dental plaque is a complex and dynamic biofilm attached to tooth surfaces, where microbial communities are embedded in a matrix of bacterial extracellular polymeric substances (EPS), proteins, salivary peptides and food scraps [1, 2]. The differential activity of these microbial communities within the dental biofilm may have profound implications in the onset and progression of periodontitis, one of the most prevalent chronic inflammatory diseases affecting humans [3]. Porphyromonas gingivalis, a Gram-negative and black-pigmented anaerobic bacterium is one of the keystone pathogens associated with the etiology of periodontitis. Its main ecological niche is the oral microbiome [4] and its pathogenic activity has been directly related to its relative high numbers and proportions within the subgingival biofilm, as well as the expression of virulence factors that facilitate its colonization within the periodontal tissues and its resistance from the host inflammatory and immune responses. [57].

Virulence factors in periodontal pathogens have been attributed to either presence of highly pathogenic strains or to the up- and down- regulation of a number of genes due to the specific ecological conditions of the bacterial communities within the biofilm. In fact, several transcriptomic studies have been conducted to elucidate the behavior of different pathogenic bacteria growing in biofilm [811]. Whiteley et al. [10] reported that about 1% of the genes from Pseudomonas aeruginosa had shown differential expression when growing in biofilm compared with planktonic. Liu et al. [9] reported that 16.2% of the genes from Clostridium acetobutylicum were differentially expressed in biofilm growth, mainly up-regulation of genes involved in amino acid biosynthesis, sporulation, extracellular polymer degradation and other various metabolic processes, what indicated that C. acetobutylicum had a distinct phenotype when growing in a biofilm.

Similarly, transcriptomic studies have reported that approximately 18.0% of the W50 genome of P. gingivalis was differentially expressed in biofilms [8]. These studies have shown down-regulation of genes encoding for cell envelope biogenesis, DNA replication, energy production and biosynthesis of co-factors and up- regulation of genes involved in transport and binding proteins. Some of these studies have focused specifically on LuxS-dependent signaling and quorum-sensing-regulated genes since they play an important role in the physiology of these micro-organisms, their communication with other bacteria, and their adaptation to the biofilm environment [1214]. Yamamoto et al. [15] reported that an increase of more than 1.5-fold in the number P. gingivalis (ATCC 33277) genes differentially regulated during the biofilm growth (312/2,090 genes, 155 genes were up-regulated and 157 genes were down-regulated).

In spite of these studies, our understanding of the regulatory processes and interactions, which allow P. gingivalis to grow within the biofilm and to develop its virulence is still limited. It is, therefore, the aim of this study to assess the differential expression of P. gingivalis genes under two different physiological states, planktonic and biofilm growth, using transcriptomic analysis in an in vitro static model.

Material and methods

Bacterial strain

Standard reference strain P. gingivalis ATCC 33277 was selected for the present study. Bacteria were grown on blood agar plates (Blood Agar Oxoid No 2; Oxoid, Basingstoke, UK), supplemented with 5% (v/v) sterile horse blood (Oxoid), 5.0 mg/L hemin (Sigma, St. Louis, MO, USA) and 1.0 mg/L menadione (Merck, Darmstadt, Germany) in anaerobic conditions (10% H2, 10% CO2, and balance N2) at 37°C for 72 hours.

Bacterial growth and experimental assays

Planktonic cultures of P. gingivalis were grown anaerobically at 37°C for 24 h in a protein-rich medium containing brain-heart infusion (BHI) (Becton, Dickinson and Company, Franklin Lakes, NJ, USA) supplemented with 2.5 g/L mucin (Oxoid), 1.0 g/L yeast extract (Oxoid), 0.1 g/L cysteine (Sigma), 2.0 g/L sodium bicarbonate (Merck), 5.0 mg/L hemin (Sigma), 1.0 mg/L menadione (Merck) and 0.25% (v/v) glutamic acid (Sigma). Upon reaching late-exponential phase [109 colony forming units (CFU)/mL, as measured spectrophotometrically by optical density at 550 nm], the cells were diluted in modified BHI medium to obtain a final concentration of 108 CFU/mL.

In order to study the gene expression of P. gingivalis, in biofilm or planktonic growth, under the same culture conditions, a volume of 1.5 mL of P. gingivalis inoculums was placed in pre-sterilized polystyrene 24-well tissue culture plates (Greiner Bio-one, Frickenhausen, Germany) with or without the presence of sterile ceramic calcium hydroxyapatite discs (HA) [7-mm diameter (standard deviation, SD = 0.2) and 1.8 mm thickness] (Clarkson Chromatography Products, Williamsport, PA, USA). To carry out the experiment, a total of 45 multiwell plates were used. In each plate 19 wells were filled with disk to develop the biofilms (each of the aggregates in each hydroxyapatite disk is considered as a biofilm) and the other five wells were used to analyze the planktonic state without hydroxyapatite disk.

Plates were incubated in anaerobic conditions at 37°C for 96 h. Wells containing only culture medium were also incubated to verify sterility and the possible contamination of bacteria growing in both planktonic and biofilm growth was frequently checked.

Confocal Laser Scanning Microscopy (CLSM) analysis to monitor P. gingivalis biofilm development

To ensure the change of P. gingivalis phenotype, from planktonic to biofilm, its growth was studied by CLSM when the biofilm reached a mature state (from 24 to 96 h). To confirm the reproducibility of the biofilm-growth, three independent experiments using trios of biofilms were carried out for each incubation time (a concentration of 108 CFU/mL P. gingivalis cells in planktonic culture were placed on sterile hydroxyapatite discs). Before the CLSM analysis, the discs were rinsed in 2 mL of sterile Buffer Phosphate Saline (PBS) three times (10 sec of immersion time per rinse), in order to remove non-adherent bacteria. Non-invasive confocal imaging of fully hydrated biofilms was carried out using a fixed-stage Ix83 Olympus inverted microscope coupled to an Olympus FV1200 confocal system and with a ×63 water-immersion lens (Olympus; Shinjuku, Tokio, Japan). Specimens were stained with LIVE/DEAD® BacLightTM Bacterial Viability Kit solution (Molecular Probes B. V., Leiden, The Netherlands) at room temperature. A 1:1 fluorocromes ratio and 9±1 min of staining time was used to obtain the optimum fluorescence signal at the corresponding wave lengths (Syto9: 515–530 nm; PI: >600 nm). At least three separate and representative locations on the HA discs covered with biofilm were selected for the study. The CLSM control software was set to take a z-series of scans (xyz) of 0.5 μm thickness (8 bits, 1024x1024 pixels). Image stacks were analyzed with the proprietary Olympus® software (Olympus).

