Brucella abortus synthesizes phosphatidylcholine from choline provided by the host

Abstract

The Brucella cell envelope is characterized by the presence of phosphatidylcholine (PC), a common phospholipid in eukaryotes that is rare in prokaryotes. Studies on the composition of Brucella abortus 2308 phospholipids revealed that the synthesis of PC depends on the presence of choline in the culture medium, suggesting that the methylation biosynthetic pathway is not functional. Phospholipid composition of pmtA and pcs mutants indicated that in Brucella, PC synthesis occurs exclusively via the phosphatidylcholine synthase pathway. Transformation of Escherichia coli with an expression vector containing the B. abortus pcs homologue was sufficient for PC synthesis upon induction with IPTG (isopropyl-beta-d-thiogalactopyranoside), while no PC formation was detected when bacteria were transformed with a vector containing pmtA. These findings imply that Brucella depends on choline provided by the host cell to form PC. We could not detect any obvious associated phenotype in the PC-deficient strain under vegetative or intracellular growth conditions in macrophages. However, the pcs mutant strain displays a reproducible virulence defect in mice, which suggests that PC is necessary to sustain a chronic infection process.

FIG. 1.

(A) Comparison of pmtA homologues from B. abortus 2308 (B.a.), B. melitensis 16 M (B.m.), B. suis 1330 (B.s.), and Sinorhizobium meliloti (S.m.). Identical residues are marked in black. The consensus motif for SAM-dependent methyltransferases is indicated on the top line. (B) ClustalW alignments of pcs from the selected species mentioned above. The characteristic residues for the CDP-alcohol phosphatidyltransferases are indicated by asterisks.

FIG. 2.

2D-TLC analysis of total lipids from B. abortus 2308 and its isogenic mutant pmtA, pcs, and pmtA pcs strains. Cells were cultured in G-W medium with or without choline in the presence of [¹⁴C]acetate, and the lipids were extracted and separated by 2D-TLC. The lipids PC, PE, OL, CL, and PG are indicated.

FIG. 3.

Lipid analysis after expression of Brucella pmtA and pcs in E. coli. E. coli BL21 transformed with pET-pmtA (lanes 2 to 5) or with pET-pcs (lanes 6 to 9) was grown in LB (lanes 2, 4, 6, and 8) or LB plus IPTG (lanes 3, 5, 7, and 9), and after extraction, lipids were separated by one-dimensional TLC. B. abortus 2308 (lane 1) grown in TSB and phosphatidylethanolamine (lane C) were run as a control. The lipids PC, PE, CL, and PG are indicated.

FIG. 4.

Virulence of B. abortus 2308 pBBR4 (black squares), the pcs mutant (open triangles), and the complemented pcs pBBR-pcs strain in BALB/c mice. Mice were infected by intraperitoneal injection. Individual spleen CFU values were plotted, and the horizontal bars represent the median CFU for each treatment group. *, P < 0.05 (compared to the group that received the wild-type strain). Statistical analysis was performed with a Kruskal-Wallis test. p.i., postinfection.