Microarray analysis of the transcriptional responses of Porphyromonas gingivalis to polyphosphate

Abstract

Background: Polyphosphate (polyP) has bactericidal activity against a gram-negative periodontopathogen Porphyromonas gingivalis, a black-pigmented gram-negative anaerobic rod. However, current knowledge about the mode of action of polyP against P. gingivalis is incomplete. To elucidate the mechanisms of antibacterial action of polyP against P. gingivalis, we performed the full-genome gene expression microarrays, and gene ontology (GO) and protein-protein interaction network analysis of differentially expressed genes (DEGs).

Results: We successfully identified 349 up-regulated genes and 357 down-regulated genes (>1.5-fold, P < 0.05) in P. gingivalis W83 treated with polyP75 (sodium polyphosphate, Na(n+2)P(n)O3(n+1); n = 75). Real-time PCR confirmed the up- and down-regulation of some selected genes. GO analysis of the DEGs identified distinct biological themes. Using 202 DEGs belonging to the biological themes, we generated the protein-protein interaction network based on a database of known and predicted protein interactions. The network analysis identified biological meaningful clusters related to hemin acquisition, energy metabolism, cell envelope and cell division, ribosomal proteins, and transposon function.

Conclusions: polyP probably exerts its antibacterial effect through inhibition of hemin acquisition by the bacterium, resulting in severe perturbation of energy metabolism, cell envelope biosynthesis and cell division, and elevated transposition. Further studies will be needed to elucidate the exact mechanism by which polyP induces up-regulation of the genes related to ribosomal proteins. Our results will shed new light on the study of the antibacterial mechanism of polyP against other related bacteria belonging to the black-pigmented Bacteroides species.

Figure 1

Differential gene expression inP. gingivalisW83 by polyP75 treatment. Differentially expressed genes with 1.5 fold change and P-value < 0.05 were plotted. X-axis presents fold difference between log₂ expression of polyP75 treatment and no treatment, and y-axis shows the –log₁₀P -value. Up-regulated genes (over-expressed in polyP75 treatment) were represented as red color and down-regulated genes were colored in blue.

Figure 2

Comparison of transcription measurements by microarray and qRT-PCR. The relative transcription levels for 10 genes are listed in Table 6. The qRT-PCR log₂ values were plotted against the microarray data log₂ values. The correlation coefficient (r) for comparison of the two datasets is 0. 92.

Figure 3

Protein-protein interaction network of differentially expressed functional genes. The network was constructed based on the STRING database. Nodes (symbolized as circles and square) and edges (linking lines) represent DEGs and interactions among DEGs, respectively. Up-regulated genes were represented as a circular shape and down-regulated genes were presented as a square shape. Node color represents the functional annotation of each gene. By applying MCODE clustering algorithm, 5 clusters with the score greater than 3 were obtained.