Scanning Electron Microscopy (SEM) analysis

Before SEM analysis, three hydroxyapatite discs covered with biofilms grown in vitro for 96 h were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde for 4h at 4°C. Then, the discs were washed twice in PBS and sterile water (immersion time 10 min) and then, dehydrated through a series of graded ethanol solutions (50, 60, 70, 80, 90 and 100%; immersion time per series, 10 min), Then, the samples were critical point dried, sputter-coated with gold and analysed with an scanning electron microscope JSM 6400 (JSM6400; JEOL, Tokyo, Japan) equipped with back-scattered electron detector and with an image resolution of 25 KV.

Harvesting of planktonic and biofilm cells for gene expression analysis

After 96 h of incubation, P. gingivalis planktonic and biofilm cells were harvested (three biological replicates of each state) for independent hybridization.

For planktonic cells 1 mL was recovered from 15 diskless well. In the same experiments a set of 300 biofilms were harvested independently, then added to 1 mL of sterile PBS, disaggregated by vortexing during 3 min. In both cases the samples were recovered as partial plucks by centrifugation at 9,000 rpm at 4°C during 5 min, in order to obtain a final 10 μg of total RNA for each replicate in each state. To preserve the bacterial total RNA intact during the time taken for the procedures, the work has always been in cold conditions.

In all cases, after the incubation period, an aliquot of each sample and 1 to 3 discs were used as quality control. They were cultivated on supplemented blood agar plates under anaerobic conditions at 37°C during two weeks to assure the absence of contamination.

Total RNA extraction

Total RNA was extracted from the harvested samples using the TRIzol® Max Bacterial RNA Isolation Kit (Ambion, Life Technologies, Carlsbad, CA, USA). Briefly, pools from planktonic and biofilm growth samples were suspended in 200 μL of preheated Max bacterial Reagent® (Ambion), incubated at 95°C for 4 min and then chilled on ice for 10 min. After, 1 mL of TRIzol® reagent (Ambion) was added to lysate the cells, incubating them at room temperature 5 min. After that, 200 μL of cold chloroform was added and incubated at room temperature for 3 min. The mixtures were then centrifuged at 13,000 rpm for 15 min at 4°C. RNA colourless aqueous phase (~ 600 μL) was collected, augmented with 0.5 mL of cold isopropanol, mixed by inversion, and incubated at room temperature for 10 min. After centrifugation at 13,000 rpm for 10 min at 4°C, the pellet of RNA was suspended in 1 mL of cold 75% ethanol, centrifuged at 9,000 rpm for 5 min, air-dried and suspended in 50 μL of RNase-free water (Roche Diagnostics, Mannheim, Germany). The samples were then treated with DNase I (Ambion, NY, USA) to remove any contaminating DNA (set of RNase-free DNase; Qiagen, CA, USA) and purified using columns of RNeasy Mini kit (Qiagen) according to the manufacturer's protocol.

RNA quantity was measured by NanoDrop ND1000 spectrophotometer (NanoDropTechnologies; Thermo Scientific, LLC, Wilmington, DE, USA). RNA quality was monitored by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). All the samples used in this study exhibited an A260/A280 ratio of at least 2.0.

cDNA synthesis and transcriptomic analysis

Three biological replicates were independently hybridized for each transcriptomic comparison. Fluorescently labeled cDNA for microarray hybridizations was obtained by using the SuperScript Indirect cDNA Labeling System (Invitrogen). In brief, 5 μg of total RNA was transformed to cDNA with Superscript III reverse transcriptase using random hexamers as primers and with aminoallyl-modified nucleotides in the reaction mixture. After cDNA purification, the Cy3 fluorescent dyes (Amersham Biosciences) were coupled to the amino-modified first-strand cDNA. Labelling efficiency was assessed using a NanoDrop ND1000 spectrophotometer (NanoDropTechnologies).

Preparation of probes and hybridization was performed as described (One-Color Microarray Based Gene Expression Analysis Manual Ver. 6.5, Agilent Technologies). Briefly, for each hybridization, 600 ng of Cy3 probes were mixed and added to 5 μL of 10x Blocking Agent and Nuclease free water in a 25 μL reaction. Then, 25 μL from 2x GExHybridization buffer was added and mixed carefully. The samples were placed on ice and quickly loaded onto arrays, hybridized at 65°C for 17 h and then washed once in GE wash buffer 1 at room temperature (1 min) and once in GE Wash Buffer 2 at 37°C (1 min).

Slides corresponded to Agilent P. gingivalis Oligo Microarrays 8x15K (074976), a genome annotation specific for strain ATCC 33277 and W83. For each culture pair, three technical replicates of array hybridizations were performed.

Microarray and data analysis

Images from Cy3 channel were equilibrated and captured with a high-resolution scanner (Agilent) and spots quantified using Feature Extraction software (Agilent). Background correction and normalization of data expression were performed using LIMMA [16, 17]. LIMMA is part of bioconductor, an R language project [18]. For local background correction and normalization, the methods "normexp" and loess in LIMMA were used, respectively [16]. To ensure similar distribution across arrays and to achieve consistency among arrays, log-ratio values were scaled using the median-absolute-value as scale estimator [17].

