The Phospholipid N-Methyltransferase and Phosphatidylcholine Synthase Pathways and the ChoXWV Choline Uptake System Involved in Phosphatidylcholine Synthesis Are Widely Conserved in Most, but Not All Brucella Species

Abstract

The brucellae are facultative intracellular bacteria with a cell envelope rich in phosphatidylcholine (PC). PC is abundant in eukaryotes but rare in prokaryotes, and it has been proposed that Brucella uses PC to mimic eukaryotic-like features and avoid innate immune responses in the host. Two PC synthesis pathways are known in prokaryotes: the PmtA-catalyzed trimethylation of phosphatidylethanolamine and the direct linkage of choline to CDP-diacylglycerol catalyzed by the PC synthase Pcs. Previous studies have reported that B. abortus and B. melitensis possess non-functional PmtAs and that PC is synthesized exclusively via Pcs in these strains. A putative choline transporter ChoXWV has also been linked to PC synthesis in B. abortus. Here, we report that Pcs and Pmt pathways are active in B. suis biovar 2 and that a bioinformatics analysis of Brucella genomes suggests that PmtA is only inactivated in B. abortus and B. melitensis strains. We also show that ChoXWV is active in B. suis biovar 2 and conserved in all brucellae except B. canis and B. inopinata. Unexpectedly, the experimentally verified ChoXWV dysfunction in B. canis did not abrogate PC synthesis in a PmtA-deficient mutant, which suggests the presence of an unknown mechanism for obtaining choline for the Pcs pathway in Brucella. We also found that ChoXWV dysfunction did not cause attenuation in B. suis biovar 2. The results of these studies are discussed with respect to the proposed role of PC in Brucella virulence and how differential use of the Pmt and Pcs pathways may influence the interactions of these bacteria with their mammalian hosts.

Keywords: brucella; cell envelope; choline; phosphatidylcholine; phospholipid.

FIGURE 1

Model for phosphatidylcholine biosynthesis in B. suis biovar 2. In the Pcs pathway, the membrane protein Pcs condenses CDP-DAG and choline to generate PC. This choline is taken up by the ChoXWV transporter. Dashed arrows indicate steps for which putative genes have not been identified in Brucella. In the PmtA pathway, three successive methylations catalyzed by PmtA convert PE to PC. PmtA, phospholipid N-methyltransferase; Pcs, phosphatidylcholine synthase; ChoXWV, high-affinity choline transport system; SAM, S-adenosylmethionine; SAH, S-adenosylhomocysteine, CMP; cytidine monophosphate; CDP-DAG, cytidine diphosphate diacylglycerol; PS, phosphatidylserine; PE, phosphatidylethanolamine; MMPE, monomethylphosphatidylethanolamine; DMPE, dimethylphosphatidylethanolamine; PC, phosphatidylcholine.

FIGURE 2

Phosphatidylcholine synthesis in Bs2WT takes place through the methylation (PmtA) and the phosphatidylcholine synthase pathway (Pcs). HPTLC analysis of the free lipids of Bs2WT, Bs2Δpcs, Bs2ΔpmtA, and Bs2ΔpcsΔpmtA grown in TSB. OL, ornithine lipids; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; MMPE, mono-methyl-phosphatidylethanolamine; DMPE, dimethyl-phosphatidylethanolamine; PC, phosphatidylcholine.

FIGURE 3

Choline accumulation in Bs2WT, Bs2ΔchoX1, Bs2ΔchoX1ΔchoX2, Bs2ΔpmtAΔchoX1ΔchoX2, and B. canis grown in mGSM. Values are the mean ± standard error of technical duplicates of a representative experiment, repeated at least two times with similar results.

FIGURE 4

High-performance thin-layer chromatography analysis of the free lipids of B. canis and BcΔpmtA grown in TSB or mGSM. OL, ornithine lipids; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; MMPE, monomethyl-phosphatidylethanolamine; DMPE, dimethyl-phosphatidylethanolamine; PC, phosphatidylcholine; UL, unknown lipid.

FIGURE 5

High-performance thin-layer chromatography analysis of the free lipids of Bs2WT- and Bs2-derived (A) mutants grown in TSB and (B) mutants grown in mGSM. OL, ornithine lipids; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; MMPE, monomethyl-phosphatidylethanolamine; DMPE, dimethyl-phosphatidylethanolamine; PC, phosphatidylcholine; UL, unknown lipid.

FIGURE 6

High-performance thin-layer chromatography analysis of Bs2WT, Bs2ΔchoX1ΔchoX2, and Bs2ΔpmtAΔchoX1ΔchoX2 after in vivo labeling with [1,2^{− 14}C] acetate in mGSM. Total free lipids were revealed by charring (A) or autoradiography (B). OL, ornithine lipids; PG, phosphatidylglycerol; PE, phosphatidylethanolamine; MMPE, monomethyl-phosphatidylethanolamine; DMPE, dimethyl-phosphatidylethanolamine; PC, phosphatidylcholine.

FIGURE 7

Phosphatidylcholine synthesis is required for Bs2WT full virulence. CFUs in spleen of infected BALB/c mice were counted after intraperitoneal inoculation with 10⁵ CFU/mouse of Bs2WT (parental strain), Bs2Δpcs, Bs2ΔpmtA, Bs2ΔpcsΔpmtA, or Bs2ΔchoX1ΔchoX2. *p ≤ 0.05, **p ≤ 0.01, n.s. not significant.