The Legionella autoinducer synthase LqsA produces an alpha-hydroxyketone signaling molecule

Abstract

The opportunistic pathogen Legionella pneumophila replicates in human lung macrophages and in free-living amoebae. To accommodate the transfer between host cells, L. pneumophila switches from a replicative to a transmissive phase. L. pneumophila harbors a gene cluster homologous to the Vibrio cholerae cqsAS quorum sensing system, encoding a putative autoinducer synthase (lqsA) and a sensor kinase (lqsS), which flank a response regulator (lqsR). LqsR is an element of the L. pneumophila virulence regulatory network, which promotes pathogen-host cell interactions and inhibits entry into the replicative growth phase. Here, we show that lqsA functionally complements a V. cholerae cqsA autoinducer synthase deletion mutant and, upon expression in L. pneumophila or Escherichia coli, produces the diffusible signaling molecule LAI-1 (Legionella autoinducer-1). LAI-1 is distinct from CAI-1 (Cholerae autoinducer-1) and was identified as 3-hydroxypentadecan-4-one using liquid chromatography coupled to high resolution tandem mass spectrometry. The activity of both LqsA and CqsA was abolished upon mutation of a conserved lysine, and covalent binding of the cofactor pyridoxal 5'-phosphate to this lysine was confirmed by mass spectrometry. Thus, LqsA and CqsA belong to a family of pyridoxal 5'-phosphate-dependent autoinducer synthases, which produce the alpha-hydroxyketone signaling molecules LAI-1 and CAI-1.

FIGURE 1.

The lqs gene cluster and model of the L. pneumophila quorum sensing circuit. A, chromosomal map of the L. pneumophila lqs gene cluster. The putative autoinducer lqsA and sensor kinase lqsS are 45 and 29% identical to V. cholerae cqsA and cqsS, respectively. LqsR encodes a putative response regulator (29), and the E. coli homologue of hdeD is possibly involved in acid resistance (55). B, model of the L. pneumophila autoinducer circuit, including LqsA, the low molecular weight diffusible signaling molecule LAI-1, the cognate sensor kinase LqsS, and the response regulator LqsR. The expression of LqsR is controlled by the alternative σ factor RpoS and the two-component system LetA/LetS. Dashed lines indicate putative pathways and links.

FIGURE 2.

Prevalence of lqsA in L. pneumophila strains and gene expression analysis. A, the presence of lqsA was assessed by PCR amplification of the genes from genomic DNA of laboratory wild-type L. pneumophila strains (JR32, AA100, Corby) and clinical (502, 509, 514) and environmental (529, 534, 535) L. pneumophila isolates as well as L. bozemanii, L. rubrilucens, and L. taurinensis. As a positive control, the 16 S rRNA gene was amplified. B, the expression of lqsA in L. pneumophila JR32 was determined by semiquantitative reverse transcription-PCR. In AYE broth lqsA was expressed preferentially in the replicative (R) rather than in the stationary (S) growth phase, and in A. castellanii lqsA was expressed during replication (17 h). gDNA, genomic DNA. Similar results were obtained in at least two independent experiments. kb, kilobase.

FIGURE 3.

Production of diffusible, volatile signaling molecules by LqsA and CqsA. A, LqsA partially complements cell density-dependent gene expression of a V. cholerae cqsA mutant. L. pneumophila lqsA or V. cholerae cqsA was expressed in the V. cholerae CAI-1 reporter strain MM920 by introducing pTS-2 (pLqsA) or pTS-6 (pCqsA), respectively. The emission of light (relative units) was quantified by luminescence. B, signal activity is produced in E. coli upon heterologous expression of LqsA or CqsA. His-LqsA or His-CqsA were produced under the control of the P_T7 promoter in E. coli BL21(DE3) harboring plasmid pTS-21 (pLqsA) or pTS-22 (pCqsA), and the bacterial supernatants were assayed for autoinducer activity by bioluminescence using V. cholerae MM920. C, the signaling molecules produced by LqsA and CqsA are partially volatile at room temperature. E. coli BL21(DE3) harboring plasmid pTS-21 (pLqsA) or pTS-22 (pCqsA) was placed in the 4 central wells of a 96-well plate surrounded by V. cholerae MM920 and incubated for 4 h before the determination of bioluminescence. As a control the wells were covered with plastic foil. Means and S.D. of triplicates are shown (A and B). Similar results were obtained in at least three independent experiments.

