Triggering Ras signalling by intracellular Francisella tularensis through recruitment of PKCα and βI to the SOS2/GrB2 complex is essential for bacterial proliferation in the cytosol.

Intracellular proliferation of Francisella tularensis is essential for manifestation of the fatal disease tularaemia, and is classified as a category A bioterrorism agent. The F. tularensis-containing phagosome (FCP) matures into a late endosome-like phagosome with limited fusion to lysosomes, followed by rapid bacterial escape into the cytosol. The Francisella pathogenicity island (FPI) encodes a type VI-like secretion system, and the FPI-encoded IglC is essential for evasion of lysosomal fusion and phagosomal escape. Many host signalling events are likely to be modulated by F. tularensis to render the cell permissive for intracellular proliferation but they are not fully understood. Here we show that within 15 min of infection, intracellular F. tularensis ssp. novicida triggers IglC-dependent temporal activation of Ras, but attached extracellular bacteria fail to trigger Ras activation, which has never been shown for other intracellular pathogens. Intracellular F. tularensis ssp. novicida triggers activation of Ras through recruitment of PKCα and PKCβI to the SOS2/GrB2 complex. Silencing of SOS2, GrB2 and PKCα and PKCβI by RNAi has no effect on evasion of lysosomal fusion and bacterial escape into the cytosol but renders the cytosol non-permissive for replication of F. tularensis ssp. novicida. Since Ras activation promotes cell survival, we show that silencing of SOS2, GrB2 and PKCα and βI is associated with rapid early activation of caspase-3 within 8 h post infection. However, silencing of SOS2, GrB2 and PKCα and βI does not affect phosphorylation of Akt or Erk, indicating that activation of the PI3K/Akt and the Erk signalling cascade are independent of the F. tularensis-triggered Ras activation. We conclude that intracellular F. tularensis ssp. novicida triggers temporal and early activation of Ras through the SOS2/GrB2/PKCα/PKCβI quaternary complex. Temporal and rapid trigger of Ras signalling by intracellular F. tularensis is essential for intracellular bacterial proliferation within the cytosol, and this is associated with downregulation of early caspase-3 activation.

Al-Khodor S ，Abu Kwaik Y 《-》

The Francisella tularensis pathogenicity island protein IglC and its regulator MglA are essential for modulating phagosome biogenesis and subsequent bacterial escape into the cytoplasm.

Santic M ，Molmeret M ，Klose KE ，Jones S ，Kwaik YA ... -  《CELLULAR MICROBIOLOGY》

A Francisella tularensis pathogenicity island protein essential for bacterial proliferation within the host cell cytosol.

Santic M ，Molmeret M ，Barker JR ，Klose KE ，Dekanic A ，Doric M ，Abu Kwaik Y ... -  《CELLULAR MICROBIOLOGY》

Intracellular fate of Francisella tularensis within arthropod-derived cells.

Santic M ，Akimana C ，Asare R ，Kouokam JC ，Atay S ，Kwaik YA ... -  《-》

Molecular complexity orchestrates modulation of phagosome biogenesis and escape to the cytosol of macrophages by Francisella tularensis.

Upon entry of Francisella tularensis to macrophages, the Francisella-containing phagosome (FCP) is trafficked into an acidified late endosome-like phagosome with limited fusion to the lysosomes followed by rapid escape into the cytosol where the organism replicates. Although the Francisella Pathogenicity Island (FPI), which encodes a type VI-like secretion apparatus, is required for modulation of phagosome biogenesis and escape into the cytosol, the mechanisms involved are not known. To decipher the molecular bases of modulation of biogenesis of the FCP and bacterial escape into the macrophage cytosol, we have screened a comprehensive mutant library of F. tularensis ssp. novicida for their defect in proliferation within human macrophages, followed by characterization of modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data show that at least 202 genes are required for intracellular proliferation within macrophages. Among the 125 most defective mutants in intracellular proliferation, we show that the FCP of at least 91 mutants colocalize persistently with the late endosomal/lysosomal marker LAMP-1 and fail to escape into the cytosol, as determined by fluorescence-based phagosome integrity assays and transmission electron microscopy. At least 34 genes are required for proliferation within the cytosol but do not play a detectable role in modulation of phagosome biogenesis and bacterial escape into the cytosol. Our data indicate a tremendous adaptation and metabolic reprogramming by F. tularensis to adjust to the micro-environmental and nutritional cues within the FCP, and these adjustments play essential roles in modulation of phagosome biogenesis and escape into the cytosol of macrophages as well as proliferation in the cytosol. The plethora of the networks of genes that orchestrate F. tularensis-mediated modulation of phagosome biogenesis, phagosomal escape and bacterial proliferation within the cytosol is novel, complex and involves an unusually large portion of the genome of an intracellular pathogen.

Asare R ，Abu Kwaik Y 《-》