Dataset Information

Molecular characterization of the Dot/Icm-translocated AnkH and AnkJ eukaryotic-like effectors of Legionella pneumophila.

ABSTRACT: Although most Dot/Icm-translocated effectors of Legionella pneumophila are not required for intracellular proliferation, the eukaryotic-like ankyrin effectors, AnkH and AnkJ are required for intracellular proliferation. In this report, we show that the IcmSW chaperones are essential for translocation of AnkJ but not AnkH. The 10 C-terminal residues and the ANK domains of AnkH and AnkJ are required for translocation. Our data indicate that the two ANK domains of AnkH are critical domains required for the function of the effector in intracellular replication of L. pneumophila. The ankH and ankJ mutants are severely defective in intrapulmonary proliferation in mice. Expression of AnkH and AnkJ fusions within HEK293 cells show a punctuate distribution in the cytosol but no association with endocytic vesicles, the Golgi apparatus or the endoplasmic reticulum. Interestingly, the defect in intracellular proliferation of the ankH or ankJ mutants is rescued in HEK293 cells expressing the respective protein. We conclude that AnkH and AnkJ are effectors translocated by the Dot/Icm system by distinct mechanisms and modulate distinct cytosolic processes in the host cell.

SUBMITTER: Habyarimana F

PROVIDER: S-EPMC2825944 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Molecular characterization of the Dot/Icm-translocated AnkH and AnkJ eukaryotic-like effectors of Legionella pneumophila.

Habyarimana Fabien F Price Chris T CT Santic Marina M Al-Khodor Souhaila S Kwaik Yousef Abu YA

Infection and immunity 20091222 3

Although most Dot/Icm-translocated effectors of Legionella pneumophila are not required for intracellular proliferation, the eukaryotic-like ankyrin effectors, AnkH and AnkJ are required for intracellular proliferation. In this report, we show that the IcmSW chaperones are essential for translocation of AnkJ but not AnkH. The 10 C-terminal residues and the ANK domains of AnkH and AnkJ are required for translocation. Our data indicate that the two ANK domains of AnkH are critical domains required ...[more]

PMID: 20028808

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Project description:The Dot/Icm type IV secretion system of Legionella pneumophila translocates numerous bacterial effectors into the host cell and is essential for bacterial proliferation within macrophages and protozoa. We have recently shown that L. pneumophila strain AA100/130b harbours 11 genes encoding eukaryotic-like ankyrin (Ank) proteins, a family of proteins involved in various essential eukaryotic cellular processes. In contrast to most Dot/Icm-exported substrates, which have little or no detectable role in intracellular proliferation, a mutation in ankB results in a severe growth defect in intracellular replication within human monocyte-derived macrophages (hMDMs), U937 macrophages and Acanthamoeba polyphaga. Single cell analyses of coinfections of hMDMs have shown that the intracellular growth defect of the ankB mutant is totally rescued in cis within communal phagosomes harbouring the wild type strain. Interestingly, distinct from dot/icm structural mutants, the ankB mutant is also rescued in trans within cells harbouring the wild type strain in a different phagosome, indicating that AnkB is a trans-acting secreted effector. Using adenylate cyclase fusions to AnkB, we show that AnkB is translocated into the host cell via the Dot/Icm secretion system in an IcmSW-dependent manner and that the last three C-terminal amino acid residues are essential for translocation. Distinct from the dot/icm structural mutants, the ankB mutant-containing phagosomes exclude late endosomal and lysosomal markers and their phagosomes are remodelled by the rough endoplasmic reticulum. We show that at the postexponential phase of growth, the LetA/S and PmrA/B Two Component Systems confer a positive regulation on expression of the ankB gene, whereas RpoS, LetE and RelA suppress its expression. Our data show that the eukaryotic-like AnkB protein is a Dot/Icm-exported effector that plays a major role in intracellular replication of L. pneumophila within macrophages and protozoa, and its expression is temporally controlled by regulators of the postexponential phase of growth.

Project description:Major differences in the transcriptional program underlying the phenotypic switch between exponential and post-exponential growth of Legionella pneumophila were formerly described characterizing important alterations in infection capacity. Additionally, a third state is known where the bacteria transform in a viable but nonculturable state under stress, such as starvation. We here describe phase-related proteomic changes in exponential phase (E), postexponential phase (PE) bacteria, and unculturable microcosms (UNC) containing viable but nonculturable state cells, and identify phase-specific proteins. We present data on different bacterial subproteomes of E and PE, such as soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins. In total, 1368 different proteins were identified, 922 were quantified and 397 showed differential abundance in E/PE. The quantified subproteomes of soluble whole cell proteins, outer membrane-associated proteins, and extracellular proteins; 841, 55, and 77 proteins, respectively, were visualized in Voronoi treemaps. 95 proteins were quantified exclusively in E, such as cell division proteins MreC, FtsN, FtsA, and ZipA; 33 exclusively in PE, such as motility-related proteins of flagellum biogenesis FlgE, FlgK, and FliA; and 9 exclusively in unculturable microcosms soluble whole cell proteins, such as hypothetical, as well as transport/binding-, and metabolism-related proteins. A high frequency of differentially abundant or phase-exclusive proteins was observed among the 91 quantified effectors of the major virulence-associated protein secretion system Dot/Icm (> 60%). 24 were E-exclusive, such as LepA/B, YlfA, MavG, Lpg2271, and 13 were PE-exclusive, such as RalF, VipD, Lem10. The growth phase-related specific abundance of a subset of Dot/Icm virulence effectors was confirmed by means of Western blotting. We therefore conclude that many effectors are predominantly abundant at either E or PE which suggests their phase specific function. The distinct temporal or spatial presence of such proteins might have important implications for functional assignments in the future or for use as life-stage specific markers for pathogen analysis.

Dataset Information

Molecular characterization of the Dot/Icm-translocated AnkH and AnkJ eukaryotic-like effectors of Legionella pneumophila.

Publications

Molecular characterization of the Dot/Icm-translocated AnkH and AnkJ eukaryotic-like effectors of Legionella pneumophila.

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