Environmental implications of <i>Francisella tularensis</i> biofilms
Francisella tularensis survives in one of the widest environmental ranges of any pathogen. Numerous mammals and arthropod vectors are infected by this highly virulent organism. How this zoonotic pathogen persists outside of its many hosts remains unexplored. We aimed to examine how F. tularensis interacts with environmental surfaces, and hypothesized that biofilm formation may enable survival of this organism in nature. By understanding the role these surface-attached bacterial communities play in F. tularensis ecology, we hope to gain insight into the mechanisms of environmental persistence and transmission of this pathogen.
We identify chitin as a potential non-host niche for F. tularensis in nature using genetic, microscopic, and biochemical techniques. This abundant polysaccharide supported F. tularensis biofilm formation in the absence of an exogenous carbon source. This interaction was dependent on putative chitinase enzymes which hydrolyze the glycosidic bonds that connect GlcNAc monomers. Using a genetic screen, we identified adherence factors, including FTN_0308 and FTN_0714, that promote attachment to chitin and colonization of chitin surfaces. We propose that biofilm formation on chitin surfaces in nature enables nutrient scavenging in nutrient-limiting environments allowing this pathogen to replicate and seed disease transmission.
We found that the effect of nutrient limitation on F. tularensis biofilm formation extended beyond chitin utilization. Genetic studies indicated that nutrient starvation triggers a biofilm stress response. We identified static growth and nutrient deprivation as cues for enhanced biofilm formation. Microarray expression studies identified genes highly expressed under these conditions, including F. tularensis biofilm determinants. Expression of nutrient transporters further indicated that biofilm formation promotes environmental persistence.
We finally examined statically grown F. tularensis microscopically to determine if altered morphology explained the enhanced biofilm phenotype of these cultures. We discovered a novel F. tularensis appendage conserved between subspecies and structurally homologous to the Caulobacter crescentus stalk. These structures were observed in association with surfaces during both biofilm formation and during intracellular infection. A genetic screen for mutants in stalk formation revealed that stalk biosynthetic components are essential. We predict this structure aids in environmental persistence by facilitating surface attachment and nutrient uptake. Through this collective work we define evidence that surface association via biofilm formation promotes survival during nutrient limitation.