Gene expression of Porphyromonas gingivalis ATCC 33277 when growing in an in vitro multispecies biofilm

Abstract

Background and objective: Porphyromonas gingivalis, an oral microorganism residing in the subgingival biofilm, may exert diverse pathogenicity depending on the presence of specific virulence factors, but its gene expression has not been completely established. This investigation aims to compare the transcriptomic profile of this pathogen when growing within an in vitro multispecies biofilm or in a planktonic state.

Materials and methods: P. gingivalis ATCC 33277 was grown in anaerobiosis within multi-well culture plates at 37°C under two conditions: (a) planktonic samples (no hydroxyapatite discs) or (b) within a multispecies-biofilm containing Streptococcus oralis, Actinomyces naeslundii, Veillonella parvula, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans deposited on hydroxyapatite discs. Scanning Electron Microscopy (SEM) and Confocal Laser Scanning Microscopy (CLSM) combined with Fluorescence In Situ Hybridization (FISH) were used to verify the formation of the biofilm and the presence of P. gingivalis. Total RNA was extracted from both the multispecies biofilm and planktonic samples, then purified and, with the use of a microarray, its differential gene expression was analyzed. A linear model was used for determining the differentially expressed genes using a filtering criterion of two-fold change (up or down) and a significance p-value of <0.05. Differential expression was confirmed by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR).

Results: SEM verified the development of the multispecies biofilm and FISH confirmed the incorporation of P. gingivalis. The microarray demonstrated that, when growing within the multispecies biofilm, 19.1% of P. gingivalis genes were significantly and differentially expressed (165 genes were up-regulated and 200 down-regulated), compared with planktonic growth. These genes were mainly involved in functions related to the oxidative stress, cell envelope, transposons and metabolism. The results of the microarray were confirmed by RT-qPCR.

Conclusion: Significant transcriptional changes occurred in P. gingivalis when growing in a multispecies biofilm compared to planktonic state.