Investigating the viable but not culturable (VBNC) state in P. gingivalis

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ABSTRACT Bacterial species have evolved multiple mechanisms to survive ecological, nutritional and chemical stresses as well as host cell defenses. Among these is to enter a viable but non-culturable (VBNC) state. As the name indicates, bacteria in the VBNC state have lost the ability to grow on routine agar, but are in fact, alive, albeit in a dormant (not spore) state. The ability of a bacterial species to enter the VBNC state and also resuscitation from this state is now recognized to be an important or even required mechanism for the survival and pathogenesis of several bacterial pathogens, especially those that are associated with chronic infections. These proposed studies are designed to genetically and metabolically characterize the VBNC state and resuscitation from the VBNC state of the oral pathogen, P. gingivalis. The goal, in addition to defining the P. gingivalis gene expression and metabolism in the VBNC state is to also identify one or more biomarkers for the VBNC state of P. gingivalis strain W83 (PgW83). In addition, knockouts of genes found to be specific for or highly expressed during the VBNC and resuscitation states will be constructed. These mutants will then be tested in both in vitro and in vivo models to determine the requirement for these genes, and thus the VBNC state, in host cell invasion and virulence in an in vivo animal model. Our hypothesis is that the existence of VBNC subpopulations with the ability to resuscitate allows P. gingivalis to survive those conditions of intracellular stress that it encounters thus allowing P. gingivalis to exist and persist in a chronic state of infection. We have designed in vitro and in vivo experiments to test this hypothesis. First, we propose to use cellular and in vitro methods to establish the VBNC and resuscitation states of PgW83. Next, we will perform RNAseq and metabolomics to identify possible biomarkers for VBNC and resuscitation states. Biomarker mutants will be constructed and tested for their abilities to enter the VBNC and resuscitate within host cells. Finally, we will treat mice with PgW83 wildtype and mutants defective in transitioning into and resuscitation from the VBNC state to determine whether these states of P. gingivalis are necessary for chronic periodontitis. These studies will provide new insights into the molecular events of P. gingivalis occurring during chronic infections such as periodontitis and identify new protein targets for future therapies.