Francisella requires dynamic type VI secretion system and ClpB to deliver effectors for phagosomal escape

Abstract

Francisella tularensis is an intracellular pathogen that causes the fatal zoonotic disease tularaemia. Critical for its pathogenesis is the ability of the phagocytosed bacteria to escape into the cell cytosol. For this, the bacteria use a non-canonical type VI secretion system (T6SS) encoded on the Francisella pathogenicity island (FPI). Here we show that in F. novicida T6SS assembly initiates at the bacterial poles both in vitro and within infected macrophages. T6SS dynamics and function depends on the general purpose ClpB unfoldase, which specifically colocalizes with contracted sheaths and is required for their disassembly. T6SS assembly depends on iglF, iglG, iglI and iglJ, whereas pdpC, pdpD, pdpE and anmK are dispensable. Importantly, strains lacking pdpC and pdpD are unable to escape from phagosome, activate AIM2 inflammasome or cause disease in mice. This suggests that PdpC and PdpD are T6SS effectors involved in phagosome rupture.

The AIM2 inflammasome: Sensor of pathogens and cellular perturbations

“…Remarkably, F. novicida developed a clustered, regularly interspaced, short palindromic repeats‐CRISPR associated‐Cas (CRISPR‐Cas) system strengthening bacterial envelope and conferring resistance to host sensors AIM2 and TLR2 . Recently, components of the type VI secretion system (T6SS) PdpC and PdpD encoded on the Francisella pathogenicity island and the chaperone ClpB were shown to be essential for phagosome escape of F. tularensis . Phagosomal rupture and AIM2 inflammasome activation depended on T6SS disassembly, which is regulated by ClpB.…”

“…Phagosomal rupture and AIM2 inflammasome activation depended on T6SS disassembly, which is regulated by ClpB. Strains lacking pdpC and pdpD , normally released by T6SS, were also unable to escape phagosome and activate AIM2 but their exact function is still not characterized …”

“…123 Recently, components of the type VI secretion system (T6SS) PdpC and PdpD encoded on the Francisella pathogenicity island and the chaperone ClpB were shown to be essential for phagosome escape of F. tularensis. 124 Phagosomal rupture and AIM2 inflammasome activation depended on T6SS disassembly, which is regulated by ClpB. Strains lacking pdpC and pdpD, normally released by T6SS, were also unable to escape phagosome and activate AIM2 but their exact function is still not characterized.…”

Diverse roles of TssA‐like proteins in the assembly of bacterial type VI secretion systems

“…This particular system does not contain a canonical transmembrane anchor and also lacks ClpV unfoldase (Böck et al, 2017). However, similarly to Francisella, contracted sheaths may be refolded by a related ATPase (Brodmann et al, 2017).…”

Cryo‐ EM reconstruction of Type VI secretion system baseplate and sheath distal end

“…Rapid sheath contraction propels the tip complex at the end of the inner tube formed from stacks of Hcp rings into the target cell periplasm or cytosol (Vettiger & Basler, 2016). In contrast to phages and many other contractile nanomachines, which translocate proteins only once by a single sheath contraction (Nakayama et al , 2000; Ge et al , 2015; Hu et al , 2015), the contracted T6SS sheath is disassembled by a cytosolic unfoldase ClpV or ClpB to allow for repeated protein secretion (Bönemann et al , 2009; Pietrosiuk et al , 2011; Basler & Mekalanos, 2012; Basler et al , 2012; Kapitein et al , 2013; Förster et al , 2014; Brodmann et al , 2017). …”