Francisella tularensis is a highly virulent gram-negative intracellular bacterium that causes the zoonotic disease tularemia. Essential for its virulence is the ability to multiply within host cells, in particular monocytic cells. The bacterium has developed intricate means to subvert host immune mechanisms and thereby facilitate its intracellular survival by preventing phagolysosomal fusion followed by escape into the cytosol, where it multiplies. Moreover, it targets and manipulates numerous host cell signaling pathways, thereby ameliorating the otherwise bactericidal capacity. Many of the underlying molecular mechanisms still remain unknown but key elements, directly or indirectly responsible for many of the aforementioned mechanisms, rely on the expression of proteins encoded by the Francisella pathogenicity island (FPI), suggested to constitute a type VI secretion system. We here describe the current knowledge regarding the components of the FPI and the roles that have been ascribed to them.

Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37°C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface.

Francisella tularensis harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS) recently identified in several gram-negative bacteria. These genes include iglA and iglB encoding IglA and IglB, homologues of which are conserved in most T6SSs. We used a yeast two-hybrid system to study the interaction of the Igl proteins of F. tularensis LVS. We identified a region of IglA, encompassing residues 33 to 132, necessary for efficient binding to IglB, as well as for IglAB protein stability and intramacrophage growth. In particular, residues 103 to 122, overlapping a highly conserved ␣-helix, played an absolutely essential role. Point mutations within this domain caused modest defects in IglA-IglB binding in the yeast Saccharomyces cerevisiae but markedly impaired intramacrophage replication and phagosomal escape, resulting in severe attenuation of LVS in mice. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity. This interaction may be universal to type VI secretion, since IglAB homologues of Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella enterica serovar Typhimurium, and Escherichia coli were also shown to interact in yeast, and the interaction was dependent on preservation of the same ␣-helix. Heterologous interactions between nonnative IglAB proteins further supported the notion of a conserved binding site. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity, and the same interaction is conserved in other human pathogens.Francisella tularensis is a gram-negative facultative intracellular bacterial pathogen capable of causing a severe disease, tularemia, in many mammalian species (22). Human infections are caused mainly by two subspecies, the more virulent organism F. tularensis subsp. tularensis (type A) found predominantly in North America and the less virulent organism F. tularensis subsp. holarctica (type B) found in North America, Europe, and Asia (34, 43). While little is known about the molecular mechanisms of Francisella pathogenesis, a key strategy appears to be its ability to survive and replicate within macrophages (42,46). Francisella-containing vacuoles have been reported to evade phagosome-lysosome fusion, followed by bacterial escape into the cytoplasm (8, 16). Several genes necessary for intramacrophage survival, as well as growth within the amoeba Acanthamoeba castellanii, a putative natural reservoir of F. tularensis, have been identified. Many of these genes, including the members of the iglABCD operon, are located in a 34-kb Francisella pathogenicity island (FPI) (reviewed in reference 31), and they are regulated by the global regulator MglA (4, 23). Almost all of the proteins of the FPI are essentially conserved across subspecies. Studies have shown that IglC and IglD are required for F. tularensis to replicate within the cytosol of macrophages (24, 37). While IglC was shown to be essential for bacterial escape from the phagosome into the cytoplasm (24, 38), the r...

The Gram-negative bacterium Francisella tularensis is the causative agent of tularemia, a disease intimately associated with the multiplication of the bacterium within host macrophages. This in turn requires the expression of Francisella pathogenicity island (FPI) genes, believed to encode a type VI secretion system. While the exact functions of many of the components have yet to be revealed, some have been found to contribute to the ability of Francisella to cause systemic infection in mice as well as to prevent phagolysosomal fusion and facilitate escape into the host cytosol. Upon reaching this compartment, the bacterium rapidly multiplies, inhibits activation of the inflammasome, and ultimately causes apoptosis of the host cell. In this study, we analyzed the contribution of the FPI-encoded proteins IglG, IglI, and PdpE to the aforementioned processes in F. tularensis LVS. The ⌬pdpE mutant behaved similarly to the parental strain in all investigated assays. In contrast, ⌬iglG and ⌬iglI mutants, although they were efficiently replicating in J774A.1 cells, both exhibited delayed phagosomal escape, conferred a delayed activation of the inflammasome, and exhibited reduced cytopathogenicity as well as marked attenuation in the mouse model. Thus, IglG and IglI play key roles for modulation of the intracellular host response and also for the virulence of F. tularensis.

