Characterization Of Adhesins And Other Virulence Factors Involved In The Interaction Of Prevotella Intermedia With Human Epithelial Cells

Periodontal disease is an oral infectious disease with a high prevalence worldwide. The oral bacterium Prevotella intermedia mediates the development of periodontal disease and other oral infections. P. intermedia is able to adhere to and invade various host cells. Although adhesins and other virulence factors are critical for bacteria to successfully adhere, invade and colonize their host, the molecular mechanisms and relative protein roles in this process remain unclear. The availability of the genomic sequence of P. intermedia 17 allowed us to apply bioinformatics, genomics and proteomic approaches to identify genes and proteins that maybe mediating in the process of the interaction between bacteria and human epitherial cells.Using bioinformatics analysis, we identified and characterized the adhesin candidates AdpD and AdpE, which are members of the leucine-rich family of proteins. Gene PI 1564 and PI 1571 were cloned into expression vector pET30a(+) and transformed into E. coli BL21(DE3) cells to generate recombinant proteins. Enzyme-linked immunosorbent assay analysis (ELISA) revealed that AdpD bound specifically to fibrinogen and AdpE bound specifically to collagen. Furthermore, Western blot showed that AdpD and AdpE localized in the outer membrane fraction of P. intermedia. Recombinant AdpD and AdpE inhibited adherence of P. intermedia to oral epithelial cells HN4. These results suggested that AdpD and AdpE play roles in P. intermedia adherence to oral epithelial cells.We used real-time quantitative reverse transcription PCR (qRT-PCR) to examine the gene expression of P. intermedia adhesins and other virulence factors upon exposure to HeLa cells. We applied relative quantitation qRT-PCR in our homemade array to 172 P. intermedia genes. Out of the 172 bacterial genes tested,31 genes were regulated by greater than two fold after P. intermedia exposure to HeLa cells for 24 h. In contrast, the transcription patterns between bacteria grown in plain HeLa medium and in conditioned medium for 24 h were not markedly different (only two genes were down-regulated). After we identified the significant regulated genes through qRT-PCR, we examined the biological function of genes involved in the interaction between P. intermedia and HeLa cells. The lectin-like precursor gene PI0137, which was up-regulated by 3.1 fold, was cloned and induced to express recombinant protein in vitro. ELISA showed that PI0137- encoded protein specifically binds to lectin. Thus, this study showed that qRT-PCR array is a rapid and effective way to screen interesting gene candidates.We prepared sets of experimental conditions designed to reflect important features of host cell environments. P. intermedia was grown in the conditioned medium of HN4 and HeLa and plain HN4 and HeLa cell medium. Two-dimensional gel electrophoresis (2-DE) with LC-MS/MS were used to analyze the proteome of P. intermedia grown in these three conditions. The 2-DE patterns of P. intermedia whole proteins and its outer membrane proteins were generally similar across three conditions. However, some proteins showed significant difference in expression in conditioned cell medium. We sampled 40 of the differentially-expressed protein spots to be analyzed by mass spectrum, and identified 15 P. intermedia outer membrane proteins from these 40 spots. Some proteins are up-regulated in the presence of HN4 and HeLa cell components, including a PI0932-encoded hemin receptor and two TPR-repeating-containing proteins, encoded by PI0779 and PI1559 respectively. These results suggest that adaptation to a host cell environment induces a shift in the expressed proteome of P. intermedia, and the regulated proteins may play important roles for the survival of P. intermedia in the host environment.