The biochemical properties of the Francisella pathogenicity island (FPI)-encoded proteins IglA, IglB, IglC, PdpB and DotU suggest roles in type VI secretion

Abstract

The Francisella pathogenicity island (FPI) encodes proteins thought to compose a type VI secretion system (T6SS) that is required for the intracellular growth of Francisella novicida. In this work we used deletion mutagenesis and genetic complementation to determine that the intracellular growth of F. novicida was dependent on 14 of the 18 genes in the FPI. The products of the iglABCD operon were localized by the biochemical fractionation of F. novicida, and Francisella tularensis LVS. Sucrose gradient separation of water-insoluble material showed that the FPI-encoded proteins IglA, IglB and IglC were found in multiple fractions, especially in a fraction that did not correspond to a known membrane fraction. We interpreted these data to suggest that IglA, IglB and IglC are part of a macromolecular structure. Analysis of published structural data suggested that IglC is an analogue of Hcp, which is thought to form long nanotubes. Thus the fractionation properties of IglA, IglB and IglC are consistent with the current model of the T6SS apparatus, which supposes that IglA and IglB homologues form an outer tube structure that surrounds an inner tube composed of Hcp (IglC) subunits. Fractionation of F. novicida expressing FLAG-tagged DotU (IcmH homologue) and PdpB (IcmF homologue) showed that these proteins localize to the inner membrane. Deletion of dotU led to the cleavage of PdpB, suggesting an interaction of these two proteins that is consistent with results obtained with other T6SSs. Our results may provide a mechanistic basis for many of the studies that have examined the virulence properties of Francisella mutants in FPI genes, namely that the observed phenotypes of the mutants are the result of the disruption of the FPI-encoded T6SS structure.

Structural Characterization and Oligomerization of the TssL Protein, a Component Shared by Bacterial Type VI and Type IVb Secretion Systems

“…In L. pneumophila, IcmH/ DotU stabilizes IcmF, whereas IcmF is absolutely required for IcmH/DotU production (38). Regarding the T6SS homologues, de Bruin et al (35) reported that TssL stabilizes TssM. Taken together, these data suggest that the TssLTssM pair is closely related to the IcmH/DotU-IcmF pair in terms of localization, topology, interaction, and stabilization.…”

“…A yeast two-hybrid approach showed that the cytoplasmic domains of the Agrobacterium tumefaciens TssL and TssM proteins are required for their association (32). An additional support for this interaction is the observation that TssL stabilizes TssM in Francisella tularensis (35). In the TssL C structure, several elements might be required to mediate the contacts with TssM.…”

“…These two components are required for the optimal function of the T4bSS in Legionella pneumophila and Coxiella burnetii (33,34). The TssL protein and its T4bSS IcmH/DotU counterpart are inner membrane proteins (32,(35)(36)(37)(38). The membrane topology of the TssL has been determined; TssL has a single trans-membrane segment (TMS) located at the C terminus of the protein (36).…”

Subversion of Host Recognition and Defense Systems by Francisella spp

“…Specific Francisella genes that play a role in this suppression of the host inflammatory response have been identified. For example, a strain with a mutation in the FPI gene iglC not only failed to escape the phagosome or replicate in macrophages but also was unable to suppress TNF-␣ production and other inflammatory responses (64,131,196,219). In addition, infection of macrophages with wild-type LVS blocked TLR2 and TLR4 activation in response to the addition of E. coli BLP and LPS, respectively, while the iglC mutant could not block this signaling (219).…”

“…(T3SS) (70,168,177,193,197). Briefly, the FPI proteins IglA and IglB share homology with proteins encoded in T6SS clusters in multiple bacterial species, are required for phagosomal escape, and may form the putative outer tube of the T6SS "needle" (64,65). IglC has been proposed to form the inner tube (64), while IglI and VgrG are secreted and therefore may interact with host proteins during infection (21).…”