The opportunistic pathogen Pseudomonas aeruginosa carries a secretable arachidonate 15-lipoxygenase

Abstract

In mammals, lipoxygenases play key roles in inflammation by initiating the transformation of arachidonic acid into potent bioactive lipid mediators such as leukotrienes and lipoxins. In general, most bacteria are believed to lack lipoxygenases and their polyunsaturated fatty acid substrates. It is therefore of interest that an ORF (PA1169) with high homology to eukaryotic lipoxygenases was discovered by analysis of the whole-genome sequence of the opportunistic bacterial pathogen Pseudomonas aeruginosa. Using TLC and liquid chromatography-UV-tandem mass spectrometry (LC-UV-MS-MS), we demonstrate that PA1169 encodes a bacterial lipoxygenase (LoxA) that converts arachidonic acid into 15-hydroxyeicosatetraenoic acid (15-HETE). Although mammalian lipoxygenases are cytoplasmic enzymes, P. aeruginosa LoxA activity is secreted. Taken together, these results suggest a mechanism by which a pathogen-secreted lipoxygenase may modulate host defense and inflammation via alteration of the biosynthesis of local chemical mediators.

Fig. 1.

(A) 5-LOs and 15-LOs dioxygenate specific carbons of arachidonic acid to form hydroperoxyeicosatetraenoic acids (HpETE), which can be rapidly reduced to hydroxyeicosatetraenoic acid (HETE, not shown) or can be transformed into other bioactive eicosanoids, e.g., leukotriene B₄ (LTB₄) or lipoxins (LX). (B) The first 50 aa of loxA were submitted to the signalp server (www.cbs.dtu.dk/services/SignalP/) (13), and the signal peptide score and signal sequence cleavage score for each amino acid were plotted. As shown, authentic signal peptides should produce high scores for the signal sequence region until the peak for the signal sequence cleavage score, after which the signal sequence score should decline. (C) Inferred amino acid sequence of P. aeruginosa PAO1 ORF PA1169 (loxA). The predicted signal sequence is indicated in boldface. Based on alignments with the rabbit reticulocyte 15-LO, a putative N-terminal domain is shaded, as are conserved amino acids likely involved in coordinating a non-heme iron (filled circles). The eight amino acid substitutions in the PA14 LoxA sequence are indicated above the corresponding amino acid in the PAO1 LoxA sequence.

Fig. 2.

(A) Low levels of loxA transcription in vitro. Overnight cultures of the indicated strains were diluted 1:100 into fresh LB and regrown with shaking at 37°C. At indicated time points, samples were taken for β-galactosidase assays. PAO1 loxA::lacZ and PA14 loxA::lacZ contain a transcriptional reporter fusion of lacZ (encodes β-galactosidase) to the chromosomal loxA gene of P. aeruginosa PAO1 and PA14, respectively. PAO1 and PA14 without the lacZ reporter were used as negative controls. PAO1 expressing a lacZ gene under the control of a constitutive lac promoter (integrated onto the chromosome at the neutral phage attachment site attB) was used as a positive control. (B) 15-HETE production by bacterial strains. Intact cells of the indicated bacterial strains were washed in PBS and incubated with [¹⁴C]arachidonic acid. Lipids were extracted and separated by TLC to detect 15-HETE. Soybean 15-LO was incubated with [¹⁴C]arachidonic acid as a positive control. A buffer-only sample (no bacteria, no enzyme) controlled for spontaneous oxidation products. pBBR-loxA^PAO1 and pBBR-loxA^PA14 are low-copy plasmids expressing the loxA gene of PAO1 and PA14, respectively, under the control of a lac promoter. The same plasmid or a control plasmid without insert (pBBR-control) was also transformed into E. coli strain SM10 as shown and tested in a separate experiment. (C) Apparent pH optimum for LoxA activity. Intact cells of P. aeruginosa PAO1 carrying the pBBR-loxA^PAO1 plasmid were resuspended in potassium phosphate buffers of varying pH and incubated with [¹⁴C]arachidonic acid. Lipids were extracted and analyzed by TLC. The percentage conversion of arachidonic acid to 15-HETE is plotted, with the percentage conversion measured in buffer of pH 6.8 taken as 100%.

Fig. 3.

LC-UV-MS-MS analysis of P. aeruginosa LoxA products. (A) The selective ion (at m/z 219 of MS-MS at m/z of 319) chromatogram showing 15-HETE and its in-line UV spectrum with absorbance maximum of 236 nm (Inset), characteristic of the conjugated diene in HETE. 15-HETE was identified based on its tandem MS-MS and UV spectra and LC retention time, as compared with authentic standards. (B) MS-MS spectra of the identified 15-HETE with diagnostic ions at m/z 219, 257 (M-H-H₂O-CO₂), 275 (M-H-CO₂), 301 (M-H-H₂O), and deprotonated parent ion 319 (M-H).

Fig. 4.

Secretion of P. aeruginosa LoxA activity. (A) P. aeruginosa strain PAO1 attB::lacZ carrying a loxA expression plasmid (pBBR-loxA^PAO1) or control plasmid was tested for LO activity by TLC as in Fig. 2. The asterisk (^*) indicates a nonspecific oxidation product routinely seen in all samples including buffer-only controls. A β-galactosidase assay was also performed on each fraction as a control for the fractionation procedure (β-galactosidase is a cytosolic enzyme). AA, arachidonic acid. (B) LoxA activity by LoxA-N-FLAG protein was assayed by TLC as in Fig. 2 and compared with the activity by untagged LoxA enzyme. Samples were sonicated where indicated. Samples from PAO1 pBBR-loxA and PAO1 pBBR-loxA-NFLAG were also subjected to Western blotting as indicated. (C) The pBBR-loxA expression plasmid was expressed in wild-type P. aeruginosa PAK or an isogenic Δxcp mutant, and LoxA activity was assayed by TLC as in Fig. 2. LO activity is expressed as the percentage of ¹⁴C-arachidonic acid converted to 15-HETE.