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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. The map mutants grew within macrophage-like U937 cells and Hartmannella amoebae to the same degree as did wild-type legionellae, indicating that this acid phosphatase is not essential for L. 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.

SUBMITTER: Aragon V

PROVIDER: S-EPMC97870 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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Legionella pneumophila major acid phosphatase and its role in intracellular infection.

Aragon V V Kurtz S S Cianciotto N P NP

Infection and immunity 20010101 1

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 hyd ...[more]

PMID: 11119504

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Project description:We have previously isolated 32 mutants of Legionella pneumophila that are defective in the infection of mammalian cells but not protozoa. The mutated loci have been designated macrophage-specific infectivity (mil) loci. In this study we characterized the mil mutant GK11. This mutant was incapable of growth within U937 macrophage-like cells and WI-26 alveolar epithelial cells. This defect in intracellular replication correlated with a defect in cytopathogenicity to these cells. Sequence analysis of the GK11 locus revealed it to be highly similar to rep helicase genes of other bacteria. Since helicase mutants of Escherichia coli are hypersensitive to thymine starvation, we examined the sensitivity of GK11 to thymineless death (TLD). In the absence of thymine and thymidine, mutant GK11 did not undergo TLD but was defective for in vitro growth, and the defect was partially restored when these compounds were added to the growth medium. In addition, supplementation with thymidine or thymine partially restored the ability of GK11 to grow within and kill U937 macrophage-like cells. The data suggested that the low levels of thymine or thymidine in the L. pneumophila phagosome contributed to the defect of GK11 within macrophages. Using confocal laser scanning microscopy, we determined the effect of the mutation in the Rep helicase homologue on the intracellular trafficking of GK11 within macrophages. In contrast to the wild-type strain, phagosomes harboring GK11 colocalized with several late endosomal/lysosomal markers, including LAMP-1, LAMP-2, and cathepsin D. In addition, only 50% of the GK11 phagosomes colocalized with the endoplasmic reticulum marker BiP 4 h postinfection. Colocalization of BiP with GK11 phagosomes was absent 6 h postinfection, while 90% of the wild-type phagosomes colocalized with this marker at both time points. We propose that the low level of thymine within the L. pneumophila phagosome in combination with simultaneous exposure to multiple stress stimuli results in deleterious mutations that cannot be repaired in the rep helicase homologue mutant, rendering it defective in intracellular replication.

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Legionella pneumophila major acid phosphatase and its role in intracellular infection.

Publications

Legionella pneumophila major acid phosphatase and its role in intracellular infection.

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