FIGURE 4.

LqsA and CqsA produce a distinct pattern of hydroxyketone molecules. Selected-ion mass spectrometry chromatograms display the patterns of hydroxyketone molecules produced by E. coli BL21(DE3) harboring pTS-21 (pLqsA) or pTS-22 (pCqsA), L. pneumophila containing pTS-2 (pLqsA), or wild-type V. cholerae MM227. Extracted supernatants were treated with O-PFB and analyzed by LC-APCI-MS. In supernatants ofE. coli or L. pneumophila producing LqsA, the C₁₅ compound was detected as the major product and termed LAI-1. Supernatants of E. coli producing CqsA or supernatants of V. cholerae contained high levels of CAI-1 (C₁₃) and its C₁₁ homologue.

FIGURE 5.

Identification of LAI-1 as 3-hydroxypentadecan-4-one. A, the chemical structures of CAI-1 and LAI-1 are shown. Treatment with O-PFB leads to the oxime adducts depicted, which will yield the proposed fragment ions upon collision-induced dissociation. B, the MS/MS spectra for the CAI-1 and LAI-1 oxime derivatives are depicted. The complete fragmentation spectra including the dominating but unspecific [M-18+H]⁺ fragment ion are shown in the insets. The major peaks of the LqsA sample correspond well to the predicted fragmentation products and are analogous to the ions produced by fragmentation of CAI-1, confirming the identity of LAI-1. Each mass peak is labeled with the exact measured m/z value, the predicted elemental composition, and the difference between the measured and calculated m/z values in ppm, respectively.

FIGURE 6.

Synthesis of active CAI-1 by CqsA and LqsA. A, selected-ion chromatograms for CAI-1 and LAI-1 display the main products of CqsA and LqsA upon expression in E. coli BL21(DE3). The peaks representing the two main autoinducer molecules are labeled, and their elution times are indicated. The identity of CAI-1 and LAI-1 as hydroxyketones was confirmed by MS/MS (data not shown). B, activity of the eluted fractions was determined with the V. cholerae CAI-1 reporter strain MM920. In both samples the activity was exclusively retained in the fraction containing CAI-1, which indicates that LAI-1 does not contribute to the activation of the reporter. The error bars represent S.D. for duplicates.

FIGURE 7.

LqsA and CqsA are pyridoxal 5′-phosphate-dependent enzymes. A, the PLP precursor pyridoxine was added at the concentrations indicated to E. coli BL21(DE3) harboring plasmid pTS-21 (His-LqsA), and autoinducer activity in cell supernatants was determined by bioluminescence using the V. cholerae CAI-1 reporter strain MM920. The data shown are the means and S.D. of 10 samples. Asterisks denote the significance of differences relative to the untreated sample (^*, p < 0.01; ^**, p < 0.001; two-tailed Student's t test). Similar results were obtained in three independent experiments. B, supernatants of E. coli strain BL21(DE3) harboring plasmid pTS-21 (pLqsA), pTS-22 (pCqsA), pTS-25 (pLqsA_K258A), pTS-26 (pCqsA_K236A), or plasmid pET-28a(+) (vector) were assayed by bioluminescence using V. cholerae MM920. The data shown are the means and S.D. of triplicates and are representative of three independent experiments. C, covalent binding of PLP to purified His-LqsA, His-LqsA_K258A, His-CqsA, or His-CqsA_K236A was determined by electrospray ionization-time of flight-MS.