Quantitative proteomics of intracellular Porphyromonas gingivalis

Abstract

Whole-cell quantitative proteomic analyses were conducted to investigate the change from an extracellular to intracellular lifestyle for Porphyromonas gingivalis, a Gram-negative intracellular pathogen associated with periodontal disease. Global protein abundance data for P. gingivalis strain ATCC 33277 internalized for 18 h within human gingival epithelial cells and controls exposed to gingival cell culture medium were obtained at sufficient coverage to provide strong evidence that these changes are profound. A total of 385 proteins were overexpressed in internalized P. gingivalis relative to controls; 240 proteins were shown to be underexpressed. This represented in total about 28% of the protein encoding ORFs annotated for this organism, and slightly less than half of the proteins that were observed experimentally. Production of several proteases, including the classical virulence factors RgpA, RgpB, and Kgp, was decreased. A separate validation study was carried out in which a 16-fold dilution of the P. gingivalis proteome was compared to the undiluted sample in order to assess the quantitative false negative rate (all ratios truly alternative). Truly null (no change) abundance ratios from technical replicates were used to assess the rate of quantitative false positives over the entire proteome. A global comparison between the direction of abundance change observed and previously published bioinformatic gene pair predictions for P. gingivalis will assist with future studies of P. gingivalis gene regulation and operon prediction.

Figure 1

Genomic representation of the proteome, each circle represents an ORF in the order it is encoded in the genome. TIGR ORF PG0001 in the upper left, PG2227 second from the left, end of the bottom row. The last ORF is P13793, the FMA protein specific to strain ATCC 33277 [41]. Colored circles show the consensus result (CS) for the combined proteome analyses of two biological replicates using two calculation methods, see supplementary Table S1. Green indicates over-expression in PG_PP1, the internalized phenotype; red, under-expression; yellow, protein was identified qualitatively, but without statistically significant abundance level change; white, no protein was detected qualitatively from this locus.

Figure 2

Scatter plots of protein abundance ratio versus total counts for technical replicates, log₂ of spectral count ratios versus log₂ of the total spectral counts from both technical replicates for nine biological samples. The two solid curves shown above and below the zero axis in each plot are the LOWESS smoothing curves [33] fit to the upper and lower boundaries of the random scatter about an abundance change of zero. The LOWESS curves shown for PG_PPC1 and PG_PPC2 were used to indicate the regions of random error in Fig. 3. PG_PP1 is the internalized biological replicate used for the abundance ratio calculations along with the two external controls, PG_PPC1 and PG_PPC2 (see text). The remaining plots are from nutrient limitation studies of the methanogenic Archaeon Methanococcus maripaludis. These plots serve as a useful graphical overview of the quantitative reproducibility of protein abundance ratios determined by spectral counting under the experimental conditions described. The similarity of these curves for different organisms under different conditions suggests that the region of random error within the upper and lower boundaries is determined largely by instrumental conditions.

Figure 3

Scatter plots of protein abundance ratio versus total counts, biological replicates; (A), Log₂ of PG_PP1/PG_PPC1 spectral count ratios vs. log₂ summed protein level spectral counts; (B), the second biological replicate, PG_PP1/PG_PPC2. The solid curves shown are the LOWESS smoothing curves of the upper and lower boundary of the log₂ ratios of PG_PPC1 run1/run2 and and PG_PPC2 run1/run2 technical replicates shown in Fig. 2.

Figure 4

Reproducibility of biological replicates, extracellular P. gingivalis; proteins found in two extracellular controls were used to generate the plots of PG_PPC1 total spectral counts versus PG_PPC2 total spectral counts for each ORF. (A), The Pearson correlation coefficient was 0.91 on a linear scale. (B), The same data as a log₂ transformation. A total of 963 proteins are plotted.

Figure 5

Spectral count (A, B) and signal intensity (C, D) correlations for two biological replicates, PG_PP1 and PG_PP2, of intracellular P. gingivalis. The Pearson correlation coefficient was 0.94 (A) for spectral counts plotted on a linear scale; on a log₂ scale (B) it was 0.88. For the signal intensity measurements the results were about the same, 0.95 on a linear scale (C), and 0.87 on a log₁₀ scale (D). A total of 649 proteins are plotted, representing the most abundant proteins detected and quantified in both replicates.