Porphyromonas ingivalis Pg) is recognized as a major pathogen of adult periodontitis and implicated in multiple systemic inflammatory conditions including cardiovascular disease. Pg's ability to invade a wide variety of host cell types and employ adaptive mechanisms to subvert specific host responses are key virulence factors critical to its survival and persistence. Whether forming tissue disseminating biofilm communities in the oral cavity and other sites in the body or residing in host cells as an intracellular pathogen, Pg must be able to sense and rapidly respond to changes in its environment. Regulation of gene expression by small non-coding regulatory RNA (sRNA) is an exciting and rapidly growing field in biology. This mechanism of gene regulation is now recognized as a common mechanism employed by bacteria to rapidly respond to environmental cues. However, the sRNA regulatory systems Pg uses to rapidly respond to dynamic environmental cues are as yet unknown. The interactions between sRNA and their mRNA targets are often mediated by Hfq, a sRNA chaperone conserved across many bacterial species. However, Pg is an Hfq negative bacterium. Therefore, elucidating the roles of virulence modulating sRNAs in Hfq negative bacteria such as Pg will expand our knowledge base of the regulation of virulence in pathogens that do not contain this sRNA chaperone. Using both NimbleGen microarray analysis and next generation Illumina sequencing of sRNA enriched cDNA libraries, we have generated Pg sRNA expression profiles in response to hemin availability and growth phase. These conditions were selected based on published studies that suggest that periodontal pathogenesis is initiated during mid-log phase under hemin limitation after hemin starvation. Significantly, employing invasion assays using human coronary artery endothelial cells (HCAECs), we found that one of the sRNA we have identified (sRNA W83-514) appears to be linked to Pg virulence. However, to fully understand the biological significance of sRNA mediated regulation, and completely characterize sRNA systems, requires identification of both the sRNA and its cognate mRNA targets. In pursuit of identifying mRNA targets of select Pg sRNAs, including sRNA W83-514, we propose to develop a novel unbiased screening model, using an E. coli surrogate host, capable of identifying both positively and negatively regulated mRNA targets (Aim 1A). This technology will enhance Pg studies of virulence because many of the methods developed to identify and characterize sRNA regulation either are specific to Hfq directed sRNA or very difficult/impossible to replicate in Pg. Furthermore, we propose to use mutational analysis and RT-PCR to generate quantitative measures of mRNA targets in wildtype and sRNA mutant Pg clones, under various environmental conditions, to determine if the mRNA targets identified in Aim 1A are regulated in Pg by its cognate sRNA (Aim 1B), while furthering our long-term objective of characterizing sRNA regulatory system(s) of this human pathogen.
The goal of our project is to study how the bacterium Porphyromonas gingivalis can rapidly respond to changes in its environment and control its ability to cause diseases, like gum disease and heart disease. We have developed a new way to find the genes involved in these systems, which can be also be used to study similar systems found in other important disease causing bacterial species.