Expression of Legionella pneumophila paralogous lipid A biosynthesis genes under different growth conditions

Abstract

Legionella pneumophila is an opportunistic pathogen that in the environment colonizes biofilms and replicates within amoebae. The bacteria employ the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system to grow intracellularly in a specific vacuole. Using Acanthamoeba castellanii as a host cell, we have previously identified lcsC (Legionella cytotoxic suppressor), a paralogue of the lipid A disaccharide synthase lpxB, as a cytotoxic factor of L. pneumophila. A bioinformatic analysis of the genome revealed that L. pneumophila is unique in harbouring two paralogues of lpxB and two and three paralogues of the lipid A biosynthesis acyltransferases lpxA and lpxD, respectively. LcsC (lpxB1) forms a transcriptional unit with gnnA, encoding a putative UDP-GlcNAc oxidase in the biosynthetic pathway leading to 3-aminoglucosamine analogues of lipid A. LpxB2 clusters with lpxD2, lpxA2 and lpxL paralogues, encoding secondary acyltransferases. LcsC/lpxB1 and lpxB2 were found to partially complement the growth defect of an Escherichia coli lpxB conditional mutant strain, indicating that both corresponding enzymes possess lipid A disaccharide synthase activity. The two L. pneumophila lpxB paralogues are not functionally equivalent, since expression of lcsC/lpxB1 but not lpxB2 in an L. pneumophila icmG mutant is cytotoxic for A. castellanii, and LPS purified from the two strains triggers CD14-dependent tumour necrosis factor (TNF)a production by macrophages with a different potency. The lpxB and lpxA paralogues are expressed under various growth conditions, including broth, biofilms and in A. castellanii. While the flagellar gene flaA is mainly expressed in late stationary phase, the lpxB and lpxA paralogues are preferentially expressed in the exponential and early stationary phases. Upon exposure to hypotonic stress and nutrient deprivation, lpxA1, and to a lesser extent lcsC/lpxB1, is upregulated. The differential regulation of lpxB or lpxA paralogues in response to changing environmental conditions might allow L. pneumophila to adapt its lipid A structure.Abbreviations: ACES, N-(2-acetamido)-2-aminoethanesulfonic acid; ACP, acyl carrier protein; Icm/Dot, intracellular multiplication/defective organelle trafficking; GlcN, 2-amino-2,3-dideoxy-a-D-glucopyranose (glucosamine); GlcN3N, 2,3-diamino-2,3-dideoxy-a-D-glucopyranose; GlcNAc, ; 2-acetamido-2,3-dideoxy-a-D-glucopyranose (N-acetylglucosamine); GlcNAc3N, 2-acetamido-3-amino-2,3-dideoxy-a-D-glucopyranose; Kdo, 3-deoxy-D-manno-oct-2-ulosonic acid; lcs, Legionella cytotoxic suppressor; PI, propidium iodide; TNF, tumour necrosis factor. 3These authors contributed equally to this work.Supplementary tables listing bacterial strains and plasmids, oligonucleotides used in this study, paralogues of LpxA-C in different bacteria and genomic arrangements of selected lipid A biosynthesis genes in three L. pneumophila strains, and a supplementary figure showing cotranscription of lcsC/lpxB1 and gnnA, are available with the online version of this...

Rab1 Guanine Nucleotide Exchange Factor SidM Is a Major Phosphatidylinositol 4-Phosphate-binding Effector Protein of Legionella pneumophila

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Synergistic Contribution of theLegionella pneumophila lqsGenes to Pathogen-Host Interactions

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The Legionella Autoinducer Synthase LqsA Produces an α-Hydroxyketone Signaling Molecule

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