DotU and VgrG, Core Components of Type VI Secretion Systems, Are Essential for Francisella LVS Pathogenicity

Abstract

The Gram-negative bacterium Francisella tularensis causes tularemia, a disease which requires bacterial escape from phagosomes of infected macrophages. Once in the cytosol, the bacterium rapidly multiplies, inhibits activation of the inflammasome and ultimately causes death of the host cell. Of importance for these processes is a 33-kb gene cluster, the Francisella pathogenicity island (FPI), which is believed to encode a type VI secretion system (T6SS). In this study, we analyzed the role of the FPI-encoded proteins VgrG and DotU, which are conserved components of type VI secretion (T6S) clusters. We demonstrate that in F. tularensis LVS, VgrG was shown to form multimers, consistent with its suggested role as a trimeric membrane puncturing device in T6SSs, while the inner membrane protein DotU was shown to stabilize PdpB/IcmF, another T6SS core component. Upon infection of J774 cells, both Δ vgrG and Δ dotU mutants did not escape from phagosomes, and subsequently, did not multiply or cause cytopathogenicity. They also showed impaired activation of the inflammasome and marked attenuation in the mouse model. Moreover, all of the DotU-dependent functions investigated here required the presence of three residues that are essentially conserved among all DotU homologues. Thus, in agreement with a core function in T6S clusters, VgrG and DotU play key roles for modulation of the intracellular host response as well as for the virulence of F. tularensis .

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

“…We followed bacterial numbers of the ⌬pdpC, ⌬iglC, ⌬iglG, and ⌬iglI mutants and the LVS strain in J774 cells after infection. In agreement with previously published data (20,(23)(24)(25)(26)(28)(29)(30), the ⌬iglC and ⌬pdpC mutants showed very marginal net replication, whereas the ⌬iglG and the ⌬iglI mutants replicated as well as LVS (Table 1). In separate experiments, we observed that the complemented strains showed the same degree of replication as did LVS (not shown).…”

“…Previously, we and others have shown that the FPI mutants display a spectrum of intracellular phenotypes, since some replicate as well as the wild type whereas others show compromised or no growth (20,(23)(24)(25)(26)(28)(29)(30). We followed bacterial numbers of the ⌬pdpC, ⌬iglC, ⌬iglG, and ⌬iglI mutants and the LVS strain in J774 cells after infection.…”

“…When mutants have been generated in the FPI, many of them show a uniform phenotype characterized by lack of phagosomal escape, no intracellular replication, and a loss of virulence in vivo. Examples of such mutants include those of the iglABCD operon and the core components VgrG and DotU (20)(21)(22)(23)(24)(25). In contrast, we have observed that the ⌬iglI and ⌬iglG mutants showed aberrant intracellular replication and very diminished cytopathogenic effects (26).…”

Repression of Inflammasome by Francisella tularensis during Early Stages of Infection

“…Francisella is different from these pathogens as it lacks type III or type IV secretion systems and exotoxins. DotU and VgrG have been identified as the core components of the type VI secretion system in F. tularensis, and null mutation of these two genes fail to activate the inflammasome (9). However, these results may apparently be due to the inability of both the dotU and vgrG mutants to escape phagosomes rather than their inhibitory effects on assembly and activation of the inflammasome components.…”

“…The virulence properties and mechanisms underlying innate immune subversion of F. tularensis are unique and remain largely undefined. F. tularensis lacks toxins or type III and IV secretion systems but possesses a type VI secretion system (9,10). Macrophages are the primary sites of predilection for F. tularensis.…”