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A type IV translocated Legionella cysteine phytase counteracts intracellular growth restriction by phytate.

ABSTRACT: The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique "Legionella-containing vacuole." The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4SS) and ?300 different "effector" proteins. Here we characterize a hitherto unrecognized Icm/Dot substrate, LppA, as a phytate phosphatase (phytase). Phytase activity of recombinant LppA required catalytically essential cysteine (Cys(231)) and arginine (Arg(237)) residues. The structure of LppA at 1.4 Å resolution revealed a mainly ?-helical globular protein stabilized by four antiparallel ?-sheets that binds two phosphate moieties. The phosphates localize to a P-loop active site characteristic of dual specificity phosphatases or to a non-catalytic site, respectively. Phytate reversibly abolished growth of L. pneumophila in broth, and growth inhibition was relieved by overproduction of LppA or by metal ion titration. L. pneumophila lacking lppA replicated less efficiently in phytate-loaded Acanthamoeba castellanii or Dictyostelium discoideum, and the intracellular growth defect was complemented by the phytase gene. These findings identify the chelator phytate as an intracellular bacteriostatic component of cell-autonomous host immunity and reveal a T4SS-translocated L. pneumophila phytase that counteracts intracellular bacterial growth restriction by phytate. Thus, bacterial phytases might represent therapeutic targets to combat intracellular pathogens.

SUBMITTER: Weber S

PROVIDER: S-EPMC4256350 | biostudies-literature | 2014 Dec

REPOSITORIES: biostudies-literature

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A type IV translocated Legionella cysteine phytase counteracts intracellular growth restriction by phytate.

Weber Stephen S Stirnimann Christian U CU Wieser Mara M Frey Daniel D Meier Roger R Engelhardt Sabrina S Li Xiaodan X Capitani Guido G Kammerer Richard A RA Hilbi Hubert H

The Journal of biological chemistry 20141022 49

The causative agent of Legionnaires' pneumonia, Legionella pneumophila, colonizes diverse environmental niches, including biofilms, plant material, and protozoa. In these habitats, myo-inositol hexakisphosphate (phytate) is prevalent and used as a phosphate storage compound or as a siderophore. L. pneumophila replicates in protozoa and mammalian phagocytes within a unique "Legionella-containing vacuole." The bacteria govern host cell interactions through the Icm/Dot type IV secretion system (T4S ...[more]

PMID: 25339170

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Project description:Legionella pneumophila is ubiquitous in freshwater environments, where it replicates within unicellular protozoa. However, L. pneumophila is also an accidental human pathogen that can cause Legionnaires' disease in immunocompromised individuals by uncontrolled replication within alveolar macrophages. To replicate within eukaryotic phagocytes, L. pneumophila utilizes a Dot/Icm type IV secretion system to translocate a large arsenal of over 300 effector proteins directly into host cells. In mammals, translocated effectors contribute to innate immune restriction of L. pneumophila We found previously that the effector LegC4 is important for L. pneumophila replication within a natural host protist but is deleterious to replication in a mouse model of Legionnaires' disease. In the present study, we used cultured mouse primary macrophages to investigate how LegC4 attenuates L. pneumophila replication. We found that LegC4 enhanced restriction of L. pneumophila replication within macrophages activated with tumor necrosis factor (TNF) or interferon gamma (IFN-?). In addition, expression of legC4 was sufficient to restrict Legionella longbeachae replication within TNF- or IFN-?-activated macrophages. Thus, this study demonstrates that LegC4 contributes to L. pneumophila clearance from healthy hosts by potentiating cytokine-mediated host defense mechanisms.IMPORTANCE Legionella spp. are natural pathogens of protozoa and accidental pathogens of humans. Innate immunity in healthy individuals effectively controls Legionella infection due in part to rapid and robust production of proinflammatory cytokines resulting from detection of Dot/Icm-translocated substrates, including effectors. Here, we demonstrate that the effector LegC4 enhances proinflammatory host restriction of Legionella by macrophages. These data suggest that LegC4 may augment proinflammatory signaling or antimicrobial activity of macrophages, a function that has not previously been observed for another bacterial effector. Further insight into LegC4 function will likely reveal novel mechanisms to enhance immunity against pathogens.

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:This study was conducted to determine effects of high phytase use on growth performance, amino acid (AA) digestibility, intestinal phytate breakdown, and nutrient transporter expression in starter broiler chickens. Male Ross 308 chicks were allocated to 24 pens, at 15 birds/pen and assigned to one of 4 dietary treatments. Treatments were: a control diet (PCa+) that contained adequate levels of calcium (Ca) and phosphorus (P) for growing broiler chicks; a reduced Ca and P diet (PCa-:-1.5 g P/kg and -1.6 g Ca/kg), and 2 additional diets in which phytase was supplemented in the PCa- diet at 1,500 (PCa-Phy1500) and 3,000 (PCa-Phy3000) FTU/kg feed. A common starter diet was fed from day 1 to 8. From day 8 to 22, birds were fed the 4 experimental diets. On day 22, birds were killed for sample collection. From day 8 to 15, average daily gain and average daily feed intake were not different across treatments (P < 0.05) but gain-to-feed ratio (G:F) was reduced (P < 0.006) in the PCa- treatment compared with other treatments. There were no further performance differences, but a tendency of phytase treatments improving the overall G:F (P = 0.079; day 8-22). Up to both the duodenum-jejunum and ileum, phytate, P, and Ca disappearance were increased (P < 0.05) in the PCa-Phy1500 and PCa-Phy3000 treatments compared with PCa- treatment. Phytase dose dependently increased myoinositol (MI) concentration in the digesta from both the duodenum-jejunum and ileum (P < 0.001). The highest concentration of MI was found in the PCa-Phy3000 treatment. Plasma MI concentration was increased by phytase supplementation (P < 0.001). Prececal disappearance of Cys was lower (P < 0.05) in the PCa- treatment than in PCa1and PCa-Phy3000 treatment. Expression of MUC2 in the duodenum-jejunum was higher (P < 0.05) in the PCa-Phy3000 treatment than in other treatments. Phytase-induced hydrolysis of phytate led to elevated digesta and plasma MI concentrations and reduced digesta concentrations of phytate breakdown intermediates.

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A type IV translocated Legionella cysteine phytase counteracts intracellular growth restriction by phytate.

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A type IV translocated Legionella cysteine phytase counteracts intracellular growth restriction by phytate.

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