The global regulatory proteins LetA and RpoS control phospholipase A, lysophospholipase A, acyltransferase, and other hydrolytic activities of Legionella pneumophila JR32

Abstract

Legionella pneumophila possesses a variety of secreted and cell-associated hydrolytic activities that could be involved in pathogenesis. The activities include phospholipase A, lysophospholipase A, glycerophospholipid:cholesterol acyltransferase, lipase, protease, phosphatase, RNase, and p-nitrophenylphosphorylcholine (p-NPPC) hydrolase. Up to now, there have been no data available on the regulation of the enzymes in L. pneumophila and no data at all concerning the regulation of bacterial phospholipases A. Therefore, we used L. pneumophila mutants in the genes coding for the global regulatory proteins RpoS and LetA to investigate the dependency of hydrolytic activities on a global regulatory network proposed to control important virulence traits in L. pneumophila. Our results show that both L. pneumophila rpoS and letA mutants exhibit on the one hand a dramatic reduction of secreted phospholipase A and glycerophospholipid:cholesterol acyltransferase activities, while on the other hand secreted lysophospholipase A and lipase activities were significantly increased during late logarithmic growth phase. The cell-associated phospholipase A, lysophospholipase A, and p-NPPC hydrolase activities, as well as the secreted protease, phosphatase, and p-NPPC hydrolase activities were significantly decreased in both of the mutant strains. Only cell-associated phosphatase activity was slightly increased. In contrast, RNase activity was not affected. The expression of plaC, coding for a secreted acyltransferase, phospholipase A, and lysophospholipase A, was found to be regulated by LetA and RpoS. In conclusion, our results show that RpoS and LetA affect phospholipase A, lysophospholipase A, acyltransferase, and other hydrolytic activities of L. pneumophila in a similar way, thereby corroborating the existence of the LetA/RpoS regulation cascade.

FIG. 1.

Lipolytic activities of L. pneumophila JR32 wild-type and ΔletA and ΔrpoS mutant, complemented ΔletA, and complemented ΔrpoS strains. Late-logarithmic (A) culture supernatants as well as late-logarithmic (B) cell lysates were incubated with DPPC, DPPG, MPLPG, MPLPC, and 1-MPG for 5 h at 37°C. The release of free fatty acids was quantified. Data are expressed as differences between the amount of free fatty acids released by culture samples and the amount released by BYE broth for supernatant samples and 40 mM Tris-HCl (pH 7.5, 25°C) for cell lysates. The results represent the means and standard deviations of duplicate cultures and four reactions and are representative of three independent experiments. For all substrates, ΔletA and ΔrpoS mutants were significantly different from the wild type (P < 0.01; Student's t test, n = 4), except for 1-MPG hydrolysis by cell lysates. (C) TLC analysis of GCAT activity of L. pneumophila JR32 wild-type, ΔletA and ΔrpoS mutant, and complemented ΔletA and ΔrpoS culture supernatants. Culture supernatants were incubated with a mixture of DPPG and cholesterol for 23 h at 37°C, and then lipids were extracted and applied to TLC. A mixture of BYE and the lipids was also incubated and served as a negative control (BYE). An apolar solvent mixture (solvent 1) was employed for the separation of the apolar lipids, in particular for cholesterol ester. For the qualitative identification of the lipid spots, lanes containing lipid standards are marked St. The results are representative of three independent experiments. (D) TLC analysis of an uncharacterized cholesterol-independent reaction product appearing in the acyltransferase assay with L. pneumophila JR32 wild-type, ΔletA and ΔrpoS mutant, and complemented ΔletA and ΔrpoS cell lysates. Cell lysates of the strains were incubated with a mixture of DPPG and cholesterol for 23 h at 37°C, and then lipids were extracted and applied to TLC. An apolar solvent mixture (solvent 2) was employed for the separation of the apolar lipids. The results are representative of three independent experiments. Abbreviations: CholE, cholesterolester; TPG, tripalmitoylglycerol; FFA, free fatty acids; Chol, cholesterol; JR32, L. pneumophila JR32; letA, L. pneumophila JR32 ΔletA; rpoS, L. pneumophila JR32 ΔrpoS; letA C, complemented L. pneumophila JR32 ΔletA; rpoS C, complemented L. pneumophila JR32 ΔrpoS.

FIG. 2.

Protease activities of late-logarithmic-phase culture supernatants of L. pneumophila JR32 wild-type, ΔletA and ΔrpoS mutant strains, and complemented ΔletA and ΔrpoS strains. Late-logarithmic-phase culture supernatants were incubated with azocasein for 1 h at 37°C and azocasein hydrolysis was evaluated. BYE was treated like culture supernatant samples and served as a negative control. Data are expressed as differences between OD₄₂₀ values of culture samples and the OD₄₂₀ value of BYE broth. The results represent the means and standard deviations of duplicate cultures and are representative of three independent experiments. ΔletA and ΔrpoS mutants were significantly different from the wild type (P < 0.01; Student's t test, n = 4). Abbreviations: JR32, L. pneumophila JR32; letA, L. pneumophila JR32 ΔletA; rpoS, L. pneumophila JR32 ΔrpoS; letA C, complemented L. pneumophila JR32 ΔletA; rpoS C, complemented L. pneumophila JR32 ΔrpoS.