Linear model methods were used for determining differentially expressed genes. Each probe was tested for changes in expression over replicates by using an empirical Bayes moderated t-statistic [17]. To control the false discovery rate p-values were corrected by using the method of Benjamani and Hochberg [16, 17]. The expected false discovery rate was controlled to be less than 5% and a filtering criterium of increase/decrease up to 2-fold differential expression between states was selected.

The National Center for Biotechnology (Genomics Unit) at Universidad Autónoma, Madrid (Spain) performed the hybridizations and statistical analysis.

Assessment of microarray data by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR)

To confirm the microarray results using RT-qPCR, nine genes differentially expressed between both situations were selected, four genes from the up-regulated group and five from the down-regulated one. Specific primers were designed using the Universal Probe Library Roche software tool (Roche Diagnostics) (Table 1). All quantifications were normalized to the P. gingivalis 16S rRNA gene.

thumbnail
Table 1. Primers used for Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR).

https://doi.org/10.1371/journal.pone.0174669.t001

To carry out the Reverse Transcription-qPCR, cDNA was generated from 1 μg of total RNA using the High Capacity cDNA Archive Kit (Applied Biosystems, ThermoFisher Scientific) in a 10 μL of final reaction volume. After that, quantitative PCR reactions were performed in triplicate by using 5 μL per well of each cDNA, and 3 μL of a mix composed by 0.4 μM of each primer, 5x HOT FIREPol® EvaGreen® qPCR Mix Plus (ROX), and nuclease-free water, to reach a final volume of 8 μL in 384-well optical plates. PCR reactions were run in an Applied Biosystems ABI PRISM 7900HT machine with SDS v2.4 software and standard protocol from Applied Biosystems (95°C 10 min, 40 cycles of 95°C 15 sec and 60°C 60 sec, and a final standard dissociation protocol). The results were analysed with the Comparative Ct Method (ΔΔCt) [19].

Results

CLSM and SEM confirmed that P. gingivalis ATCC 33277 changed its phenotypic state, from planktonic to a mono-species biofilm. Fig 1 shows representative CLSM (depicting viable bacteria as green and nonviable as red stained cells.) and SEM images of the obtained biofilms at 96 h of incubation,

thumbnail
Fig 1. Representative confocal (A) and scanning electron (B) micrographs representing Porphyromonas gingivalis ATCC 33277 biofilm after 96h of growth.

BacLight Live/Dead strain was used to assess the viability of cells in CLSM distinguishing viable bacteria depicted as green and non-viable as red stained cells.

https://doi.org/10.1371/journal.pone.0174669.g001

With the use of the filtering criteria threshold of two-fold change in differential expression (up or down) of the contained in P. gingivalis ATCC 33277 arrays, a total of 92 out of 1,909 (4.8%) genes were differentially expressed in the biofilm phenotype compared to planktonic growth. These differences were statistically significant (p<0.05).

Fig 2 shows the genes differentially expressed in P. gingivalis ATCC 33277 biofilms compared to planktonic cells. From the identified genes, the 54 up-regulated genes in the biofilm were mainly related to cell envelope, transport and binding proteins, outer membranes proteins, DNA repair enzymes, ribosomal proteins, or genes related to transcription initiation. Conversely, the 38 genes that were down-regulated in biofilm cells were mostly genes encoding proteins related to transposases, the CRISPRs system (cluster regularly inter-spaced short palindromic repeats) or oxidative stress.

thumbnail
Fig 2. Differential gene expression in Porphyromonas gingivalis ATCC 33277 biofilm as opposed to planktonic cells.

Differentially expressed genes with 2.0 fold change (up or down) and p-value < 0.05 were plotted. X-axis presents fold difference between log expression of planktonic, and y-axis shows the log expression of biofilm. Up-regulated genes (over-expressed in biofilm) were represented as red color and down-regulated genes were colored in green.

https://doi.org/10.1371/journal.pone.0174669.g002

In Table 2, these genes are grouped by functional categories, such as the genes encoding for the cationic outer membrane proteins (OmpH-1 and OmpH-2, PG_0448, and PG_0987), which have shown up-regulated expression in this model of P. gingivalis biofilm. These genes codifying proteins located specifically in the outer membrane vesicles, have been recognized as important virulence factors of P. gingivalis. Moreover, the gene coding lipoprotein PGN_0151 appeared up regulated by a factor of 3.16 (SD 0.28) compared to planktonic state (Table 2). Similarly, the genes related with the Por Secretion System (PorSS) (porP, PGN_1514 and PG_0448), involved in the biosynthesis of cell surface polysaccharides and implicated in the translocation of gingipains were up-regulated in biofilm growth. These proteins are well known virulence factors and serve as anchors for Rgp, Kgp, hemagglutinins, and the hemoglobin receptor protein. Only one gene, implicated in predicted exporter proteins (PGN_0946) was found significantly down regulated.

thumbnail
Table 2. Genes differently expressed in Porphyromonas gingivalis ATCC 33277 biofilm (cutoff ratio ≥ ±2.0 fold change, p-value < 0.05) for the microarray analysis, grouped by functional role categories.

https://doi.org/10.1371/journal.pone.0174669.t002

An additional group of genes related to oxidative stress and metabolism was differentially expressed in P. gingivalis, as shown in Table 2. This group of genes, represented by PGN_2076 and PG_2213, are involved in oxidative and/or regulatory mechanisms, as Nitric oxide (NO) stress resistance and were significantly suppressed in biofilm growth. These genes enable bacteria to survive within the inflammatory microenvironment of the periodontal pocket. Similarly, alkyl hydroperoxidase reductase subunits genes (AhpC-F (PG_0618, PGN_0660, PG_0619 and PGN_0661) were down regulated in P. gingivalis biofilms. These genes are involved in the primary defense against reactive oxygen species (ROS), and therefore affecting the bacterium aero-tolerance. In fact, PG_0619 was the gene most differentially suppressed (-13.13 (SD 0.70)). On the other hand, the putative genes related to metabolism NADPH-NAD transhydrogenases (PGN_1120, PGN_1122 and pntB) were up-regulated.

