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.

FIG. 1

Acid phosphatase activity produced by L. pneumophila strains. Late-log-phase (OD₆₆₀ = 1.8 to 1.9) supernatants (A) and cell lysates (B) of wild-type strain 130b (WT), pilD mutant NU243, NU254, NU255, and NU256 were examined for their ability to release pNP from pNPP at pH 5. Bars represent the means ± standard deviation of the activity found in three cultures and are representative of the results seen in three independent experiments.

FIG. 2

Lipase/esterase and pNPPC hydrolase activity secreted by L. pneumophila strains. Late-log-phase (OD₆₆₀ = 1.8 to 1.9) supernatants of wild-type 130b (WT), NU254, NU255, and NU256 were tested for their ability to release pNP from p-nitrophenyl palmitate (A) and pNPPC (B). Bars represent the means ± standard deviation of the activity found in three cultures and are representative of the results obtained in three independent experiments. Similar to the results in panel A, the three mutants were not defective in the hydrolysis of p-nitrophenyl caprylate (data not shown)

FIG. 3

Nucleotide sequence of the L. pneumophila 130b map locus. The deduced amino acid sequences of the various ORFs and the termination codons (#) are indicated. For the predicted Map protein, the putative signal peptide is indicated in bold, and conserved protein domains are shadowed. Within the conserved domains, asterisks indicate conserved amino acids, and bold asterisks indicate amino acids that are essential for activity in the E. coli periplasmic acid phosphatase. Putative promoter sequences are indicated by the −10 and −35 designations, and the Shine-Dalgarno sequence is indicated (SD). The mini-Tn10 insertions are indicated by an arrow alongside the name of the corresponding mutant. The positions of the insertions are approximate for NU254 and NU256. Primers 2 and 3 (see Materials and Methods) are shown in italics at their binding regions.

FIG. 4

Acid and alkaline phosphatase activity in L. pneumophila cultures. Supernatants and cell lysates from the wild type (bar 1) and map mutants NU254 (bar 2), NU255 (bar 3), and NU256 (bar 4) were examined for their ability to hydrolyze pNPP at pH 5 and 10. Bars represent the means ± standard deviation of three cultures. The only significant difference between the wild type and mutants was observed with supernatants tested at pH 5 (P < 0.01).

FIG. 5

L. pneumophila map mutants lack a tartrate-sensitive acid phosphatase. Supernatants from wild-type 130b (●), the pilD mutant NU243 (□), and the mutants NU254 (▴) and NU255 (▾) were examined for their ability to hydrolyze pNPP in the presence of different concentrations of molybdate or tartrate. The isolated symbols represent the level of activity without inhibitor. The results are the means ± standard deviation of the activity found in three cultures. Similar results were obtained in two independent experiments using concentrated supernatants (data not shown).

FIG. 6

Effect of molybdate and tartrate on the cloned L. pneumophila Map activity. Whole cultures of E. coli DH5α(pVA12) (◊) and DH5α(pVA13) (⧫) were tested for acid phosphatase activity in acetate buffer (pH 5.5) in the presence of different concentrations of sodium molybdate (top panel) or sodium tartrate (bottom panel). The results are expressed as the percentage of refractive activity and are the means ± standard deviation of the activity found in three cultures and representative of two independent experiments. The error bars are too small to be seen.

FIG. 7

Cytopathic effect of L. pneumophila on U937 cells. At different times after inoculation, the viability of the macrophage monolayer that had been infected at a multiplicity of infection of 0.1 with 130b (●), pilD mutant NU243 (■), NU254 (▵), or NU255 (▿) was measured by fluorescence associated with alamar blue reduction. As a control, an uninfected monolayer (○) was processed in parallel. Results represent the means ± standard deviation of triplicate wells and are representative of two independent experiments.

FIG. 8

Intracellular infection by wild-type and mutant L. pneumophila. U937 cells (A) and H. vermiformis amoebae (B) were infected at multiplicities of infection of 0.1 and 1, respectively, with wild-type 130b (●), NU254 (▵), or NU255 (▿). The number of bacteria in each well was quantitated at 0, 24, 48, and 72 h by plating aliquots on BCYE agar. Results represent the means ± standard deviation of triplicate wells and are representative of two independent experiments. In a third U937 experiment, the numbers of bacteria were also determined at 6 and 18 h (inset).