Structural similarity of tailed phages and pathogenic bacterial secretion systems.

T he transport of proteins across the bacterial cell membrane is a fundamental process carried out by all groups of Gramnegative bacteria. Especially in the case of pathogenic bacteria, transport systems are used at a number of different steps in the bacterial infection pathway, such as in the export of toxins, cell adhesion, and direct penetration of effectors into the host cell. There are at least 6 distinct extracellular protein secretion systems reported as types I through VI (T1SS–T6SS) that can deliver proteins through the multilayered bacterial cell membrane and sometimes directly into the target host cell (1). The molecular organization and secretion mechanisms of type I–V secretion systems are relatively well characterized (2). However, less is known about the organization, function, and mechanism of the T6SS system first discovered by Mekalanos and coworkers in 2006 (3). After their initial report, a number of bioinformatics-based comparative analyses of the T6SS gene clusters between bacterial strains have been made to assess possible function (4–10). In this issue of PNAS, both Leiman et al. (11) and Pell et al. (12) use structure-based analysis to demonstrate the existence of a structure/function relationship between the molecular components of T6SS and the tail proteins of bacteriophages T4 (11) and (12). T6SS components usually are encoded by a single gene cluster consisting of 15 conserved ORFs. This gene cluster is found in a wide range of pathogenic bacteria, including Vibrio cholerae, Pseudomonas aeruginosa, Escherichia coli, Agrobacterium tumefaciens, and Rhizobium leguminosarum (13). A common observation for all T6SS-carrying bacteria is the presence of Hcp and VgrG proteins in the culture media (3, 4, 14). Both Hcp and VgrG proteins are required for the function of the T6SS (3, 5, 14). The article by Leiman et al. (11) compares the crystal structures of VgrG homologs from the E. coli C3393 strain and the (gp27)3–(gp5)3 complex derived from bacteriophage T4. Although there is almost no sequence identity (13%) between VgrG and (gp27)3–(gp5)3, the N-terminal portion of VgrG is well superimposable onto the (gp27)3–(gp5)3 as a single connected polypeptide chain. This similarity was first predicted by careful bioinformatic analysis (3) from the same group in ref. 11. Leiman et al. (11) have also found that the structure of the hexameric ring formed by Hcp protein (14) is similar to the gp27 trimer and that the sequences of at least 2 T6SS proteins are highly similar to the T4 phage tail components (baseplate protein gp25 and the tail tube protein gp19). In the article by Pell et al. (12) they demonstrate a strong similarity between a phage major tail protein and the Hcp protein of the T6SS. They carried out NMR-based determination of the structure of the N-terminal 153 residues of gpV major tail protein from phage followed by subsequent comparison with both monomeric and hexameric structure of the Hcp protein. Penetration of the tail tube into the multilayered envelope of Gram-negative bacteria by bacteriophage T4 and ‘‘T4related’’ phages is well controlled by the T4 tail complex (15, 16). The tail complex first recognizes the host bacterial surface by using the tip of the long tail fibers. This recognition event then triggers a conformational change in the tail baseplate that leads to the tail sheath contraction and tail tube penetration (Fig. 1A). At the tip of the tail tube, a needle-like cell-puncturing device, (gp27)3–(gp5)3 complex, is located. The structure at the tip of the needle complex is uniquely intertwined into a 3-stranded -helix that has tandem 8 amino acid repeats of VXGXXXXX (17). Although the first Val is replaced with Ile or Leu in some instances, this 8-residue repeat is widely conserved in T4-related phages. The same type of repeat was found in Vgr-G proteins (3). Combining this phage infection mechanism and the known T6SS protein– protein interaction, Leiman et al. (11) propose a protein organization/assembly model of T6SS. Namely, (i) ClpV, IcmF, DotU, and other proteins that may be required for ATPase activity of ClpV assemble near the inner membrane, (ii) this protein complex transports Hcp, VgrG, and T4 gp25-like proteins into the periplasmic space, (iii) these proteins then assemble in a manner similar to the phage tail in the periplasmic space, and (iv) this assembly makes a tubular channel through the outer membrane in the direction of the host cell (Fig. 1B). In summary, a protein complex analogous to an upside-down bacteriophage tail complex constitutes the T6SS apparatus. The increase in the amount of genomic sequence data in combination with the development of bioinformatic analysis methods has allowed for the prediction of the function of unknown gene products. In an analogous manner,