Cyclic Di-GMP Stimulates Biofilm Formation and Inhibits Virulence of Francisella novicida

Abstract

Francisella tularensisis a Gram-negative bacterium that is highly virulent in humans, causing the disease tularemia.F. novicidais closely related toF. tularensisand exhibits high virulence in mice, but it is avirulent in healthy humans. AnF. novicida-specific gene cluster (FTN0451 to FTN0456) encodes two proteins with diguanylate cyclase (DGC) and phosphodiesterase (PDE) domains that modulate the synthesis and degradation of cyclic di-GMP (cdGMP). No DGC- or PDE-encoding protein genes are present in theF. tularensisgenome.F. novicidastrains lacking either the two DGC/PDE genes (cdgAandcdgB) or the entire gene cluster (strain KKF457) are defective for biofilm formation. In addition, expression of CdgB or a heterologous DGC in strain KKF457 stimulatedF. novicidabiofilms, even in a strain lacking the biofilm regulator QseB. Genetic evidence suggests that CdgA is predominantly a PDE, while CdgB is predominantly a DGC. TheF. novicida qseBstrain showed reducedcdgAandcdgBtranscript levels, demonstrating anF. novicidabiofilm signaling cascade that controls cdGMP levels. Interestingly, KKF457 with elevated cdGMP levels exhibited a decrease in intramacrophage replication and virulence in mice, as well as increased growth yields and biofilm formationin vitro. Microarray analyses revealed that cdGMP stimulated the transcription of a chitinase (ChiB) known to contribute to biofilm formation. Our results indicate that elevated cdGMP inF. novicidastimulates biofilm formation and inhibits virulence. We suggest that differences in human virulence betweenF. novicidaandF. tularensismay be due in part to the absence of cdGMP signaling inF. tularensis.