Functional Analysis of a LuxS Regulated Protein

Objectives: Our previous microarray experiments demonstrated that one gene, PG1102, among others, was upregulated in a luxS mutant versus the wild type strain W83. This hypothetical protein has no apparent homology to other proteins. In order to perform functional analysis on this gene, a mutant, designated YB1971, was constructed in W83. The phenotype of the mutant versus the WT strain was then determined. Methods: Real time PCR was done to confirm the microarray results. To quantitate cell adhesion and invasion, a standard antibiotic protection assay was performed using KB cells, HCAECs (human coronary artery endothelial cells), and GECs (gingival epithelial cells). To test for virulence in an abscess model, a competition assay was done by injecting a 1:1 ratio of W83 and YB1971 into BALB/c mice, followed by collecting samples each day for 5 days to determine the percentage survival for each strain. A second virulence assay was also conducted to determine the death rate of mice resulting from subcutaneous injection of the mutant versus wild type. Results: Real time PCR experiments confirmed Pg1102 was expressed 2 fold higher in the luxS knock out strain. Adhesion and invasion assays did not reveal any differences when KB cells were used; however W83 showed significantly higher adhesion than did YB1971 to HCAECs and GECs. The mouse abscess models showed no statistical difference between W83 and YB1971 in terms of survival. Conclusions: Mutation of Pg1102 caused a reduction in adhesion to HCAECs and GECs, indicating that the Pg1102 gene product is directly or indirectly involved in adherence to certain human cells. Since the mouse abscess model used may abrogate a need for adherence, in retrospect, it is not surprising that no difference in virulence was detected using this assay. (supported by NIDCR DE007496).