Type VI Secretion System and Its Effectors PdpC, PdpD, and OpiA Contribute to Francisella Virulence in Galleria mellonella Larvae

Francisella tularensis causes the deadly zoonotic disease tularemia in humans and is able to infect a broad range of organisms including arthropods, which are thought to play a major role in Francisella transmission. However, while mammalian in vitro and in vivo infection models are widely used to investigate Francisella pathogenicity, a detailed characterization of the major Francisella virulence factor, a noncanonical type VI secretion system (T6SS), in an arthropod in vivo infection model is missing. Here, we use Galleria mellonella larvae to analyze the role of the Francisella T6SS and its corresponding effectors in F. tularensis subsp. novicida virulence. We report that G. mellonella larvae killing depends on the functional T6SS and infectious dose. In contrast to other mammalian in vivo infection models, even one of the T6SS effectors PdpC, PdpD, or OpiA is sufficient to kill G. mellonella larvae, while sheath recycling by CIpB is dispensable. We further demonstrate that treatment by polyethylene glycol (PEG) activates Francisella T6SS in liquid culture and that this is independent of the response regulator PmrA. PEG-activated IgIC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK, OpiB(1), OpiB(2), and OpiB(3). The results of larvae infection and secretion assay suggest that AnmK, a putative T6S5 component with unknown function, interferes with OpiA-mediated toxicity but not with general 76SS activity. We establish that the easy-to-use G. mellonella larvae infection model provides new insights into the function of T6SS and pathogenesis of Francisella.