Unique substrates secreted by the type VI secretion system of Francisella tularensis during intramacrophage infection

PLoS One. 2012;7(11):e50473. doi: 10.1371/journal.pone.0050473. Epub 2012 Nov 20.


Gram-negative bacteria have evolved sophisticated secretion machineries specialized for the secretion of macromolecules important for their life cycles. The Type VI secretion system (T6SS) is the most widely spread bacterial secretion machinery and is encoded by large, variable gene clusters, often found to be essential for virulence. The latter is true for the atypical T6SS encoded by the Francisella pathogenicity island (FPI) of the highly pathogenic, intracellular bacterium Francisella tularensis. We here undertook a comprehensive analysis of the intramacrophage secretion of the 17 FPI proteins of the live vaccine strain, LVS, of F. tularensis. All were expressed as fusions to the TEM β-lactamase and cleavage of the fluorescent substrate CCF2-AM, a direct consequence of the delivery of the proteins into the macrophage cytosol, was followed over time. The FPI proteins IglE, IglC, VgrG, IglI, PdpE, PdpA, IglJ and IglF were all secreted, which was dependent on the core components DotU, VgrG, and IglC, as well as IglG. In contrast, the method was not directly applicable on F. novicida U112, since it showed very intense native β-lactamase secretion due to FTN_1072. Its role was proven by ectopic expression in trans in LVS. We did not observe secretion of any of the LVS substrates VgrG, IglJ, IglF or IglI, when tested in a FTN_1072 deficient strain of F. novicida, whereas IglE, IglC, PdpA and even more so PdpE were all secreted. This suggests that there may be fundamental differences in the T6S mechanism among the Francisella subspecies. The findings further corroborate the unusual nature of the T6SS of F. tularensis since almost all of the identified substrates are unique to the species.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Bacterial Vaccines / immunology
  • Bacterial Vaccines / pharmacology*
  • Cell Line
  • Francisella tularensis / pathogenicity
  • Francisella tularensis / physiology*
  • Genes, Reporter
  • Genomic Islands / genetics
  • Macrophages / immunology
  • Macrophages / microbiology*
  • Mice
  • Molecular Sequence Data
  • Recombinant Fusion Proteins / genetics*
  • Recombinant Fusion Proteins / immunology
  • Sequence Alignment
  • Tularemia / prevention & control
  • Vaccines, Attenuated
  • beta-Lactamases / genetics
  • beta-Lactamases / metabolism


  • Bacterial Proteins
  • Bacterial Vaccines
  • Recombinant Fusion Proteins
  • Vaccines, Attenuated
  • beta-Lactamases

Grant support

This work was supported by grants 2009–5026 (to AS) from the Swedish Research Council (www.vr.se) and a grant from the Medical Faculty, Umeå University, Umeå, Sweden. The work was performed in part at the Umeå Centre for Microbial Research (UCMR) (www.ucmr.umu.se). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.