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.

The pathogenic species of the genus Yersinia (Yersinia pestis, Y. enterocolitica, and Y. pseudotuberculosis) cause infections of highly varied severity in humans. Y. pestis causes plague and is transmitted by flea bites or infectious aerosols, whereas Y. enterocolitica and Y. pseudotuberculosis are enteric pathogens that cause gastroenteritis after the ingestion of contaminated food or water (for reviews, see references 9 and 35). Still, the virulence mechanisms of the different species show a lot of similarities. One such similarity is the ability to inhibit phagocytosis, which enables the pathogens to replicate in lymphoid tissues. This is conferred by an ca. 70-kb plasmid that is required for virulence in all three species. The plasmid encodes a type III secretion system (TTSS) that delivers antihost proteins or virulence effectors called Yops (Yersinia outer proteins) into the cytosol of eukaryotic cells (12, 13). Yop secretion is normally triggered by eukaryotic cell contact (36, 37), but it can also be induced in vitro by growing the bacteria in calcium-depleted medium at 37°C (13).TTSSs are found in several gram-negative animal and plant pathogens (20, 39). The overall mechanism of secretion appears to be conserved in the different systems. Typically, 20 to 25 proteins are required to assemble a functional secretion system. Nine of these proteins are conserved not only in the TTSSs of different pathogens but also in the bacterial flagellar export apparatus (for reviews, see references 1, 20, and 25). For several animal pathogens, the type III secretion organelle, also referred to as the secreton, has been isolated and analyzed (5,6,21,22,40,44). The basal body of this structure possesses two sets of rings resembling the flagellar basal body. The components common to the virulence associated and the flagellar TTSS are believed either to associate with the cytoplasmic face of the basal body-like structure or to form a pore in the inner membrane ring (15,42). A common feature of secretons isolated so far is a needle-like structure that protrudes from the ring structure located in the outer membrane. This needle is required for secretion, suggesting that the combination of the basal portion and needle extension (needle complex) constitutes an intact secretion organelle (5,6,21,22,40,44). In Yersinia spp. the needle-like structure is comprised of the YscF protein and localizes to the bacterial cell surface prior to eukaryotic cell contact (18; P. Edqvist, J. Olsson, M. Lavander, L. Sundberg, Å. Forsberg, H. Wolf-Watz, and S. Lloyd, unpublished data).The proteins forming the actual secretion apparatus are believed to somehow identify the type III secretion substrates to enable their secretion through the basal body-like structure. One key protein in the export of flagellar components is FlhB, a membrane protein with a large cytoplasmic C-terminal domain (29,30). YscU, the corresponding protein of the TTSS of Yersinia spp. has also been shown to localize to the cytoplasmic membrane (3). The flagellum is a tripar...

Yersinia species pathogenic to humans have been extensively characterized with respect to type III secretion and its essential role in virulence. This study concerns the twin arginine translocation (Tat) pathway utilized by gram-negative bacteria to secrete folded proteins across the bacterial inner membrane into the periplasmic compartment. We have shown that the Yersinia Tat system is functional and required for motility and contributes to acid resistance. A Yersinia pseudotuberculosis mutant strain with a disrupted Tat system (tatC) was, however, not affected in in vitro growth or more susceptible to high osmolarity, oxidative stress, or high temperature, nor was it impaired in type III secretion. Interestingly, the tatC mutant was severely attenuated via both the oral and intraperitoneal routes in the systemic mouse infection model and highly impaired in colonization of lymphoid organs like Peyer's patches and the spleen. Our work highlights that Tat secretion plays a key role in the virulence of Y. pseudotuberculosis.As the integrity of the bacterial cytoplasm is protected by membranes, the necessity of means for transport of molecules between the bacterial compartments, periplasm and cytoplasm, and the surrounding milieu is obvious. Not only do bacteria require the ability to take up nutrients and get rid of waste, transport across the membranes is also highly involved in the ability of bacteria to cause disease. A number of specialized protein secretion systems are essential for delivery of virulence factors across the two membranes of gram-negative pathogens (39). The importance of these systems is reflected in the multitude of mechanisms that have evolved not only for protein translocation across the bacterial membranes but also for targeting of anti-host factors across the host cell membrane. The means of translocation across the bacterial outer membrane have been grouped into five different pathways

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 .