Detection of Francisella tularensis using recombinant reporter fusion proteins

Francisella tularensis causes tularemia in animals and humans. Although rare, the potential severity of human disease and the possibility of misuse as a biological agent of terrorism or warfare necessitates rapid and accurate diagnosis. Diagnostic laboratory methods commonly depend on culture, serology and molecular genetic approaches (e.g., polymerase chain reaction, PCR). All of these have limitations and require infrastructure which is frequently limited in non-traditional laboratory environments such as military field operation scenarios.

In this work we employed a F. tularensis-specific single-chain variable fragment antibody (scFv) to develop non-PCR identification methods for the sensitive and rapid detection of F. tularensis cells. For this, synthetic genes of a F. tularensis-specific scFv and a green fluorescent protein were fused by PCR-amplification and cloned into the expression vector pASG-105 harboring a N-terminal twin-strep-tag-epitope. The resulting reporter fusion protein (RFP) was heterologously produced in Escherichia coli and purified by affinity chromatography. Binding of the RFP to a range of Francisella spp. and other bacteria was assessed by fluorescence microscopy. Additionally, human macrophage cell lines were infected with F. tularensis to determine the intracellular binding capability of the RFP. In order to assess the impact of a relevant clinical matrix on efficient labeling of F. tularensis bacteria with RFP, blood was used.

All three subspecies of F. tularensis incubated with the RFP were successfully labeled; more distantly related bacteria were not. Importantly, most close relatives of F. tularensis could be differentiated from F. tularensis with the exception of F. hispaniensis and certain strains of F. novicida. Detection of F. tularensis in blood posed a challenge. Labeling of intracellular F. tularensis was possible using the RFP. Overall, the accurate detection of
F. tularensis by a recombinant RFP was successful and our results indicate the possibility to further develop this approach to facilitate identification of the tularemia agent in the field and laboratory.