Characterization of the viable but non-culturable state of Francisella tularensis
Abstract
This innovative basic research project is focused on the viable but non-culturable (VBNC) state of Francisella tularensis (Ft). Understanding the VBNC state of this potential biowarfare agent (BWA) is critical because evidence suggests that this state is important for the long-term persistence of Ft in the environment, wind-borne dispersal of this bacterium, and its ability to cause local repeated outbreaks among humans. Understanding the VBNC state of Ft would also be necessary in order to design effective elimination and detection strategies if Ft is used as a weapon of mass destruction (WMD). Our team was the first to describe the VBNC state in Ft and we possess all of the necessary skills and expertise to generate greater knowledge and understanding about it. In Task 1 we will determine the environmental factors that cause Ft to enter the VBNC state. This will be accomplished using our existing laboratory model system for VBNC Ft. We will examine factors associated with both host associated growth – growth in a mammalian host prior to it entering directly into the environment, as well as growth in the environment outside a host – the conditions that Ft encounters once it is in the environment. In Task 2 we will determine the behavior of Ft while it is in the VBNC state. This will be examined in three different microcosms: 1) natural freshwater to study the combined effects of competition, predation from predatory protozoa, and nutrient availability; 2) natural soil to study potential associations of Ft while in the VBNC state; and 3) wet straw that will naturally generate a culture with a high density of protozoa and provide the opportunity to monitor VBNC Ft behavior in a complex environment that includes interactions with protozoa, growth as free living cells, and/or cells in mixed biofilms. Ft behavior in a biofilm state will be further tested by an in vitro biofilm experiment using 40 Ft strains. In Task 3 we will determine the conditions that resuscitate Ft from the VBNC state. This will be accomplished by introducing VBNC Ft to various eukaryotic hosts and environmental conditions. In Task 4 we will determine gene expression changes that occur when Ft enters the VBNC state in order to identify specific genes that may be regulating this state. We will generate and validate these data using RNA sequencing (RNA-seq), proteomic profiling, and amplicon sequencing (amp-seq). Importantly, for all of the work that we will carry out under Tasks 1-4 we will utilize fully virulent strains from both Ft subsp. tularensis (Type A) and Ft. subsp. holarctica (Type B). We will also validate the virulence of VBNC Ft grown in many diverse experimental treatments using a mouse model (C57Bl/6). In Task 5 we will disseminate the results of this study to DTRA and others in research presentations, a final report of our findings, and peer-reviewed publications, including a review paper on Ft ecology and persistence in the environment. In Task 6 we will manage all aspects of this project, including monthly video-conferences with all of the team members, regular email communications, and face-to-face annual meetings with DTRA and team members.
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Jul 16, 2019
- Source ID
- HDTRA11810037
Entities
People
- David M Wagner
Organizations
- Defense Threat Reduction Agency
- Northern Arizona University