Proteomics of Porphyromonas gingivalis within a model oral microbial community

Abstract

Background: Porphyromonas gingivalis is a periodontal pathogen that resides in a complex multispecies microbial biofilm community known as dental plaque. Confocal laser scanning microscopy showed that P. gingivalis can assemble into communities in vitro with Streptococcus gordonii and Fusobacterium nucleatum, common constituents of dental plaque. Whole cell quantitative proteomics, along with mutant construction and analysis, were conducted to investigate how P. gingivalis adapts to this three species community.

Results: 1156 P. gingivalis proteins were detected qualitatively during comparison of the three species model community with P. gingivalis incubated alone under the same conditions. Integration of spectral counting and summed signal intensity analyses of the dataset showed that 403 proteins were down-regulated and 89 proteins up-regulated. The proteomics results were inspected manually and an ontology analysis conducted using DAVID. Significant decreases were seen in proteins involved in cell shape and the formation of the cell envelope, as well as thiamine, cobalamin, and pyrimidine synthesis and DNA repair. An overall increase was seen in proteins involved in protein synthesis. HmuR, a TonB dependent outer membrane receptor, was up-regulated in the community and an hmuR deficient mutant was deficient in three species community formation, but was unimpaired in its ability to form mono- or dual-species biofilms.

Conclusion: Collectively, these results indicate that P. gingivalis can assemble into a heterotypic community with F. nucleatum and S. gordonii, and that a community lifestyle provides physiologic support for P. gingivalis. Proteins such as HmuR, that are up-regulated, can be necessary for community structure.

Figure 1

Confocal laser scanning microscopy of P. gingivalis-F. nucleatum-S. gordonii community. S. gordonii cells (red, stained with hexidium iodide) were cultured on a glass plate. FITC-labeled F. nucleatum cells (green), followed by DAPI labeled P. gingivalis cells (blue), were reacted sequentially with the S. gordonii substratum. Bacterial accumulations were examined on a Bio-Rad Radiance 2100 confocal laser scanning microscope. A series of fluorescent optical x-y sections in the z-plane to the maximum vertical extent of the accumulation were collected with Laser Sharp software. Images were digitally reconstructed with Imaris software. Image is representative of three independent experiments.

Figure 2

Pseudo M versus A plot [28,29] of the average protein abundance ratios over all replicates for the P. gingivalis-F. nucleatum-S. gordonii/P. gingivalis comparison versus total abundance as estimated by spectral counting. Color codes: red, P. gingivalis protein is over-expressed in the P. gingivalis-F. nucleatum-S. gordonii community relative to P. gingivalis alone; green, P. gingivalis protein is under-expressed in the community relative to P. gingivalis alone; black, no significant abundance change. Solid black lines represent a LOWESS curve fit [30] to the biological replicates of P. gingivalis alone, and represent the upper and lower boundaries of the experimentally observed error regions or null distributions associated with the relative abundance ratio calculations. Proteins coded as either red or green were determined to be significantly changed at the q-value [24] cut-off value of 0.01. Thus, the G-test predictions [56] were in good agreement with the curve fitting procedure. Details regarding hypothesis testing procedures can be found in Methods and in the explanatory notes to the data tables [see Additional File 1].

Figure 3

Genomic representation of the P. gingivalis proteome, showing changes in relative abundance for the P. gingivalis-F. nucleatum-S. gordonii/P. gingivalis comparison by spectral counting. Each dot represents a PGN ORF number in the order followed by the ATCC 33277 strain annotation. Color codes: red, over-expression in the P. gingivalis-F. nucleatum-S. gordonii community relative to P. gingivalis alone; green, under-expression in the community relative to P. gingivalis alone; yellow, protein was detected qualitatively, but did not change in abundance; gray, proteins that were qualitative non-detects; gaps indicate ORFs that were not common to both the ATCC 33277 and W83 annotations according to a master cross-reference compiled by LANL (G. Xie, personal communication).

Figure 4

Thiamine biosynthetic pathway, showing protein abundance changes for the P. gingivalis-F. nucleatum-S. gordonii/P. gingivalis comparison. Proteins catalyzing each step in the pathway are shown by their P. gingivalis ATCC 33277 gene designation (PGN number) and protein name, where applicable. Green downward arrows indicate decreased abundance in the three species community. Yellow squares indicate no statistically significant abundance change. Empty squares indicate that the protein was not detected in the proteomic analysis. Thiamine diphosphate is shown in bold.

Figure 5

Pyrimidine biosynthetic pathway, showing protein abundance changes for the P. gingivalis-F. nucleatum-S. gordonii/P. gingivalis comparison. The protein names follow the same conventions as in Fig. 4. Green downward arrows indicate decreased abundance in the three species community. Red upward arrows indicate increased abundance. Yellow squares indicate no statistically significant abundance change. Empty squares indicate that the protein was not detected in the proteomic analysis. RNA and DNA are shown in bold.

Figure 6

Purine biosynthetic pathway, showing protein abundance changes for the P. gingivalis-F. nucleatum-S. gordonii/P. gingivalis comparison. The protein names and arrows/squares follow the same conventions as in Fig. 5. RNA and DNA are shown in bold. GAR: 5-Phosphoribosyl glycinamide; FGAM: 5-phosphoribosyl-N-formylglycineamidine; FGAR: 1-(5'-Phosphoribosyl)-N-formylglycinamide; AICAR: 5'-phosphoribosyl-4-(N-succinocarboxamide)-5-aminoimidazole; AIR: 1-(5'-Phophoribosyl)-5-aminoimidazole; CAIR: 5'P-Ribosyl-4-carboxy-5-aminoimidazole; SAICAR: 5'P-Ribosyl-4-(N-succinocarboximide)-5-aminoimidazole; FAICAR: 1 (5'-Phosphoribosyl)-5-formamido-4-imidazole carboxamide.

Figure 7

HmuR mutant of P. gingivalis is deficient in community accumulation. A) Confocal microscopy showing x-y and x-z projections of communities of S. gordonii (red), F. nucleatum (green) and P. gingivalis (blue) wild type (WT) or ΔhmuR mutant strains. Representative image from three independent experiments. B) Confocal microscopy showing x-y and x-z projections of single species P. gingivalis WT or ΔhmuR mutant accumulations. Representative image from three independent experiments. C) Biovolume analysis of P. gingivalis WT or ΔhmuR mutant accumulation in the P. gingivalis-F. nucleatum-S. gordonii communities shown in A. D) Biovolume analysis of P. gingivalis WT or ΔhmuR single species accumulations shown in B. E) Biomass of P. gingivalis WT or ΔhmuR single species accumulations measured by crystal violet staining and release. F) Biovolume analysis of P. gingivalis WT or ΔhmuR accumulation in two species P. gingivalis-S. gordonii communities. G) Biomass of P. gingivalis WT or ΔhmuR two species accumulation with F. nucleatum measured with P. gingivalis antibodies. ** denotes p < 0.01 (n = 3) compared to WT.