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The Distinct Immune-Stimulatory Capacities of Porphyromonas gingivalis Strains 381 and ATCC 33277 Are Determined by the fimB Allele and Gingipain Activity.

ABSTRACT: The Porphyromonas gingivalis strain ATCC 33277 (33277) and 381 genomes are nearly identical. However, strain 33277 displays a significantly diminished capacity to stimulate host cell Toll-like receptor 2 (TLR2)-dependent signaling and interleukin-1? (IL-1?) production relative to 381, suggesting that there are strain-specific differences in one or more bacterial immune-modulatory factors. Genomic sequencing identified a single nucleotide polymorphism in the 33277 fimB allele (A?T), creating a premature stop codon in the 33277 fimB open reading frame relative to the 381 fimB allele. Gene exchange experiments established that the 33277 fimB allele reduces the immune-stimulatory capacity of this strain. Transcriptome comparisons revealed that multiple genes related to carboxy-terminal domain (CTD) family proteins, including the gingipains, were upregulated in 33277 relative to 381. A gingipain substrate degradation assay demonstrated that cell surface gingipain activity is higher in 33277, and an isogenic mutant strain deficient for the gingipains exhibited an increased ability to induce TLR2 signaling and IL-1? production. Furthermore, 33277 and 381 mutant strains lacking CTD cell surface proteins were more immune-stimulatory than the parental wild-type strains, consistent with an immune-suppressive role for the gingipains. Our data show that the combination of an intact fimB allele and limited cell surface gingipain activity in P. gingivalis 381 renders this strain more immune-stimulatory. Conversely, a defective fimB allele and high-level cell surface gingipain activity reduce the capacity of P. gingivalis 33277 to stimulate host cell innate immune responses. In summary, genomic and transcriptomic comparisons identified key virulence characteristics that confer divergent host cell innate immune responses to these highly related P. gingivalis strains.

SUBMITTER: Coats SR

PROVIDER: S-EPMC6867864 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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The Distinct Immune-Stimulatory Capacities of Porphyromonas gingivalis Strains 381 and ATCC 33277 Are Determined by the fimB Allele and Gingipain Activity.

Coats Stephen R SR Kantrong Nutthapong N To Thao T TT Jain Sumita S Genco Caroline A CA McLean Jeffrey S JS Darveau Richard P RP

Infection and immunity 20191118 12

The Porphyromonas gingivalis strain ATCC 33277 (33277) and 381 genomes are nearly identical. However, strain 33277 displays a significantly diminished capacity to stimulate host cell Toll-like receptor 2 (TLR2)-dependent signaling and interleukin-1β (IL-1β) production relative to 381, suggesting that there are strain-specific differences in one or more bacterial immune-modulatory factors. Genomic sequencing identified a single nucleotide polymorphism in the 33277 fimB allele (A→T), ...[more]

PMID: 31570556

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Project description:Porphyromonas gingivalis is a Gram-negative intracellular pathogen associated with periodontal disease. We have previously reported on whole-cell quantitative proteomic analyses to investigate the differential expression of virulence factors as the organism transitions from an extracellular to intracellular lifestyle. The original results with the invasive strain P. gingivalis ATCC 33277 were obtained using the genome sequence available at the time, strain W83 [GenBank: AE015924]. We present here a re-processed dataset using the recently published genome annotation specific for strain ATCC 33277 [GenBank: AP009380] and an analysis of differential abundance based on metabolic pathways rather than individual proteins.Qualitative detection was observed for 1266 proteins using the strain ATCC 33277 annotation for 18 hour internalized P. gingivalis within human gingival epithelial cells and controls exposed to gingival cell culture medium, an improvement of 7% over the W83 annotation. Internalized cells showed increased abundance of proteins in the energy pathway from asparagine/aspartate amino acids to ATP. The pathway producing one short chain fatty acid, propionate, showed increased abundance, while that of another, butyrate, trended towards decreased abundance. The translational machinery, including ribosomal proteins and tRNA synthetases, showed a significant increase in protein relative abundance, as did proteins responsible for transcription.Use of the ATCC 33277 specific genome annotation resulted in improved proteome coverage with respect to the number of proteins observed both qualitatively in terms of protein identifications and quantitatively in terms of the number of calculated abundance ratios. Pathway analysis showed a significant increase in overall protein synthetic and transcriptional machinery in the absence of significant growth. These results suggest that the interior of host cells provides a more energy rich environment compared to the extracellular milieu. Shifts in the production of cytotoxic fatty acids by intracellular P. gingivalis may play a role in virulence. Moreover, despite extensive genomic re-arrangements between strains W83 and 33277, there is sufficient sequence similarity at the peptide level for proteomic abundance trends to be largely accurate when using the heterologous strain annotated genome as the reference for database searching.

Project description:Background and objectivePorphyromonas 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 methodsP. 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).ResultsSEM 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.ConclusionSignificant transcriptional changes occurred in P. gingivalis when growing in a multispecies biofilm compared to planktonic state.

Dataset Information

The Distinct Immune-Stimulatory Capacities of Porphyromonas gingivalis Strains 381 and ATCC 33277 Are Determined by the fimB Allele and Gingipain Activity.

Publications

The Distinct Immune-Stimulatory Capacities of Porphyromonas gingivalis Strains 381 and ATCC 33277 Are Determined by the <i>fimB</i> Allele and Gingipain Activity.

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