The genes involved in transposon functions, demonstrated heterogeneous results (Table 2). While genes corresponding to partial transposase in ISPg1 (PGN_0219, PGN_0575, PGN 1216 and PGN_1420) and PGN_0579 were down-regulated, genes belonging to the partial transposase in ISPg4 (PGN_0478) and PGN_0578 were up-regulated.

Genes related to the CRISPRs and associated CAS proteins system (CRISPR/Cas), like (PGN_1924-Cas2, PGN_1925-Cas1) were down-regulated in biofilm growth, while the gene PGN_1286, thought to be a lysozyme, was up regulated.

Among the genes related to fimbriae, only one gene, fimD, one of the minor components of the fimbriae A, appeared down-regulated by a factor of -2.30 (SD 0.26) in biofilm versus planktonic cells.

Among the genes involved in the biogenesis of components of ribosomal subunits, the genes rpmH, rpsF and rpII were up-regulated while KsgA were down-regulated when in comparing biofilm with planktonic growth.

The array data (Table 2) indicated that several RNA polymerase sigma factors of the σ70 family (PG_0214, PG_0985, PGN_0319, PGN_0450, PGN_0970), involved in the regulation of biofilm formation and diverse physiological processes, particularly virulence, were up-regulated in biofilm versus planktonic cells. On the other hand, PGN_0082, a probable transcriptional regulator in the AraC family, was down-regulated in biofilms cells.

The riboflavin-related gene encoded to the 3,4-dihydroxy-2-butanone 4-phosphate synthase/ GTP cyclohydrolase II protein (PGN_0643) was found up-regulated in P. gingivalis biofilm. This gene has been implicated in quorum sensing signaling and extracellular electron transfer. On the contrary, the gene VimF was down-regulated. This gene has been involved in the maturation/activation/anchorage of gingipains and other virulence factors of P. gingivalis. Lastly, 45% of the 92 differentially regulated P. gingivalis genes were of unknown or poorly characterized functions, most of them encoding unknown proteins.

The microarray results were validated by RT-qPCR on four of the genes from the up-regulated group and five from the down-regulated group. Fig 3 illustrates the high correlation between the gene expression of logarithm-transformed of RT-qPCR plotted against the average log2 ratio values obtained by microarray analysis (R² = 0.9716).

thumbnail
Fig 3. Correlation between microarray and Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR) gene expression ratios determined for biofilm versus planktonic cells.

The RT-qPCR log2 values were plotted against the microarray data log2 values (R2 = 0.9716).

https://doi.org/10.1371/journal.pone.0174669.g003

Discussion

This microarray-based comparative transcriptomic study has shown the up- and down- regulation of specific genes of P. gingivalis during the early stages of biofilm maturation (96 h of incubation). These gene expression patterns showed that 4.8% (92/1,909) of the genes of P. gingivalis significantly changed in biofilm, when compared to planktonic growth. Although this does not represent a huge difference between the two lifestyles [10, 20, 21], small changes in the level of expression of one gene can be amplified through regulatory networks and result in significant phenotypic alterations [2224]. These results are in agreement with previous reports on other pathogens, such as P. aeruginosa or Escherichia coli grown under similar differential growth conditions, in which less of 5% of differential expression was demonstrated [10, 11, 25, 26].

When assessing the different functional categories affected by the differentially regulated genes, a wide diversity was observed, which may indicate that the adaptation of P. gingivalis to a community lifestyle required a broad-based transcriptional modulation. This adaptation involved different virulence factors, as proteins codifying for outer membrane proteins or for fimbriae. Outer membrane vesicles (OMVs) of P. gingivalis, which are formed by “blebbing” portions of their outer membrane, have been recognized as important virulence factors of this pathogen in relation to periodontitis [6]. These vesicles contained specific proteases, termed gingipains (Arg-gingipain [Rgp] and Lys-gingipain [Kgp]) [5] associated with the capacity of P. gingivalis to invade host epithelial cells [27, 28]. This transcriptomic study has revealed four genes, which codify proteins located in the OMVs of P. gingivalis being over-expressed (OmpH-1 and OmpH-2, PG_0448, and PG_0987). This finding was already described by Veith et al. (2014) [29]. Similarly, Kuboniwa et al. (2009) [30] using proteomic technology studied P. gingivalis in biofilm growth and reported significantly increased cell envelope proteins, such as OmpH protein PGN_0301, whose encoding gene has been shown over expressed in this investigation.

The up-regulation of these proteins in biofilm versus planktonic state has also been reported in others studies demonstrating that OMVs and related genes play an important role in bacterial co-aggregation [31] and attachment to epithelial cells [32]. Although differential expression of genes has been shown at in vivo polymicrobial biofilms (Díaz and Kolenbrander [33], this study has confirmed that up-regulation could also occur when growing in an in vitro mono-species P. gingivalis biofilm.

Fimbriae of P. gingivalis have also been recognized as a major virulence factor, since they mediate in cell adhesion and may facilitate their capacity to invade periodontal tissues [3438]. Only one gene, fim D, was found down-regulated in this study. This gene is a minor component of a seven gene cluster, fimX, pgmA and fim ABCDE, which encode type 1 fimbriae, and it is characterized by mannose-sensitive hemagglutination and being assembled via the chaperone/usher pathway [39, 40]. These genes participate in the biogenesis of the fimbriae, regulating their number and length, as well as their adherence function [41, 42]. Nevertheless, Krogfelt and Klemm (1988) observed that a clone of E. coli, not containing the genes encoding the minor component proteins, still produced fimbriae consisting of pure Fim A protein, (main structural component of the fimbriae type I), indicating that, at least in the case of E. coli, the minor components were not necessary for the structural integrity of the fimbriae, although these fimbriae were non-adhesive and did not confer hemaglutination [4245]. Similarly, Whiteley et al. (2001) suggested that these appendages may not be required at the later stages of biofilm formation for maintenance of a mature biofilm, since fimbria, pili or flagella were only involved at initial steps of attachment [10, 15].

