Abstract
P. gingivalis (Pg), a causative agent of chronic generalized periodontitis, has been implicated in promoting cardiovascular disease. Expression of lipoprotein gene PG0717 of Pg strain W83 was found to be transiently upregulated during invasion of human coronary artery endothelial cells (HCAEC), suggesting this protein may be involved in virulence. We characterized the virulence phenotype of a PG0717 deletion mutant of pg W83. There were no differences in the ability of W83Δ717 to adhere and invade HCAEC. However, the increased proportion of internalized W83 at 24 hours post-inoculation was not observed with W83∆717. Deletion of PG0717 also impaired the ability of W83 to usurp the autophagic pathway in HCAEC and to induce autophagy in Saos-2 sarcoma cells. HCAEC infected with W83Δ717 also secreted significantly greater amounts of MCP-1, IL-8, IL-6, GM-CSF, and soluble ICAM-1, VCAM-1, and E-selectin when compared to W83. Further characterization of W83Δ717 revealed that neither capsule nor lipid A structure was affected by deletion of PG0717. Interestingly, the activity of both arginine (Rgp) and lysine (Kgp) gingipains was reduced in whole-cell extracts and culture supernatant of W83Δ717. RT-PCR revealed a corresponding decrease in transcription of rgpB but not rgpA or kgp. Quantitative proteome studies of the two strains revealed that both RgpA and RgpB, along with putative virulence factors peptidylarginine deiminase and Clp protease were significantly decreased in the W83Δ717. Our results suggest that PG0717 has pleiotropic effects on W83 that affect microbial induced manipulation of host responses important for microbial clearance and infection control.
Highlights
The Gram negative anaerobic bacterium Porphyromonas gingivalis is a predominant periodontal pathogen that has been implicated in cardiovascular disease [1,2,3]
In order to adjust for any variations in the ability of W83 and W83Δ717 to persist in human coronary artery endothelial cells (HCAEC), we normalized the number of internalized bacteria at 6 and 24 hours PI by dividing the colony forming unit (CFU) of bacteria enumerated at these time points by the CFU of 2.5 hour PI cultures, which we designated as time zero (Figure 1)
These features are largely executed through well characterized virulence factors such as cysteine proteases, fimbriae, lipopolysaccharide, capsule, and hemagglutinins that exert their effects through direct interactions with the host
Summary
The Gram negative anaerobic bacterium Porphyromonas gingivalis is a predominant periodontal pathogen that has been implicated in cardiovascular disease [1,2,3]. Several comparative genomic approaches have been used to identify novel virulence genes of P. gingivalis [4,6,7]. These studies have identified multiple insertion sequences, hypothetical genes, and functionally assigned genes in the pathogenic W83 strain that are altered or missing in the genome of the less virulent strain 33277 [7,8]. P. gingivalis has several two-component sensor histidine kinase systems, which have been shown to enhance virulence by regulating the processing or expression of various virulence factors including major fimbriae [10], biofilm production [11], and the maturation and proper localization of gingipains [12]. We hypothesized that PG0717 may modulate the virulence of W83 through a similar mechanism, namely, regulation of virulence factor expression or processing
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