FIG. 3.

Phosphatase activities of late-logarithmic-phase culture samples of L. pneumophila JR32 wild-type, ΔletA and ΔrpoS mutant strains, and complemented ΔletA and ΔrpoS strains. Late-logarithmic (A) culture supernatants, as well as late-logarithmic (B) cell lysates were incubated with p-NPP for 1 h at 37°C and hydrolysis of p-NPP was evaluated. BYE and 40 mM Tris-HCl (pH 7.5, 25°C) were treated in the same way as the bacterial samples and served as negative controls for culture supernatants and cell lysates, respectively. Data are expressed as differences between the amount of p-NP released by culture samples and the amount released by BYE broth for supernatant samples and 40 mM Tris-HCl (pH 7.5, 25°C) for cell pellet lysates. These results represent the means and standard deviations of duplicate cultures and are representative of three independent experiments. ΔletA and ΔrpoS mutants were significantly different from the wild type in all experiments (P < 0.01; Student's t test, n = 4) except the ΔletA culture supernatant. Abbreviations: JR32, L. pneumophila JR32; letA, L. pneumophila JR32 ΔletA; rpoS, L. pneumophila JR32 ΔrpoS; letA C, complemented L. pneumophila JR32 ΔletA; rpoS C, complemented L. pneumophila JR32 ΔrpoS.

FIG. 4.

p-NPPC hydrolase activities of late-logarithmic-phase culture samples of L. pneumophila JR32 wild-type, ΔletA and ΔrpoS mutant strains, and complemented ΔletA and ΔrpoS strains. Late-logarithmic (A) culture supernatants, as well as late-logarithmic (B) cell lysates were incubated with p-NPPC for 44 h at 37°C and hydrolysis of p-NPPC was evaluated. BYE and 40 mM Tris-HCl (pH 7.5 25°C) were treated in the same way as the bacterial samples and served as negative controls for culture supernatants and cell pellet lysates, respectively. Data are expressed as differences between the amount of p-NP released by culture samples and the amount released by BYE broth for supernatant samples and 40 mM Tris-HCl (pH 7.5 25°C) for cell lysates. The results shown here represent the means and standard deviations of duplicate cultures and are representative of three independent experiments. ΔletA and ΔrpoS mutants were significantly different from the wild type in all experiments (P < 0.01; Student's t test, n = 4). Abbreviations: JR32, L. pneumophila JR32; letA, L. pneumophila JR32 ΔletA; rpoS, L. pneumophila JR32 ΔrpoS; letA C, complemented L. pneumophila JR32 ΔletA; rpoS C, complemented L. pneumophila JR32 ΔrpoS.

FIG. 5.

RNase activities of L. pneumophila JR32, ΔletA and ΔrpoS mutant strains, and complemented ΔletA and ΔrpoS strains. Bacterial cell suspensions of the strains were applied to RNA-containing BSYE agar plates and grown for 2 days at 37°C. Then, plates were covered with 10% trichloroacetic acid in order to visualize clear zones in which RNA was hydrolyzed. L. pneumophila strain 130b and its derivate ΔlspDE which is impaired in RNase secretion were used as a negative control by which the effect of the lacking RNase should be demonstrated. The results shown here are representative of three independent experiments. Abbreviations: JR32, L. pneumophila JR32; letA, L. pneumophila JR32 ΔletA; rpoS, L. pneumophila JR32 ΔrpoS; letA C, complemented L. pneumophila JR32 ΔletA; rpoS C, complemented L. pneumophila JR32 ΔrpoS; 130b, L. pneumophila 130b; lspDE, L. pneumophila 130b ΔlspDE.

FIG. 6.

Expression of plaC in L. pneumophila JR32, ΔletA and ΔrpoS mutant strains, and complemented ΔletA and ΔrpoS strains. RNA was isolated from L. pneumophila late logarithmic growth phase cultures and used for quantitative RT-PCR with specific primers for L. pneumophila plaC. Specific primers for L. pneumophila gyr, the constitutively expressed gene coding for subunit B of gyrase, were used as an internal control. The quantity of RNA applied in the experiment was 0.25 μg. A: 24 PCR cycles; B: 28 PCR cycles. The results shown here are representative of two independent experiments. JR32, L. pneumophila JR32; letA, L. pneumophila JR32 ΔletA; rpoS, L. pneumophila JR32 ΔrpoS; letA C, complemented L. pneumophila JR32 ΔletA; rpoS C, complemented L. pneumophila JR32 ΔrpoS; DNA, JR32 genomic DNA.