The lipoprotein-related gene PGN_0151 was over-expressed in biofilm. Hirano et al. (2013) reported that a mutant of this gene was reduced in its ability to form biofilms compared to wild type [46] what suggests that these genes were significantly involved in the biofilm lifestyles of P.gingivalis. In regards to those genes involved in the adaptation to new local environmental conditions, this investigation showed a differential expression of those genes involved in the transposition system (PGN_0219, PGN_0575, PGN 1216, PGN_1420, PGN_0579, PGN_0478 and PGN_0578), some of them codifying insertion sequences (IS). Since transposition is generally known to be triggered by cellular stress [4749], this finding suggests that these transposable elements, moving from one site within the genome to another, could have an important role in the genomic re-arrangement and recombination in P. gingivalis growing in biofilm. This adaptation to stressful local environmental conditions has been previously reported [7, 5053]. Furthermore, the CRISPR-Cas and associated CAS proteins system represents a unique system that provides prokaryotic cells, as P. gingivalis, adaption and protection from host defenses [54, 55]. Down-regulation of the genes PGN_1924-Cas2, PGN_1925-Cas1 may suggest a decrease in the defensive capability of P. gingivalis ATCC 33277 when growing as single-species biofilm in vitro or its adaptation to an environment without competing species.

Gene PG_2213, encoding a putative nitrite reductase-related protein and implicated in nitric oxide (NO) stress resistance was repressed in P. gingivalis biofilm growth [56, 57]. The ability to down-regulate nitrite reduction [58], involves the expression of several genes known to be induced by nitrogen oxides and low oxygen tension [59, 60]. Whether P. gingivalis PG_2213 has a similar role is unknown. Boutrin et al. (2012) suggested that NO stress resistance in P.gingivalis was facilitated by a complex and tightly regulated network of genes involved in multiple pathways, including, energy metabolism, gene regulation, detoxification, and virulence [56].

Furthermore, although P. gingivalis seems to lack a protective NADH oxidase, Alkyl hydroperoxide reductase (genes PG_0618, PGN_0660, PG_0619 and PGN_0661), C subunit (AhpC), have been reported to be involved in P. gingivalis aero-tolerance processes. The up-regulation of genes related to NADPH-NAD transhydrogenases (PGN_1120, PGN_1122, pntB) suggests that P. gingivalis growing in biofilm has elevated metabolic activities, as shown with C. acetobutylicum, by Liu et al. (2016) [9]. In this investigation, several genes related to ribosome function (rpmH, rpsF, rpII and KsgA) were over expressed in the biofilm, what may indicate that the metabolic increase was associated to ribosome function, that may require up to 40% of the cell's energy in growing bacteria [52].

The observed differential up regulated expression of sigma factors in biofilm cells (PG_0214, PG_0985, PGN_0450, PGN_0970, PGN_0319) might indicate that these genes are important regulators of P. gingivalis during biofilm growth [8]. Similar results have been reported for E.coli [61]. Besides, members of the AraC family of transcriptional regulators (PGN_0082), with decreased expression in the biofilm, have been shown to be important in carbon metabolism (degradation of sugars such as arabinose), stress response to virulence in other species [62], and in the regulation of quorum sensing signaling in P. aeruginosa [63]. Also, related to quorum sensing signaling, the up regulated gene PGN_0643, has been involved in the biosynthesis of riboflavin, a substance associated in a number of extracellular processes by bacteria, especially Gram-negative organisms [6466].

There are, however, important limitations associated to this study, since the biofilm used was an in vitro single-species model. The obtained results, however, may serve as a resource for future studies in oral biofilms aimed to further understand the genetic basis of the regulatory mechanisms of P.gingivalis and other pathogenic bacteria involved in subgingival biofilm growth and maturation.

Conclusions

By means of transcriptomic analysis, this study has shown that 4.8% of the P. gingivalis ATCC 33277 genome exhibited differential expression profiles when grown in biofilm. In such biofilm growth, the up-regulated genes were mainly those related to the cell envelope, as the genes encoding for the cationic OMPs or gene PGN_0151, which appear as a novel P. gingivalis gene that seems to have a role in the biofilm state. Also, the genes implicated in PorSS system and RNA polymerase sigma factors of the σ70 family, which are genes related to virulence/proliferation factors were up-regulated. On the contrary, the expression of most of the genes involved in oxidative stress or CRISPRs system were suppressed.

Therefore the adaptive response of P. gingivalis in biofilm growth demonstrated changes in gene expression profiles.

Acknowledgments

We thank G. García and L. Almonacid from the Centro Nacional de Biotecnología (Universidad Autónoma, Madrid, Spain) for hybridizations and statistical analysis of the microarrays and RT-qPCR performed by the Genomics Facility. We thank C. Pérez from the Centre of Microscopy and Cytometry from Universidad Complutense (Madrid, Spain). And also AM. Vicente for her technical microscopy services at the ICTS National Centre of Electron Microscopy, Universidad Complutense (Madrid, Spain). And finally we want to express our gratitude to Dr. Vanessa Blanc y Rubén León from the company DENTAID (Cerdanyola del Vallés, Barcelona, Spain).

Author Contributions

  1. Conceptualization: PR-L MCS EF DH MS.
  2. Funding acquisition: DH MS.
  3. Investigation: PR-L MCS HR-V AL-P.
  4. Methodology: PR-L MCS HR-V AL-P.
  5. Supervision: MCS EF DH MS.
  6. Writing – original draft: PR-L MCS.
  7. Writing – review & editing: EF DH MS.

