Identification of key determinants in Porphyromonas gingivalis host-cell invasion assays.

The periodontal pathogen Porphyromonas gingivalis can invade host cells, a virulence trait which may contribute to the persistence of infection at subgingival sites. Whilst the antibiotic protection assay has been commonly employed to investigate and quantify P. gingivalis invasion, data obtained have varied widely and a thorough investigation of the factors influencing this is lacking. We investigated the role of a number of bacterial and host-cell factors and report that the growth phase of P. gingivalis, source (laboratory strain vs. clinical strain), host-cell identity (cell line vs. primary), host-cell lysis method, and host-cell passage number had no significant effect on bacterial invasion. However, incubation time, host-cell seeding density, method of quantification (viable count vs. DNA), and whether host cells were plated or in suspension, were shown to influence invasion. Also, cells isolated by rapid adhesion to fibronectin exhibited higher levels of P. gingivalis invasion, possibly as a result of increased levels of active α5β1 integrin. Interestingly, this may represent a population of cells with stem cell-like properties. This study provides important new information by identifying the most important factors that influence P. gingivalis invasion assays and may help to explain variations in the levels previously reported.