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 ΔvgrG and ΔdotU 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.
Highlights
Gram-negative bacteria rely on protein secretion systems to mediate successful colonization of hosts [1]
According to Phyre2 [36], the highest similarity to F. tularensis VgrG is exhibited by the C-terminal domain of the tail-associated lysozyme of the T4 bacteriophage, whereas HHpred [37] suggests a structural similarity to the tail spike protein of the bacteriophage P2, as well as a putative adhesin present in Bacteroides fragilis
F. tularensis VgrG was shown to align to the central part of VgrG from V. cholerae, which overlaps with parts of the C-terminal region of gp27, and most of the C-terminal domain of the gp5 protein [22]
Summary
Gram-negative bacteria rely on protein secretion systems (denoted type I to type VII) to mediate successful colonization of hosts [1]. The type VI secretion system (T6SS) was first discovered in Vibrio cholerae in 2006 [2], but has since been identified in more than one fourth of all sequenced bacterial genomes [3,4,5]. Many of these T6SS-containing bacteria are known pathogens that rely on T6SSs to mediate infection of eukaryotic hosts (reviewed in [6]), type VI secretion (T6S) play an important role in interbacterial interactions [7]. The importance of VipA/B for substrate secretion has been experimentally demonstrated [11,16,17,18]
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