References

  1. 1. Socransky SS, Haffajee AD. Dental biofilms: difficult therapeutic targets. Periodontology 2000. 2002;28:12–55. Epub 2002/05/16. pmid:12013340
  2. 2. Marsh PD. Dental plaque as a microbial biofilm. Caries research. 2004;38(3):204–11. Epub 2004/05/22. pmid:15153690
  3. 3. Filoche S, Wong L, Sissons CH. Oral biofilms: emerging concepts in microbial ecology. Journal of dental research. 2010;89(1):8–18. Epub 2009/11/18. pmid:19918089
  4. 4. Ahn J, Yang L, Paster BJ, Ganly I, Morris L, Pei Z, et al. Oral microbiome profiles: 16S rRNA pyrosequencing and microarray assay comparison. PloS one. 2011;6(7):e22788. Epub 2011/08/11. pmid:21829515
  5. 5. Zhu Y, Dashper SG, Chen YY, Crawford S, Slakeski N, Reynolds EC. Porphyromonas gingivalis and Treponema denticola synergistic polymicrobial biofilm development. PloS one. 2013;8(8):e71727. Epub 2013/08/31. pmid:23990979
  6. 6. O'Brien-Simpson NM, Pathirana RD, Walker GD, Reynolds EC. Porphyromonas gingivalis RgpA-Kgp proteinase-adhesin complexes penetrate gingival tissue and induce proinflammatory cytokines or apoptosis in a concentration-dependent manner. Infection and immunity. 2009;77(3):1246–61. Epub 2008/12/31. pmid:19114547
  7. 7. Tribble GD, Kerr JE, Wang BY. Genetic diversity in the oral pathogen Porphyromonas gingivalis: molecular mechanisms and biological consequences. Future microbiology. 2013;8(5):607–20. Epub 2013/05/07. pmid:23642116
  8. 8. Lo AW, Seers CA, Boyce JD, Dashper SG, Slakeski N, Lissel JP, et al. Comparative transcriptomic analysis of Porphyromonas gingivalis biofilm and planktonic cells. BMC microbiology. 2009;9:18. Epub 2009/01/30. pmid:19175941
  9. 9. Liu D, Xu J, Wang Y, Chen Y, Shen X, Niu H, et al. Comparative transcriptomic analysis of Clostridium acetobutylicum biofilm and planktonic cells. Journal of biotechnology. 2016;218:1–12. Epub 2015/12/02. pmid:26621081
  10. 10. Whiteley M, Bangera MG, Bumgarner RE, Parsek MR, Teitzel GM, Lory S, et al. Gene expression in Pseudomonas aeruginosa biofilms. Nature. 2001;413(6858):860–4. Epub 2001/10/26. pmid:11677611
  11. 11. Waite RD, Papakonstantinopoulou A, Littler E, Curtis MA. Transcriptome Analysis of Pseudomonas aeruginosa Growth: Comparison of Gene Expression in Planktonic Cultures and Developing and Mature Biofilms. Journal of bacteriology. 2005;187(18):6571–6. pmid:16159792
  12. 12. McNab R, Lamont RJ. Microbial dinner-party conversations: the role of LuxS in interspecies communication. Journal of medical microbiology. 2003;52(Pt 7):541–5. Epub 2003/06/17. pmid:12808073
  13. 13. Yuan L, Hillman JD, Progulske-Fox A. Microarray Analysis of Quorum-Sensing-Regulated Genes in Porphyromonas gingivalis. Infection and immunity. 2005;73(7):4146–54. pmid:15972504
  14. 14. Hirano T, Beck DAC, Demuth DR, Hackett M, Lamont RJ. Deep Sequencing of Porphyromonas gingivalis and Comparative Transcriptome Analysis of a LuxS Mutant. Frontiers in Cellular and Infection Microbiology. 2012;2:79. pmid:22919670
  15. 15. Yamamoto R, Noiri Y, Yamaguchi M, Asahi Y, Maezono H, Ebisu S. Time Course of Gene Expression during Porphyromonas gingivalis Strain ATCC 33277 Biofilm Formation. Applied and Environmental Microbiology. 2011;77(18):6733–6. pmid:21803908
  16. 16. Smyth GK, Speed T. Normalization of cDNA microarray data. Methods (San Diego, Calif). 2003;31(4):265–73. Epub 2003/11/05.
  17. 17. Smyth GK. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Statistical applications in genetics and molecular biology. 2004;3:Article3. Epub 2006/05/02. pmid:16646809
  18. 18. Ihaka R, Gentleman R. R: A language for data analysis and graphics. Journal of Computational and Graphical Statistics. 1996;5(3):299–314.
  19. 19. Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative C(T) method. Nature protocols. 2008;3(6):1101–8. Epub 2008/06/13. pmid:18546601
  20. 20. Costerton JW, Lewandowski Z, Caldwell DE, Korber DR, Lappin-Scott HM. Microbial biofilms. Annual review of microbiology. 1995;49:711–45. Epub 1995/01/01. pmid:8561477
  21. 21. Costerton JW, Lewandowski Z, DeBeer D, Caldwell D, Korber D, James G. Biofilms, the customized microniche. Journal of bacteriology. 1994;176(8):2137–42. Epub 1994/04/01. pmid:8157581
  22. 22. Shemesh M, Tam A, Kott-Gutkowski M, Feldman M, Steinberg D. DNA-microarrays identification of Streptococcus mutans genes associated with biofilm thickness. BMC microbiology. 2008;8:236-. pmid:19114020
  23. 23. Simionato MR, Tucker CM, Kuboniwa M, Lamont G, Demuth DR, Tribble GD, et al. Porphyromonas gingivalis genes involved in community development with Streptococcus gordonii. Infection and immunity. 2006;74(11):6419–28. Epub 2006/08/23. pmid:16923784
  24. 24. VanBogelen RA, Greis KD, Blumenthal RM, Tani TH, Matthews RG. Mapping regulatory networks in microbial cells. Trends in microbiology. 1999;7(8):320–8. Epub 1999/08/04. pmid:10431205
