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Evolutionary Dissection of the Dot/Icm System Based on Comparative Genomics of 58 Legionella Species.

ABSTRACT: The Dot/Icm type IVB secretion system of Legionella pneumophila is essential for its pathogenesis by delivering >300 effector proteins into the host cell. However, their precise secretion mechanism and which components interact with the host cell is only partly understood. Here, we undertook evolutionary analyses of the Dot/Icm system of 58 Legionella species to identify those components that interact with the host and/or the substrates. We show that high recombination rates are acting on DotA, DotG, and IcmX, supporting exposure of these proteins to the host. Specific amino acids under positive selection on the periplasmic region of DotF, and the cytoplasmic domain of DotM, support a role of these regions in substrate binding. Diversifying selection acting on the signal peptide of DotC suggests its interaction with the host after cleavage. Positive selection acts on IcmR, IcmQ, and DotL revealing that these components are probably participating in effector recognition and/or translocation. Furthermore, our results predict the participation in host/effector interaction of DotV and IcmF. In contrast, DotB, DotO, most of the core subcomplex elements, and the chaperones IcmS-W show a high degree of conservation and not signs of recombination or positive selection suggesting that these proteins are under strong structural constraints and have an important role in maintaining the architecture/function of the system. Thus, our analyses of recombination and positive selection acting on the Dot/Icm secretion system predicted specific Dot/Icm components and regions implicated in host interaction and/or substrate recognition and translocation, which will guide further functional analyses.

SUBMITTER: Gomez-Valero L

PROVIDER: S-EPMC6761968 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Multiple substrates of the Legionella pneumophila Dot/Icm system identified by interbacterial protein transfer.

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Induction of caspase 3 activation by multiple Legionella pneumophila Dot/Icm substrates.

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Project description:The bacterium Legionella pneumophila is a causative agent of Legionnaires' disease. It utilizes the Dot/Icm type IV secretion system (T4SS) to deliver over 300 effector proteins into the host cell, leading to modification of cellular processes and creating a safe environment for bacterial proliferation. Dot/Icm is a multi-subunit molecular machine. The effectors are recognized by the inner membrane-embedded coupling complex (T4CC), which then delivers them to the translocation apparatus. This T4CC subcomplex is made up of DotL, DotM, DotN, IcmS, IcmW, LvgA, DotY and DotZ, and its structure was recently determined by cryo-EM. DotY is a highly mobile component of this subcomplex and its structure was only partially defined. DotY is a unique component of the T4SS that is only found in the Legionella genus. Here, the crystal structure of DotY on its own is presented and its fold and the connectivity of its secondary-structure elements are established. The protein is divided into three segments. The first and last segments form a four-helix bundle domain, while the middle segment forms an α/β domain that has a unique fold. The flexibility of the interdomain linkers allows the reorientation of the two domains between that observed in the crystal structure and that assumed within the T4CC subcomplex.

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Cryo-EM reveals new species-specific proteins and symmetry elements in the <i>Legionella pneumophila</i> Dot/Icm T4SS.

Project description:Legionella pneumophila is an opportunistic pathogen that causes the potentially fatal pneumonia known as Legionnaires' disease. The pathology associated with infection depends on bacterial delivery of effector proteins into the host via the membrane spanning Dot/Icm type IV secretion system (T4SS). We have determined sub-3.0 Å resolution maps of the Dot/Icm T4SS core complex by single particle cryo-EM. The high-resolution structural analysis has allowed us to identify proteins encoded outside the Dot/Icm genetic locus that contribute to the core T4SS structure. We can also now define two distinct areas of symmetry mismatch, one that connects the C18 periplasmic ring (PR) and the C13 outer membrane cap (OMC) and one that connects the C13 OMC with a 16-fold symmetric dome. Unexpectedly, the connection between the PR and OMC is DotH, with five copies sandwiched between the OMC and PR to accommodate the symmetry mismatch. Finally, we observe multiple conformations in the reconstructions that indicate flexibility within the structure.

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Legionella pneumophila DotU and IcmF are required for stability of the Dot/Icm complex.

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