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

Maj Brodmann,Sophie T Schnider,Marek Basler,Denise Monack

doi:10.1128/iai.00579-20

Abstract

ABSTRACTFrancisella 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 ClpB 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 IglC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK, OpiB1, OpiB2, and OpiB3. The results of larvae infection and secretion assay suggest that AnmK, a putative T6SS component with unknown function, interferes with OpiA-mediated toxicity but not with general T6SS 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.

Highlights

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
We demonstrate that Francisella T6SS can be activated in vitro by polyethylene glycol (PEG) in a PmrA-independent manner and use this to show that AnmK affects OpiA-mediated killing of G. mellonella larvae without altering T6SS activity or IglC secretion
In order to characterize G. mellonella larvae as an in vivo infection model for Francisella, we first tested if F. tularensis subsp. novicida establishes infection in a T6SS-dependent

Summary

Introduction

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. We use Galleria mellonella larvae to analyze the role of the Francisella T6SS and its corresponding effectors in F. tularensis subsp. 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 ClpB is dispensable. PEG-activated IglC secretion is dependent on T6SS structural component PdpB but independent of putative effectors PdpC, PdpD, AnmK, OpiB1, OpiB2, and OpiB3. Novicida encodes one FPI, while the more virulent subspecies F. tularensis subsp. Novicida, six secreted T6SS effectors were identified as follows: PdpC, PdpD, OpiA, and OpiB1, OpiB2, and OpiB3 (OpiB1–3) [14]. The FPI encodes additional components PdpE and AnmK, which are dispensable for T6SS assembly and dynamics, and may be putative effectors [5]. The corresponding deletion mutants were indistinguishable from the parental strains in various infection models [5, 15, 18, 19]

Methods

Results

Conclusion