Evidence that the intra-amoebal Legionella drancourtii acquired a sterol reductase gene from eukaryotes

Abstract

Background: Free-living amoebae serve as a natural reservoir for some bacteria that have evolved into "amoeba-resistant" bacteria. Among these, some are strictly intra-amoebal, such as Candidatus "Protochlamydia amoebophila" (Candidatus "P. amoebophila"), whose genomic sequence is available. We sequenced the genome of Legionella drancourtii (L. drancourtii), another recently described intra-amoebal bacterium. By comparing these two genomes with those of their closely related species, we were able to study the genetic characteristics specific to their amoebal lifestyle.

Findings: We identified a sterol delta-7 reductase-encoding gene common to these two bacteria and absent in their relatives. This gene encodes an enzyme which catalyses the last step of cholesterol biosynthesis in eukaryotes, and is probably functional within L. drancourtii since it is transcribed. The phylogenetic analysis of this protein suggests that it was acquired horizontally by a few bacteria from viridiplantae. This gene was also found in the Acanthamoeba polyphaga Mimivirus genome, a virus that grows in amoebae and possesses the largest viral genome known to date.

Conclusion: L. drancourtii acquired a sterol delta-7 reductase-encoding gene of viridiplantae origin. The most parsimonious hypothesis is that this gene was initially acquired by a Chlamydiales ancestor parasite of plants. Subsequently, its descendents transmitted this gene in amoebae to other intra-amoebal microorganisms, including L. drancourtii and Coxiella burnetii. The role of the sterol delta-7 reductase in prokaryotes is as yet unknown but we speculate that it is involved in host cholesterol parasitism.

Figure 1

Phylogenetic analysis of the L. drancourtii sterol delta-7 reductase matches. Unrooted phylogenetic tree of protein sequences matching the L. drancourtii sterol delta-7 reductase protein. Three distinct clusters were identified: a cluster contained delta-7 sterol reductases implicated in cholesterol biosynthesis in eukaryotes (blue), a cluster contained C24 sterol reductases implicated in ergosterol biosynthesis in fungi (green) and a third cluster contained C14 sterol reductases implicated in both biosyntheses (red). Bootstrap values are indicated at the nodes.

Figure 2

PCR amplification and RT-PCR of the sterol delta-7 reductase-encoding gene. Lanes 1 and 6: DNA molecular weight marker VI (Roche); lanes 2 and 4: DNA; lanes 3 and 5: RNA

Figure 3

Sequence alignment of RT-PCR product and sterol delta-7 reductase-encoding gene. RT-PCR products were sequenced and aligned with the sterol delta-7 reductase-encoding gene of L. drancourtii using the Muscle software. "Reverse_1" and "reverse_2" were obtained using the reverse primer, "forward_1" and "forward_2" using the forward primer, "LD_reducta" is the reference sequence obtained from the L. drancourtii genome.

Figure 4

Two hypothetical scenarios to explain the sterol delta-7 reductase gene acquisition by L. drancourtii

Figure 5

Eukaryotic sterol biosynthesis. Ergosterol and cholesterol are synthesized in fungi and all eukaryotes respectively. Enzymes present in L. drancourtii (L), C. burnetii (C) and Candidatus "P. amoebophila" (P) are represented in italics and enzymes present in the previous phylogenetic tree are framed.