Unique Substrates Secreted by the Type VI Secretion System of Francisella tularensis during Intramacrophage Infection

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.

IglE Is an Outer Membrane-Associated Lipoprotein Essential for Intracellular Survival and Murine Virulence of Type A Francisella tularensis

“…The presence of an N-terminal signal sequence and our combined biochemical and genetic data suggesting that IglE is an outer membrane-anchored lipoprotein are inconsistent with the results of a second study which showed that IglE coupled to a beta-lactamase reporter is secreted from the cell in a manner that is dependent on other core FPI proteins (37). The same authors also noted that PdpE (a second secreted FPI-encoded protein) also possesses a signal peptide (37).…”

“…The same authors also noted that PdpE (a second secreted FPI-encoded protein) also possesses a signal peptide (37). This raises the intriguing possibility either that the Sec and T6SS pathways are connected in Francisella or that some FPI-secreted substrates (i.e., IglE) have two biological functions, one which requires a membrane-anchoring lipid moiety (e.g., T6SS assembly) and another where soluble protein is required (e.g., as a T6SS substrate of unknown function).…”

“…However, these authors did not address potential polar effects on downstream vgrG gene transcription, and attempts to complement the iglE mutation with a multicopy extrachromosomal plasmid were unsuccessful (36); thus, the phenotype of the resultant mutants could not be unequivocally attributed to the loss of iglE alone. IglE was also identified as one of eight secreted proteins on the basis of a comprehensive reporter assay of FPI-encoded proteins coupled to TEM (a ␤-lactamase) (37). IglE has a putative signal sequence and lipobox signature motif, suggesting that its gene may encode a bacterial lipoprotein, but it lacks significant homology to other known proteins.…”

Importance of PdpC, IglC, IglI, and IglG for Modulation of a Host Cell Death Pathway Induced by Francisella tularensis

“…Most likely these features are not modulated directly by IglC but result from the lack of a functional T6SS due to the essential role of the protein for the secretion system. It should be noted, however, that no defined role of IglC for the secretion machinery has been demonstrated, and in fact, a recent publication demonstrated that it is secreted during macrophage infection (46).…”

Francisella tularensisas a potential agent of bioterrorism?

“…The molecular mechanisms allowing F. tularensis to reach and multiply within the eukaryotic cell cytosol are increasingly well understood. The Type 6 secretion system effectors could be new targets to inhibit intracellular growth of this pathogen [25]. Enhancing the host response to F. tularensis infection may be more hazardous because of the potential deleterious effects of inflammation.…”