ABSTRACT
Microarrays were used to identify genes of Porphyromonas gingivalis W83 differentially expressed during invasion of primary human coronary artery endothelial cells. Analyses of microarray images indicated that 62 genes were differentially regulated. Of these, 11 genes were up-regulated and 51 were down-regulated. The differential expression of 16 selected genes was confirmed by real-time PCR.
Several epidemiological studies have led to the hypothesis of an infection theory of atherosclerosis (31, 39). An accumulation of evidence suggests that periodontopathogenic bacterial species, among others, may be involved in cardiovascular diseases (1, 8, 22, 29, 30, 32). In addition to these data, there is also biological evidence for such a relationship. For example, periodontal pathogens can be detected in atheromas dissected from vascular tissues (17), and Porphyromonas gingivalis has been shown to accelerate atherosclerosis in apolipoprotein E-deficient mice (16, 26, 28). In addition, several studies have demonstrated that P. gingivalis internalizes within arterial endothelial cells and smooth muscle cells in vitro (7, 10) and can also induce foam cell formation and secretion of monocyte chemoattractants, both important phenomena in atherosclerotic lesion formation (25). Most recently, a direct correlation between the presence of P. gingivalis in periodontal plaque and the progression of atherosclerosis (9), as well as the isolation of viable P. gingivalis from atherosclerotic tissue (24), has been reported. P. gingivalis is known to have a direct route to the circulatory system in periodontitis patients (3, 38). Therefore, invasion of coronary artery cells by P. gingivalis may be involved in atherosclerosis.
To identify genes differentially expressed during the course of P. gingivalis invasion of human coronary artery endothelial cells (HCAEC), T-75 flasks with 90% confluence of HCAEC were infected with P. gingivalis strain W83 for 2.5 h as described previously (11, 27). Total RNA was isolated from both 10 ml of broth culture (prior to invasion) and internalized bacteria by using 10 ml of Trizol reagent followed by RNA isolation as described by the manufacturer (Invitrogen Life Technologies, Carlsbad, CA). All RNA samples were DNase treated and purified using the RNeasy kit (QIAGEN Inc., Valencia, CA). To separate bacterial total mRNA from poly(A) mRNA, cellular and internalized bacterial RNAs were also treated with the Oligotex kit (QIAGEN) according to the manufacturer's instructions and the supernatant (invasion RNA) was again treated with Trizol LS reagent (Invitrogen Life Technologies). Reverse transcription (RT) and microarray reactions were performed either with 2.0 μg of total bacterial RNA (control) or with invasion RNA (200 μg of total RNA containing 2.0 μg of bacterial RNA), collected from one T-75 flask of invaded HCAEC (per microarray slide), as previously described (14, 37). Details of the microarrays can be found at http://www.tigr.org . The resulting images were analyzed by TIGR Spotfinder 1.0 and TIGR Multiple Experiment Viewer software 1.2 (The Institute for Genomic Research [TIGR] [http://www.tigr.org ]). The generated files were imported into Microsoft Excel (Microsoft Corporation, Redmond, WA) for subsequent analyses. The results represent the common findings of three independent biological replicate arrays performed with three different RNA samples. Genes were identified as differentially expressed if there was a 2.0-fold difference in their average expression values. To confirm the microarray data, 16 different genes were subjected to RT-PCR (Table 1) using an iCycler Thermal Cycler and iQ SYBR green supermix according to the manufacturer's instructions (Bio-Rad Laboratories). DNA fragments of each gene were used as internal controls and standard curves. Subsequent data normalization and analysis were performed by using the iCycler and Microsoft Excel softwares. All locus numbers and operon predictions were obtained from the website for TIGR.
