Glycerol-3-phosphate acquisition in spirochetes: distribution and biological activity of glycerophosphodiester phosphodiesterase (GlpQ) among Borrelia species

Abstract

Relapsing-fever spirochetes achieve high cell densities (>10(8)/ml) in their host's blood, while Lyme disease spirochetes do not (<10(5)/ml). This striking contrast in pathogenicity of these two groups of bacteria suggests a fundamental difference in their ability to either exploit or survive in blood. Borrelia hermsii, a tick-borne relapsing-fever spirochete, contains orthologs to glpQ and glpT, genes that encode glycerophosphodiester phosphodiesterase (GlpQ) and glycerol-3-phosphate transporter (GlpT), respectively. In other bacteria, GlpQ hydrolyzes deacylated phospholipids to glycerol-3-phosphate (G3P) while GlpT transports G3P into the cytoplasm. Enzyme assays on 17 isolates of borreliae demonstrated GlpQ activity in relapsing-fever spirochetes but not in Lyme disease spirochetes. Southern blots demonstrated glpQ and glpT in all relapsing-fever spirochetes but not in the Lyme disease group. A Lyme disease spirochete, Borrelia burgdorferi, that was transformed with a shuttle vector containing glpTQ from B. hermsii produced active enzyme, which demonstrated the association of glpQ with the hydrolysis of phospholipids. Sequence analysis of B. hermsii identified glpF, glpK, and glpA, which encode the glycerol facilitator, glycerol kinase, and glycerol-3-phosphate dehydrogenase, respectively, all of which are present in B. burgdorferi. All spirochetes examined had gpsA, which encodes the enzyme that reduces dihydroxyacetone phosphate (DHAP) to G3P. Consequently, three pathways for the acquisition of G3P exist among borreliae: (i) hydrolysis of deacylated phospholipids, (ii) reduction of DHAP, and (iii) uptake and phosphorylation of glycerol. The unique ability of relapsing-fever spirochetes to hydrolyze phospholipids may contribute to their higher cell densities in blood than those of Lyme disease spirochetes.

FIG. 1.

Spirochetemia of B. anserina with a very high cell density in the blood of a 12-day-old chicken 6 days after i.p. inoculation. The spirochetes far outnumber the nucleated red blood cells. Scale bar, 25 μm.

FIG. 2.

GlpQ-specific activity in Borrelia species. Each assay was performed six times, and the average activity is shown above each bar. Vertical lines represent one standard deviation above and below the mean activity. Only spirochetes in the relapsing-fever group had specific activity.

FIG. 3.

Comparison of the glp (glycerol transport and metabolism) locus in B. hermsii and B. burgdorferi. Both species contain glpF (glycerol facilitator), glpK (glycerol kinase), and glpA (G3PDH). B. hermsii has glpQ (glycerophosphodiester phosphodiesterase) and glpT (G3P transporter). These genes are replaced in B. burgdorferi with a small ORF (BB0242) that matches nothing in the database. These results suggest that B. hermsii, but not B. burgdorferi, has the potential to acquire G3P through the catabolism of deacylated phospholipids and to use this G3P in glycolysis and the synthesis of new phospholipids.

FIG. 4.

Distribution of glpQ among Borrelia species demonstrated by Southern blot analysis. (A) Agarose gel with genomic DNA digested with EcoRI and stained with ethidium bromide. (B) Hybridization pattern with the glpQ probe, which was identical to the hybridization pattern with the glpT probe (data not shown). Molecular size standards in kilobase pairs are on the left. The relapsing-fever (RF) spirochetes were positive with both probes, while the Lyme disease (LD) group of spirochetes were negative. All samples hybridized with the gpsA probe (data not shown).

FIG. 5.

Immunoblot analysis of B. burgdorferi transformed with the shuttle vector containing glpTQ from B. hermsii. (A) SDS-PAGE analysis of whole-cell lysates of B. hermsii, two transformed clones (clones 21 and 25) of B. burgdorferi (glpTQ+), and B. burgdorferi with only the shuttle vector (SV) (glpTQ−). Proteins were stained with Coomassie blue, and molecular mass standards (MMS) are shown at the left in kilodaltons. (B) Immunoblot analysis with rabbit anti-GlpQ antibody. B. hermsii and the glpTQ-transformed clones of B. burgdorferi produced GlpQ (arrow), while B. burgdorferi with only the shuttle vector did not.

FIG. 6.

Immunogold labeling for GlpQ and Vsp33 in B. hermsii. (A) Negative stain with no GlpQ detected on the outer surface with anti-GlpQ antibody and secondary antibody conjugated to gold particles. (B) Negative stain with Vsp33 detected on the outer surface with anti-Vsp33 antibody. (C) No GlpQ detected in thin sections with the outer membrane intact. (D) GlpQ detected in thin sections when the membranes were disrupted from the protoplasmic cylinder. Scale bars, 0.25 μm.

FIG. 7.

The three pathways for the acquisition of G3P in relapsing-fever and Lyme disease spirochetes. Evidence for pathways 1 and 2 is present in both groups of spirochetes, while pathway 3 is restricted to the relapsing-fever spirochetes.