Legionella pneumophila Major Acid Phosphatase and Its Role in Intracellular Infection

ABSTRACT Legionella pneumophila is an intracellular pathogen of protozoa and alveolar macrophages. This bacterium contains a gene (pilD) that is involved in both type IV pilus biogenesis and type II protein secretion. We previously demonstrated that the PilD prepilin peptidase is crucial for intracellular infection by L. pneumophila and that the secreted pilD-dependent proteins include a metalloprotease, an acid phosphatase, an esterase/lipase, a phospholipase A, and a p-nitrophenyl phosphorylcholine hydrolase. Since mutants lacking type IV pili, the protease, or the phosphorylcholine hydrolase are not defective for intracellular infection, we sought to determine the significance of the secreted acid phosphatase activity. Three mutants defective in acid phosphatase activity were isolated from a population of mini-Tn10-mutagenized L. pneumophila. Supernatants as well as cell lysates from these mutants contained minimal acid phosphatase activity while possessing normal levels of other pilD-dependent exoproteins. Genetic studies indicated that the gene affected by the transposon insertions encoded a novel bacterial histidine acid phosphatase, which we designated Map for major acid phosphatase. Subsequent inhibitor studies indicated that Map, like its eukaryotic homologs, is a tartrate-sensitive acid phosphatase. Themap mutants grew within macrophage-like U937 cells andHartmannella amoebae to the same degree as did wild-type legionellae, indicating that this acid phosphatase is not essential forL. pneumophila intracellular infection. However, in the course of characterizing our new mutants, we gained evidence for a second pilD-dependent acid phosphatase activity that, unlike Map, is tartrate resistant.

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ISOZYMES OF ACID PHOSPHATASE IN NORMAL AND CALMETTE-GUÉRIN BACILLUS-INDUCED RABBIT ALVEOLAR MACROPHAGES

Journal of Experimental Medicine ◽

10.1084/jem.128.5.1031 ◽

1968 ◽

Vol 128 (5) ◽

pp. 1031-1048 ◽

Cited By ~ 39

Author(s):

S. G. Axline

Keyword(s):

Acid Phosphatase ◽

Phosphatase Activity ◽

Alveolar Macrophages ◽

Acid Phosphatase Activity ◽

Enzyme Inhibitors ◽

Freezing And Thawing ◽

Ph Optimum ◽

Lysosomal Fraction ◽

Repeated Freezing ◽

Major Acid

The acid phosphatase activity of normal alveolar and BCG-induced alveolar macrophages has been examined. Five electrophoretically distinct forms of acid phosphatase have been identified in both normal and BCG-induced macrophages. The acid phosphatases can be divided into two major categories. One category, containing four distinct forms, is readily solubilized after repeated freezing and thawing or mechanical disruption The second category, containing one form, is firmly bound to the lysosomal membrane and can be solubilized by treatment of the lysosomal fraction with Triton X-100. The Triton-extractable acid phosphatase and the predominant aqueous soluble acid phosphatase have been shown to differ in the degree of membrane binding, in solubility, in net charge, and in molecular weight. The two pre-dominant phosphatases possess identical pH optimum and do not differ in response to enzyme inhibitors. BCG stimulation has been shown to result in a nearly twofold increase in acid phosphatase activity. A nearly proportionate increase in the major acid phosphatase forms has been observed.

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Secreted Enzymatic Activities of Wild-Type andpilD-Deficient Legionella pneumophila

Infection and Immunity ◽

10.1128/iai.68.4.1855-1863.2000 ◽

2000 ◽

Vol 68 (4) ◽

pp. 1855-1863 ◽

Cited By ~ 75

Author(s):

Virginia Aragon ◽

Sherry Kurtz ◽

Antje Flieger ◽

Birgid Neumeister ◽

Nicholas P. Cianciotto

Keyword(s):