Analysis of microarray images showed that a total of 63 genes were differentially regulated (Fig. 1). Of these genes, 11 were up-regulated (Table 2) and 52 were down-regulated (Table 3) during invasion of HCAEC, compared with those growing in broth culture. Among the up-regulated genes are several that may be involved in intracellular trafficking and/or interactions with autophagosomal vesicles or other virulence functions. Examples are as follows. (i) PG1682 encodes a glycosyl transferase, and PG1683 encodes a conserved hypothetical protein which has homology to α-amylases. These enzymes have been suggested to be involved in the attachment of P. gingivalis to epithelial cells (glycosyl transferase) (4) and coaggregation of P. gingivalis with other oral bacterial species (hypothetical protein) (15, 23). Genes PG1682 and PG1683 might also be involved in the coaggregation of P. gingivalis with cell membranes (autophagosomes). (ii) PG0280 encodes a putative ABC transporter permease protein that is organized as a channeling pore complex through the membrane (34). The ABC transporter superfamily is responsible for the translocation of a wide variety of substances into or out of cells. However, the substrate of this particular ABC transporter has not yet been described. (iii) PG0092 encodes a putative transporter of unknown substrate which belongs to the HlyD secretion protein family (34). The HlyD family of secretion proteins is involved in the activation and release of hemolysins in Escherichia coli (19, 41, 42) as well as in the secretion of toxins in other bacterial species (18, 21). Perhaps related, PG1286 (ftn) encodes a ferritin and PG1172 encodes a putative iron-sulfur cluster binding protein, a prosthetic group present in a diverse set of proteins involved in environmental sensing, gene regulation, and substrate activation. (iv) PG1896 (metk) encodes an S-adenosylmethionine synthase, the product of which is S-adenosylmethionine (SAM), a major methyl donor in metabolism. SAM is an essential metabolite in yeasts (5), and the lack of SAM in E. coli cells has been shown to result in a cell division defect (35). In previous work in our laboratory, Dorn et al. (12) observed profiles of P. gingivalis dividing inside late autophagosomes. Therefore, PG1896 could be involved in intracellular replication of P. gingivalis. However, its up-regulation may be due to other metabolic processes necessary for the survival of P. gingivalis inside of HCAEC.
In contrast to genes up-regulated during invasion assays, a larger number of genes (52 of 63) were down-regulated (Table 3). Several of the down-regulated genes (12 of 52) are hypothetical proteins; however, a substantial number of down-regulated genes (21 of 52) are likely involved in protein synthesis, transcription, and energy metabolism. This reduced level of expression may indicate a reduced intracellular bacterial growth rate and/or that intracellular P. gingivalis organisms at this time point have limited but more specific metabolic activity when compared with laboratory-grown late-log-phase bacteria.
This is the first report of a global genomic expression profile of intracellular P. gingivalis during invasion of endothelial host cells. The results presented here may provide new insights at the molecular level of P. gingivalis gene expression once inside human cells. It is expected that the gene expression profiles will differ at earlier or later times during invasion of HCAEC cultures. Similarly, P. gingivalis genetic expression profiles would be expected to differ in different cell lines, since P. gingivalis traffics intracellularly differently in different cell types (2, 6, 12, 13, 20, 33, 36, 40). We are currently studying these genes and their products to better understand the invasive mechanism of P. gingivalis.
Distribution of differentially expressed genes grouped by functional classification according to the TIGR P. gingivalis genome database. Numbers above the bars indicate the number of genes differentially expressed in each functional group.
Comparison of RT-PCR and microarray expression values of selected genes
P. gingivalis genes up-regulated during invasion of human coronary artery endothelial cells
P. gingivalis genes down-regulated during invasion of human coronary artery endothelial cells
ACKNOWLEDGMENTS
This work was supported by grant NIH DE013545.
We thank The Institute for Genomic Research (Rockville, MD) for kindly providing the microarray slides supported by NIH DE10510 and Henry Baker and Cecilia Lopez for advice and assistance with array screening.
FOOTNOTES
- Received 26 October 2004.
- Returned for modification 18 November 2004.
- Accepted 20 April 2005.
- Copyright © 2005 American Society for Microbiology
