Abstract
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.
Highlights
Gram-negative bacteria have evolved various types of sophisticated machineries specialized for the secretion of macromolecules as a means to promote bacterial fitness and/or establish colonization or attachment to host cells
It was concluded that IglI and VgrG were both secreted in F. novicida and LVS, the data was conflicting regarding the requirement for other Francisella pathogenicity island (FPI) proteins since the findings in F. novicida suggested that only secretion of IglI was FPI-dependent, whereas the findings on LVS concluded that IglI as well as VgrG secretion was FPI-independent [34,35]
While the TEM-tag in several instances was found to interfere with FPI protein function in terms of its ability to support intracellular growth and/or Lactate dehydrogenase (LDH) release of the corresponding LVS mutant, it did not have a general impact on the ability of the protein to be secreted as 4 out of 5 identified substrates tested in these additional assays failed to promote growth but were still secreted
Summary
Gram-negative bacteria have evolved various types of sophisticated machineries specialized for the secretion of macromolecules as a means to promote bacterial fitness and/or establish colonization or attachment to host cells. In V. cholerae, VipA/VipB have been shown to form tubular structures that structurally resemble bacteriophage T4 contractile tail sheath [14,16] and a recent, elegant microscopy study revealed that the similarity extends to the level of function, as the tubules were shown to cycle between assembly, quick contraction, disassembly, and re-assembly [17] This suggests that the sheath may energize the translocation of substrates into the extracellular milieu or into adjacent target cells by a phage tail-like contraction mechanism
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