Fatty Acids ◽

Free Fatty Acids ◽

Legionella Pneumophila ◽

Wild Type ◽

Intracellular Infection ◽

Type Iv ◽

Nitrophenyl Phosphate ◽

Pilus Biogenesis ◽

Legionnaires Disease

ABSTRACT Legionella pneumophila, the agent of Legionnaires' disease, is an intracellular pathogen of protozoa and macrophages. Previously, we had determined that the Legionella pilD gene is involved in type IV pilus biogenesis, type II protein secretion, intracellular infection, and virulence. Since the loss of pili and a protease do not account for the infection defect exhibited by apilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release ofp-nitrophenol (pNP) from p-nitrophenyl phosphate, acid phosphatase activity was detected in wild-type but not in pilD mutant supernatants. Mutant supernatants also did not release either pNP from p-nitrophenyl caprylate and palmitate or free fatty acid from 1-monopalmitoylglycerol, suggesting that they lack a lipase-like activity. However, since wild-type samples failed to release free fatty acids from 1,2-dipalmitoylglycerol or to cleave a triglyceride derivative, this secreted activity should be viewed as an esterase-monoacylglycerol lipase. The mutant supernatants were defective for both release of free fatty acids from phosphatidylcholine and degradation of RNA, indicating that PilD-negative bacteria lack a secreted phospholipase A (PLA) and nuclease. Finally, wild-type but not mutant supernatants liberated pNP from p-nitrophenylphosphorylcholine (pNPPC). Characterization of a new set of mutants defective for pNPPC-hydrolysis indicated that this wild-type activity is due to a novel enzyme, as opposed to a PLC or another known enzyme. Some, but not all, of these mutants were greatly impaired for intracellular infection, suggesting that a second regulator or processor of the pNPPC hydrolase is critical for L. pneumophila virulence.

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Type II Protein Secretion Is a Subset of the PilD-Dependent Processes That Facilitate Intracellular Infection byLegionella pneumophila

Infection and Immunity ◽

10.1128/iai.69.4.2092-2098.2001 ◽

2001 ◽

Vol 69 (4) ◽

pp. 2092-2098 ◽

Cited By ~ 82

Author(s):

Ombeline Rossier ◽

Nicholas P. Cianciotto

Keyword(s):

Legionella Pneumophila ◽

Enzymatic Activities ◽

Colony Morphology ◽

Type Ii Secretion ◽

Type Ii ◽

Intracellular Infection ◽

Type Iv ◽

Fold Reduction ◽

Intracellular Multiplication ◽

Prepilin Peptidase

ABSTRACT Previously, we had demonstrated that a Legionella pneumophila prepilin peptidase (pilD) mutant does not produce type IV pili and shows reduced secretion of enzymatic activities. Moreover, it displays a distinct colony morphology and a dramatic reduction in intracellular growth within amoebae and macrophages, two phenotypes that are not exhibited by a pilin (pilEL ) mutant. To determine whether thesepilD-dependent defects were linked to type II secretion, we have constructed two new mutants of L. pneumophila strain 130b. Mutations were introduced into either lspDE, which encodes the type II outer membrane secretin and ATPase, orlspFGHIJK, which encodes the pseudopilins. Unlike the wild-type and pilEL strains, bothlspDE and lspG mutants showed reduced secretion of six pilD-dependent enzymatic activities; i.e., protease, acid phosphatase, p-nitrophenol phosphorylcholine hydrolase, lipase, phospholipase A, and lysophospholipase A. However, they exhibited a colony morphology different from that of thepilD mutant, suggesting that their surfaces are distinct. The pilD, lspDE, and lspG mutants were similarly and greatly impaired for growth within Hartmannella vermiformis, indicating that the intracellular defect of the peptidase mutant in amoebae is explained by the loss of type II secretion. When assessed for infection of U937 macrophages, bothlsp mutants exhibited a 10-fold reduction in intracellular multiplication and a diminished cytopathic effect. Interestingly, thepilD mutant was clearly 100-fold more defective than the type II secretion mutants in U937 cells. These results suggest the existence of a novel pilD-dependent mechanism for promotingL. pneumophila intracellular infection of human cells.

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Erythrophagocytosis in the Liver in Hemolytic Anemias

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100100664 ◽

1968 ◽

Vol 26 ◽

pp. 184-185

Author(s):

O. T. Minick ◽

E. Orfei ◽

F. Volini ◽

G. Kent

Keyword(s):

Acid Phosphatase ◽

Oxidative Damage ◽

Phosphatase Activity ◽

Kupffer Cells ◽

Acid Phosphatase Activity ◽

Common Type ◽

Red Cell ◽

Cell Fragments ◽

Reticulo Endothelial System ◽

Important Site

Hemolytic anemias were produced in rats by administering phenylhydrazine or anti-erythrocytic (rooster) serum, the latter having agglutinin and hemolysin titers exceeding 1:1000.Following administration of phenylhydrazine, the erythrocytes undergo oxidative damage and are removed from the circulation by the cells of the reticulo-endothelial system, predominantly by the spleen. With increasing dosage or if animals are splenectomized, the Kupffer cells become an important site of sequestration and are greatly hypertrophied. Whole red cells are the most common type engulfed; they are broken down in digestive vacuoles, as shown by the presence of acid phosphatase activity (Fig. 1). Heinz body material and membranes persist longer than native hemoglobin. With larger doses of phenylhydrazine, erythrocytes undergo intravascular fragmentation, and the particles phagocytized are now mainly red cell fragments of varying sizes (Fig. 2).

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