The core genome of the anaerobic oral pathogenic bacterium Porphyromonas gingivalis

Abstract

Background: The Gram negative anaerobic bacterium Porphyromonas gingivalis has long been recognized as a causative agent of periodontitis. Periodontitis is a chronic infectious disease of the tooth supporting tissues eventually leading to tooth-loss. Capsular polysaccharide (CPS) of P. gingivalis has been shown to be an important virulence determinant. Seven capsular serotypes have been described. Here, we used micro-array based comparative genomic hybridization analysis (CGH) to analyze a representative of each of the capsular serotypes and a non-encapsulated strain against the highly virulent and sequenced W83 strain. We defined absent calls using Arabidopsis thaliana negative control probes, with the aim to distinguish between aberrations due to mutations and gene gain/loss.

Results: Our analyses allowed us to call aberrant genes, absent genes and divergent regions in each of the test strains. A conserved core P. gingivalis genome was described, which consists of 80% of the analyzed genes from the sequenced W83 strain. The percentage of aberrant genes between the test strains and control strain W83 was 8.2% to 13.7%. Among the aberrant genes many CPS biosynthesis genes were found. Most other virulence related genes could be found in the conserved core genome. Comparing highly virulent strains with less virulent strains indicates that hmuS, a putative CobN/Mg chelatase involved in heme uptake, may be a more relevant virulence determinant than previously expected. Furthermore, the description of the 39 W83-specific genes could give more insight in why this strain is more virulent than others.

Conclusion: Analyses of the genetic content of the P. gingivalis capsular serotypes allowed the description of a P. gingivalis core genome. The high resolution data from three types of analysis of triplicate hybridization experiments may explain the higher divergence between P. gingivalis strains than previously recognized.

Figure 1

Hybridization signals of P. gingivalis strains - dead probes. A. The total intensity distribution of probe signals of W83 DNA hybridized to the W83 array. The density peak around 7.5 contains the negative controls (empty spots and A. thaliana probes). The peak around 12 should contain all present genes in strain W83. B Probe signal intensities of each P. gingivalis test strain are represented in light blue dots; medium blue dots, slightly below that, symbolize A. thaliana negative control genes. Dark blue dots represent P gingivalis probes, which show the same low intensity as the negative control probes. These 22 probes are called dead probes as they do not give any significant hybridization signal.

Figure 2

P. gingivalis core genome. Pie diagram representing all probes included in the results divided into pieces representing the conserved core genome, aberrant core genome and the variable genes. The percentages show the proportions of the total of functional probes. 80% of the strain W83 genes is present and conserved among the test strains. 6% of the W83 genes is present but aberrant and 13% of the genes is absent in at least one of the test strains. Two probes with very low signals were found as non-aberrant but absent.

Figure 3

Virulence associated genes in the conserved core genome of P. gingivalis. A. 153 potential virulence genes from the genome annotation of W83 combined with the conserved core genome of P. gingivalis [29]. B 39 genes known to be up-regulated during infection combined with the conserved core genome of P. gingivalis [46,47]. The number in the overlapping part of the circles is the number of potential virulence associated genes that was found in the conserved core genome of P. gingivalis.

Figure 4

CPS biosynthesis locus diversity. A. Heat map showing presence (green), aberrance (orange) and absence (red) of each gene in each test strain, showing the variation within the CPS biosynthesis locus. The CPS locus of the serotype K7 strain 34-4 shows the highest similarity with the K1 serotype strain W83. B. For each probe in the CPS biosynthesis locus and for each test strain a log-ratio value compared to strain W83 is depicted by a data point, supporting the heat map results as shown in figure 4A.

Figure 5

Highly variable regions of P. gingivalis. Breakpoint analysis of test strains describing potential lacking genomic regions as positioned on the W83 genome sequence. Black lines depict breakpoint data. As long as the line is flat there is low variability of the test strain compared to W83. Dips in the line indicate variability. Blue lines/rectangles below depict potential absent regions. At the top the probe positions are given as described in the W83 genome [29]. The numbers at the bottom label the 10 highly variable regions in each strain which are explained in the text. CRISPR represents a region of interest with CRISPR associated genes as described in the text.