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Differential protein expression by Porphyromonas gingivalis in response to secreted epithelial cell components.

ABSTRACT: The human oral pathogen Porphyromonas gingivalis colonizes the gingival crevice and invades gingival epithelial cells. Multidimensional capillary high-performance liquid chromatography coupled with tandem mass spectrometry and two-dimensional gel electrophoresis were used to analyze the proteome of P. gingivalis as it adapts to a set of experimental conditions designed to reflect important features of an epithelial cell environment. 1014 proteins (46% of the total theoretical proteome) were identified in four independent analyses; 479 of these proteins showed evidence of differential expression after exposure of P. gingivalis to either conditioned epithelial cell growth medium or control conditions: i.e., they were only detected under one set of conditions. Moreover, 276 genes annotated as hypothetical were found to encode expressed proteins. Among the proteins up-regulated in the presence of epithelial cell components were a homolog of the internalin proteins of Listeria monocytogenes and subunits of the ATP-dependent Clp protease complex. Insertional inactivation of clpP, encoding the Clp proteolytic subunit, resulted in approximately a 50% reduction in invasion of P. gingivalis. These results suggest that adaptation to an epithelial cell environment induces a major shift in the expressed proteome of the organism. Furthermore, ClpP, that is up-regulated in this environment, is required for optimal invasive activity of P. gingivalis.

SUBMITTER: Zhang Y

PROVIDER: S-EPMC2651681 | biostudies-other | 2005 Jan

REPOSITORIES: biostudies-other

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Differential protein expression by Porphyromonas gingivalis in response to secreted epithelial cell components.

Zhang Yi Y Wang Tiansong T Chen Weibin W Yilmaz Ozlem O Park Yoonsuk Y Jung Il-Young IY Hackett Murray M Lamont Richard J RJ

Proteomics 20050101 1

The human oral pathogen Porphyromonas gingivalis colonizes the gingival crevice and invades gingival epithelial cells. Multidimensional capillary high-performance liquid chromatography coupled with tandem mass spectrometry and two-dimensional gel electrophoresis were used to analyze the proteome of P. gingivalis as it adapts to a set of experimental conditions designed to reflect important features of an epithelial cell environment. 1014 proteins (46% of the total theoretical proteome) were iden ...[more]

PMID: 15619293

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Project description:Porphyromonas gingivalis is a pathogen in severe periodontal disease. Able to exploit an intracellular lifestyle within primary gingival epithelial cells (GECs), a reservoir of P. gingivalis can persist within the gingival epithelia. This process is facilitated by manipulation of the host cell signal transduction cascades which can impact cell cycle, cell death and cytokine responses. Using microarrays, we investigated the ability of P. gingivalis 33277 to regulate microRNA (miRNA) expression in GECs. One of several miRNAs differentially regulated by GECs in the presence of P. gingivalis was miR-203, which was upregulated 4-fold compared with uninfected controls. Differential regulation of miR-203 was confirmed by qRT-PCR. Putative targets of miR-203, suppressors of cytokine signaling (SOCS) 3 and 6, were evaluated by qRT-PCR. SOCS3 and SOCS6 mRNA levels were reduced >5-fold and >2-fold, respectively, in P. gingivalis-infected GECs compared with controls. Silencing miR-203 using a si-RNA construct reversed the inhibition of SOCS3 expression. A dual luciferase assay confirmed binding of miR-203 to the putative target binding site of SOCS3 3’ UTR. Western blot analysis demonstrated that activation of Stat3, a downstream target of SOCS, was diminished following miR-203 silencing. This study shows that induction of miRNAs by P. gingivalis can modulate important host signaling responses. MicroRNAs differentially expressed in human gingival epithelial cells in response to P. gingivalis infection were identified. P. gingivalis were reacted with primary gingival epithelial cells at an MOI of 100 for 6 hours at 37C in 5% CO2. Co-cultures were carried out in triplicate. The GECs were lysed with Trizol (Invitrogen) prior to RNA extraction and total RNA isolation. The microarray assay was performed using a service provider (LC Sciences). The assay started from 4 to 8 µg total RNA sample which was size fractionated using a YM-100 Microcon centrifugal filter (Millipore) and the small RNAs (< 300 nt) isolated were 3’-extended with a poly(A) tail using poly(A) polymerase. An oligonucleotide tag was then ligated to the poly(A) tail for later fluorescent dye staining. Hybridization was performed overnight on a µParaflo microfluidic chip using a micro-circulation pump (Atactic Technologies). The hybridization melting temperatures were balanced by chemical modifications of the detection probes. Hybridization used 100 uL 6xSSPE buffer (0.90 M NaCl, 60 mM Na2HPO4, 6 mM EDTA, pH 6.8) containing 25% formamide at 34 °C. After RNA hybridization, tag-conjugating Cy3 dyes were circulated through the microfluidic chip for dye staining. Fluorescence images were collected using a laser scanner (GenePix 4000B, Molecular Device) and digitized using Array-Pro image analysis software (Media Cybernetics). Microarray data were analyzed by first subtracting the background and then normalizing the signals using a LOWESS filter (Locally-weighted Regression). For a transcript to be listed as detectable it had a signal intensity higher than 3×(background standard deviation) and spot coefficient of variance (CV) < 0.5. CV was calculated by (standard deviation)/(signal intensity). When repeating probes are present on an array, a transcript is listed as detectable only if the signals from at least 50% of the repeating probes are above detection level. t-Test is performed between “control” and “test” sample groups. T-values are calculated for each miRNA, and p-values are computed from the theoretical t-distribution. miRNAs with p-values below a critical p-value (typically 0.01) are selected for cluster analysis. The clustering is done using hierarchical method and is performed with average linkage and Euclidean distance metric. This experiment includes a total of 6 samples which are divided in to 2 groups, each with 3 biological repeats: 3 untreated normal samples as controls and 3 infected samples as the treated condition.

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Differential protein expression by Porphyromonas gingivalis in response to secreted epithelial cell components.

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Differential protein expression by Porphyromonas gingivalis in response to secreted epithelial cell components.

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