  25. 25. Schembri MA, Kjaergaard K, Klemm P. Global gene expression in Escherichia coli biofilms. Molecular microbiology. 2003;48(1):253–67. Epub 2003/03/27. pmid:12657059
  26. 26. Beloin C, Valle J, Latour-Lambert P, Faure P, Kzreminski M, Balestrino D, et al. Global impact of mature biofilm lifestyle on Escherichia coli K-12 gene expression. Molecular microbiology. 2004;51(3):659–74. Epub 2004/01/21. pmid:14731270
  27. 27. Ellis TN, Kuehn MJ. Virulence and Immunomodulatory Roles of Bacterial Outer Membrane Vesicles. Microbiology and molecular biology reviews: MMBR. 2010;74(1):81–94. pmid:20197500
  28. 28. Kulp A, Kuehn MJ. Biological Functions and Biogenesis of Secreted Bacterial Outer Membrane Vesicles. Annual review of microbiology. 2010;64:163–84. pmid:20825345
  29. 29. Veith PD, Chen YY, Gorasia DG, Chen D, Glew MD, O'Brien-Simpson NM, et al. Porphyromonas gingivalis outer membrane vesicles exclusively contain outer membrane and periplasmic proteins and carry a cargo enriched with virulence factors. Journal of proteome research. 2014;13(5):2420–32. Epub 2014/03/14. pmid:24620993
  30. 30. Kuboniwa M, Hendrickson EL, Xia Q, Wang T, Xie H, Hackett M, et al. Proteomics of Porphyromonas gingivalis within a model oral microbial community. BMC microbiology. 2009;9:98. Epub 2009/05/21. pmid:19454014
  31. 31. Kamaguchi A, Nakayama K, Ichiyama S, Nakamura R, Watanabe T, Ohta M, et al. Effect of Porphyromonas gingivalis vesicles on coaggregation of Staphylococcus aureus to oral microorganisms. Current microbiology. 2003;47(6):485–91. Epub 2004/02/06. pmid:14756532
  32. 32. Inagaki S, Onishi S, Kuramitsu HK, Sharma A. Porphyromonas gingivalis vesicles enhance attachment, and the leucine-rich repeat BspA protein is required for invasion of epithelial cells by "Tannerella forsythia". Infection and immunity. 2006;74(9):5023–8. Epub 2006/08/24. pmid:16926393
  33. 33. Díaz PI, Kolenbrander PE. Subgingival Biofilm Communities in Health and Disease. Revista Clínica de Periodoncia, Implantología y Rehabilitación Oral. 2009;2(3):187–92. http://dx.doi.org/10.1016/S0718-5391(09)70033-3.
  34. 34. Yoshimura F, Murakami Y, Nishikawa K, Hasegawa Y, Kawaminami S. Surface components of Porphyromonas gingivalis. Journal of periodontal research. 2009;44(1):1–12. Epub 2008/11/01. pmid:18973529
  35. 35. Weinberg A, Belton CM, Park Y, Lamont RJ. Role of fimbriae in Porphyromonas gingivalis invasion of gingival epithelial cells. Infection and immunity. 1997;65(1):313–6. Epub 1997/01/01. pmid:8975930
  36. 36. Malek R, Fisher JG, Caleca A, Stinson M, van Oss CJ, Lee JY, et al. Inactivation of the Porphyromonas gingivalis fimA gene blocks periodontal damage in gnotobiotic rats. Journal of bacteriology. 1994;176(4):1052–9. Epub 1994/02/01. pmid:8106316
  37. 37. Jenkinson HF, Lamont RJ. Oral microbial communities in sickness and in health. Trends in microbiology. 2005;13(12):589–95. Epub 2005/10/11. pmid:16214341
  38. 38. Kolenbrander PE, Andersen RN, Blehert DS, Egland PG, Foster JS, Palmer RJ Jr. Communication among oral bacteria. Microbiology and molecular biology reviews: MMBR. 2002;66(3):486–505, table of contents. Epub 2002/09/05. pmid:12209001
  39. 39. Nagano K, Hasegawa Y, Abiko Y, Yoshida Y, Murakami Y, Yoshimura F. Porphyromonas gingivalis FimA fimbriae: fimbrial assembly by fimA alone in the fim gene cluster and differential antigenicity among fimA genotypes. PloS one. 2012;7(9):e43722. Epub 2012/09/13. pmid:22970139
  40. 40. Nagano K, Abiko Y, Yoshida Y, Yoshimura F. Porphyromonas gingivalis FimA fimbriae: Roles of the fim gene cluster in the fimbrial assembly and antigenic heterogeneity among fimA genotypes. Journal of Oral Biosciences. 2012;54(3):160–3. http://dx.doi.org/10.1016/j.job.2012.07.002.
  41. 41. Klemm P, Christiansen G. The fimD gene required for cell surface localization of Escherichia coli type 1 fimbriae. Molecular & general genetics: MGG. 1990;220(2):334–8. Epub 1990/01/01.
  42. 42. Minion FC, Abraham SN, Beachey EH, Goguen JD. The genetic determinant of adhesive function in type 1 fimbriae of Escherichia coli is distinct from the gene encoding the fimbrial subunit. Journal of bacteriology. 1986;165(3):1033–6. Epub 1986/03/01. pmid:2419305
  43. 43. Krogfelt KA, Klemm P. Investigation of minor components of Escherichia coli type 1 fimbriae: protein chemical and immunological aspects. Microbial pathogenesis. 1988;4(3):231–8. Epub 1988/03/01. pmid:2904111
  44. 44. Klemm P, Christiansen G. Three fim genes required for the regulation of length and mediation of adhesion of Escherichia coli type 1 fimbriae. Molecular & general genetics: MGG. 1987;208(3):439–45. Epub 1987/07/01.
  45. 45. Adams JL, McLean RJ. Impact of rpoS deletion on Escherichia coli biofilms. Appl Environ Microbiol. 1999;65(9):4285–7. Epub 1999/09/03. pmid:10473455
