Screen for fitness and virulence factors of Francisella sp. strain W12-1067 using amoebae.

Francisella tularensis is the causative agent of the human disease referred to as tularemia. Other Francisella species are known but less is understood about their virulence factors. The role of environmental amoebae in the life-cycle of Francisella is still under discussion. Francisella sp. strain W12-1067 (F-W12) is an environmental Francisella isolate recently identified in Germany which is negative for the Francisella pathogenicity island, but exhibits a putative alternative type VI secretion system. Putative virulence factors have been identified in silico in the genome of F-W12. In this work, we established a "scatter screen", used earlier for pathogenic Legionella, to verify experimentally and identify candidate fitness factors using a transposon mutant bank of F-W12 and Acanthamoeba lenticulata as host organism. In these experiments, we identified 79 scatter clones (amoeba sensitive), which were further analyzed by an infection assay identifying 9 known virulence factors, but also candidate fitness factors of F-W12 not yet described as fitness factors in Francisella. The majority of the identified genes encoded proteins involved in the synthesis or maintenance of the cell envelope (LPS, outer membrane, capsule) or in the metabolism (glycolysis, gluconeogenesis, pentose phosphate pathway). Further C-flux analysis of the Tn5 glucokinase mutant strain revealed that the identified gene indeed encodes the sole active glucokinase in F-W12. In conclusion, candidate fitness factors of the new Francisella species F-W12 were identified using the scatter screen method which might also be usable for other Francisella species.