Secreted enzymatic activities of wild-type and pilD-deficient Legionella pneumophila

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 a pilD-deficient strain, we sought to define other secreted proteins absent in the mutant. Based upon the release of p-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.

FIG. 1

Acid phosphatase, protease, and pNPPC-hydrolase activity within L. pneumophila wild-type and pilD mutant supernatants. (A) Wild-type 130b (●) and mutant NU243 (□) were inoculated into BYE broth, and then bacterial growth was monitored spectrophotometrically. After various times of incubation, filtered culture supernatants were examined for phosphatase activity at pH 5 (B), protease activity against azocasein (C), and hydrolysis of pNPPC (D). The values given represent the mean and standard deviations from triplicate cultures. Significant differences in enzyme activity were most evident during late-log and early-stationary phases, i.e., P < 0.001 and 0.005 for the 10- and 13-h time points, respectively, in panels B and C, and P < 0.001 for the 13 h time point in panel D (Student's t test). The increasing levels of enzymatic activity in the older mutant cultures likely reflect inevitable cell lysis (see Fig. 2). Comparable results were obtained in an additional, identical experiment. Single readings of late-log cultures were also obtained on at least five additional occasions (see Fig. 2 and data not shown).

FIG. 2

Acid phosphatase secretion by and accumulation within L. pneumophila wild-type and pilD mutant bacteria. Wild-type 130b (black columns) and mutant NU243 (white columns) were grown in BYE broth, and then, at late log phase, culture supernatants (left) and cell lysates (right) were examined for phosphatase activity at pH 5. The values presented are the mean and standard deviations from triplicate cultures. For both the supernatant and lysate comparisons, the differences in strain activity were significant (P < 0.001, Student's t test). Similar results were obtained on six additional occasions.

FIG. 3

Secretion, accumulation, and substrate specificity of a L. pneumophila esterase-lipase. (A) 130b (black columns) and NU243 (white columns) were grown in BYE broth, and then, at late log phase, culture supernatants (left) and cell lysates (right) were examined for their ability to cleave p-nitrophenyl caprylate. (B) In a separate experiment, late-log supernatants from wild-type and pilD mutant cultures were compared for their ability to cleave p-nitrophenyl caprylate (pNPC) (left) versus p-nitrophenyl palmitate (pNPPa) (right). The values presented are the mean and standard deviations from triplicate cultures. For both the supernatant and lysate comparisons, the differences between 130b and NU243 activity were significant (P < 0.001, Student's t test). The results obtained in panel A were observed on six other occasions, while those in panel B were seen in two other experiments.

FIG. 4

Activity of the L. pneumophila esterase upon monoacylglycerol. On two occasions (i.e., I and II), supernatants from 130b (black columns) and NU243 (white columns) late-log-phase cultures were examined for their ability to release free fatty acid (FFA) from monoacylglycerol as measured by the NEFA-C-Kit. The values presented are the mean and standard deviation from two cultures. Differences between 130b and NU243 activity were significant (P < 0.001, Student's t test).

FIG. 5

PLA secretion by wild-type and pilD mutant L. pneumophila. In two trials (I and II), supernatants from 130b (black column) and NU243 (white column) late-log cultures were tested for their ability to release free fatty acid (FFA) from phosphatidylcholine as measured by the NEFA-C-Kit. The values presented are the mean and standard deviations from duplicate cultures. The differences between 130b and NU243 activity were significant (P < 0.01 and < 0.001 for I and II, respectively; Student's t test). Similar conclusions were obtained by using thin-layer chromatography.

FIG. 6

Secreted activities of L. pneumophila pNPPC-hydrolase mutants. Wild-type (black columns) and mutant bacteria (hatched columns) were grown in BYE broth, and then, at late log phase, culture supernatants were examined for their relative abilities to cleave pNPPC (A), hide powder azure (B), p-nitrophenylphosphate (pNPP) (C), and p-nitrophenyl palmitate (pNPPa) (D). The activities for wild type are set at 100%. In each figure, the mutants' results are arranged in the same order, beginning with NU245 on the left and ending with NU253 on the right. For NU247 and NU253, similar results were obtained in one additional experiment. For all others, similar results were obtained on two other occasions.

FIG. 7

Macrophage infection by wild-type and mutant L. pneumophila. U937 cell monolayers were infected with approximately 5 × 10⁵ CFU of wild-type 130b (●), pilD mutant NU243 (□), and pNPPC-hydrolase mutants NU247 (◊) and NU253 (▵). CFU per well were quantitated at 0, 24, 48, and 72 h. Each datum point represents the mean and standard deviation for three monolayers. Significant differences in recovery between 130b and its mutant derivatives were evident at 24 h (P < 0.05) and beyond (P < 0.001). These differences were seen in three additional experiments (data not shown).

FIG. 8

Cytopathic effect of L. pneumophila strains on U937 cells. Replicate monolayers (n = 6) were either not infected (▴) or were infected with 10³ CFU of strain 130b (●), 5 × 10⁴ CFU of NU243 (□), 8 × 10⁴ CFU of NU247 (◊), or 6.5 × 10⁴ CFU of NU253 (▵). After various periods of incubation, the viability of the host cells was measured by neutral red uptake. Since the pilD mutant does not elicit any cytopathic effect within the typical 72-h infection assay (41), the monolayers were monitored for 96 h and were purposely infected with greater numbers of mutant relative to wild-type bacteria. Datum points represent the mean OD₅₄₀, and vertical bars indicate the standard deviations. Differences in cytopathic effect between 130b and its mutant derivatives were significant at 72 and 96 h after inoculation (P < 0.001, Student's t test). Similar conclusions were obtained from two additional experiments with neutral red and a third trial with alamar blue (data not shown).

FIG. 9

Infection of H. vermiformis by L. pneumophila strains. Wells containing Hartmannella amoebae were infected with approximately 5 × 10³ CFU of wild-type 130b (●), pilD mutant NU243 (□), and pNPPC-hydrolase mutants NU247 (◊) and NU253 (▵). Bacterial CFU per well were quantitated at 0, 24, 48, and 72 h after inoculation. Each datum point represents the mean and standard deviations for three wells. Significant differences in recovery between 130b and its mutant derivatives were evident at 48 and 72 h (P < 0.005, Student's t test). These differences were observed in two additional experiments (data not shown).