  46. 46. Hirano T, Beck DA, Wright CJ, Demuth DR, Hackett M, Lamont RJ. Regulon controlled by the GppX hybrid two component system in Porphyromonas gingivalis. Molecular oral microbiology. 2013;28(1):70–81. Epub 2012/12/01. pmid:23194602
  47. 47. Zhang Z, Saier MH Jr. Transposon-mediated adaptive and directed mutations and their potential evolutionary benefits. Journal of molecular microbiology and biotechnology. 2011;21(1–2):59–70. Epub 2012/01/18. pmid:22248543
  48. 48. Wheeler BS. Small RNAs, big impact: small RNA pathways in transposon control and their effect on the host stress response. Chromosome research: an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology. 2013;21(6–7):587–600. Epub 2013/11/21.
  49. 49. Arnault C, Dufournel I. Genome and stresses: reactions against aggressions, behavior of transposable elements. Genetica. 1994;93(1–3):149–60. Epub 1994/01/01. pmid:7813912
  50. 50. Hendrickson EL, Xia Q, Wang T, Lamont RJ, Hackett M. Pathway analysis for intracellular Porphyromonas gingivalis using a strain ATCC 33277 specific database. BMC microbiology. 2009;9:185. Epub 2009/09/03. pmid:19723305
  51. 51. Xia Q, Wang T, Taub F, Park Y, Capestany CA, Lamont RJ, et al. Quantitative proteomics of intracellular Porphyromonas gingivalis. Proteomics. 2007;7(23):4323–37. Epub 2007/11/06. pmid:17979175
  52. 52. Moon JH, Lee JH, Lee JY. Microarray analysis of the transcriptional responses of Porphyromonas gingivalis to polyphosphate. BMC microbiology. 2014;14:218. Epub 2014/08/26. pmid:25148905
  53. 53. Enersen M, Olsen I, Kvalheim O, Caugant DA. fimA genotypes and multilocus sequence types of Porphyromonas gingivalis from patients with periodontitis. Journal of clinical microbiology. 2008;46(1):31–42. Epub 2007/11/06. pmid:17977992
  54. 54. Burmistrz M, Dudek B, Staniec D, Rodriguez Martinez JI, Bochtler M, Potempa J, et al. Functional Analysis of Porphyromonas gingivalis W83 CRISPR-Cas Systems. Journal of bacteriology. 2015;197(16):2631–41. Epub 2015/05/28. pmid:26013482
  55. 55. Hovik H, Yu WH, Olsen I, Chen T. Comprehensive transcriptome analysis of the periodontopathogenic bacterium Porphyromonas gingivalis W83. Journal of bacteriology. 2012;194(1):100–14. Epub 2011/11/01. pmid:22037400
  56. 56. Boutrin MC, Wang C, Aruni W, Li X, Fletcher HM. Nitric oxide stress resistance in Porphyromonas gingivalis is mediated by a putative hydroxylamine reductase. Journal of bacteriology. 2012;194(6):1582–92. Epub 2012/01/17. pmid:22247513
  57. 57. Krishnan K, Duncan MJ. Role of sodium in the RprY-dependent stress response in Porphyromonas gingivalis. PloS one. 2013;8(5):e63180. Epub 2013/05/15. pmid:23671672
  58. 58. Zumft WG. Cell biology and molecular basis of denitrification. Microbiology and molecular biology reviews: MMBR. 1997;61(4):533–616. Epub 1997/12/31. pmid:9409151
  59. 59. Ye RW, Averill BA, Tiedje JM. Denitrification: production and consumption of nitric oxide. Appl Environ Microbiol. 1994;60(4):1053–8. Epub 1994/04/01. pmid:8017903
  60. 60. Zumft WG. Nitric oxide signaling and NO dependent transcriptional control in bacterial denitrification by members of the FNR-CRP regulator family. Journal of molecular microbiology and biotechnology. 2002;4(3):277–86. Epub 2002/04/05. pmid:11931559
  61. 61. Sheldon JR, Yim M-S, Saliba JH, Chung W-H, Wong K-Y, Leung KT. Role of rpoS in Escherichia coli O157:H7 Strain H32 Biofilm Development and Survival. Applied and Environmental Microbiology. 2012;78(23):8331–9. pmid:23001657
  62. 62. Gallegos MT, Schleif R, Bairoch A, Hofmann K, Ramos JL. Arac/XylS family of transcriptional regulators. Microbiology and molecular biology reviews: MMBR. 1997;61(4):393–410. Epub 1997/12/31. pmid:9409145
  63. 63. Dong YH, Zhang XF, Xu JL, Tan AT, Zhang LH. VqsM, a novel AraC-type global regulator of quorum-sensing signalling and virulence in Pseudomonas aeruginosa. Molecular microbiology. 2005;58(2):552–64. Epub 2005/10/01. pmid:16194239
  64. 64. Rajamani S, Bauer WD, Robinson JB, Farrow JM 3rd, Pesci EC, Teplitski M, et al. The vitamin riboflavin and its derivative lumichrome activate the LasR bacterial quorum-sensing receptor. Molecular plant-microbe interactions: MPMI. 2008;21(9):1184–92. Epub 2008/08/15. pmid:18700823
  65. 65. Marsili E, Baron DB, Shikhare ID, Coursolle D, Gralnick JA, Bond DR. Shewanella secretes flavins that mediate extracellular electron transfer. Proceedings of the National Academy of Sciences of the United States of America. 2008;105(10):3968–73. Epub 2008/03/05. pmid:18316736
  66. 66. Yurgel SN, Rice J, Domreis E, Lynch J, Sa N, Qamar Z, et al. Sinorhizobium meliloti flavin secretion and bacteria-host interaction: role of the bifunctional RibBA protein. Molecular plant-microbe interactions: MPMI. 2014;27(5):437–45. Epub 2014/01/11. pmid:24405035