Sequencing of the smallest Apicomplexan genome from the human pathogen Babesia microti

Abstract

We have sequenced the genome of the emerging human pathogen Babesia microti and compared it with that of other protozoa. B. microti has the smallest nuclear genome among all Apicomplexan parasites sequenced to date with three chromosomes encoding ∼3500 polypeptides, several of which are species specific. Genome-wide phylogenetic analyses indicate that B. microti is significantly distant from all species of Babesidae and Theileridae and defines a new clade in the phylum Apicomplexa. Furthermore, unlike all other Apicomplexa, its mitochondrial genome is circular. Genome-scale reconstruction of functional networks revealed that B. microti has the minimal metabolic requirement for intraerythrocytic protozoan parasitism. B. microti multigene families differ from those of other protozoa in both the copy number and organization. Two lateral transfer events with significant metabolic implications occurred during the evolution of this parasite. The genomic sequencing of B. microti identified several targets suitable for the development of diagnostic assays and novel therapies for human babesiosis.

Figure 1.

Babesia microti strain R1 characterization. (A) Light-microscopy analysis of B. microti infected blood. Left: blood smear. Right: immunofluorescence analysis using serum from a hamster infected with the B. microti Gray standard laboratory strain. Similar serum was used for serological assays. (B) PCR amplification of the ssu gene using PIRO-A and PIRO-B primers (77). These primers amplify a 436 bp fragment in B. microti Gray and R1 strains and a 408 bp fragment in B. divergens Rouen strain. The integrity of the PCR fragment was confirmed by DNA sequencing. (C) B. microti karyotype. PFGE conditions used are: left, 0.7% agarose, 400 s pulses for 55 h; right, 1% agarose, 200 s pulses for 65 h. Length polymorphism between the R1 isolate and the Gray strain is observed on chromosome 1 and 2. (D) 2D-PFGE NotI Restriction Fragment Length Polymorphism (RFLP) analysis of B. microti. Each chromosome length polymorphism results from a single RFLP of 15 kbp (see triangle and star for chromosome I and II respectively). The genome structure of Gray and R1 strain may differ from each other only with a single recombination event. PFGE conditions used are: 1.2 % agarose gel, pulse conditions, 5 s for 8 h, 15 s for 7 h and 30 s for 6 h, tension, 6.5 V/cm.

Figure 2.

Mosaic organization of the Babesia microti chromosome extremities. Chromosome ends are labeled according to Figure S2. These regions are characterized by the presence of duplicated sequences scattered among the different chromosome extremities (S1–S8). Limits of sequence homologies have been calculated using miropeat and BLASTN analyses. Annotated genes are indicated on the figure. Most repeated genes are part of the bmn gene family and included truncated genes and pseudogenes. Several bmn genes are in transition regions between two duplicated sequences. The S2 sequence encoding a putative VESA antigen is repeated three times. The S4 sequence encodes Tpr orthologues and is repeated four times, two copies of which on chromosome ends IIb and IIIa are significantly shorter. The GC content in the chromosome ends is significantly lower than in the coding core. The sequence between S1 and S4 at extremity Ib encodes a putative sugar transporter (TM). The sequence is not duplicated but does not show any base composition bias compared to adjacent regions. It was not possible to precisely map the recombination sites associated with the rearrangements that took place at chromosome ends (average resolution of 100 bp).

Figure 3.

Circular organization of the Babesia microti mitochondrial genome. IR: inverted repeats; cox1: cytochrome c oxidase subunit 1, cox3: cytochrome c oxidase subunit 3, cytb: cytochrome bc complex subunit. The numbering of the ribosomal lsu and ssu genes fragments is performed according to the P. falciparum nomenclature.

Figure 4.

Babesia microti defines a new clade in the Apicomplexan phylum. The tree is inferred using a maximum likelihood approach on a concatenated alignment of 316 single-copy genes. Tetrahymena thermophila was included as outgroup. The same tree topology is inferred by a supertree approach compiling the 316 trees inferred from the 316 genes. Labels indicate the boostrap support from both the Supermatrix and Supertree analyses and the level of tree supporting the clade (%).

Figure 5.

An integrated model for central metabolism of Babesia microti. Arrows show the direction of net fluxes. Lactate and malate are expected to be the major end products of the central metabolism. The gene encoding the lactate dehydrogenase is likely obtained by lateral transfer from a mammalian host. The apicoplast is devoted to the production of isoprenoids precursors. Numbers in brackets represent the biochemical steps in the reaction. Dashed arrows connect two reactions using the same metabolite. For simplicity, the membranes of the apicoplast and the outer membrane of the mitochondrion is not shown. Abbreviation used are: (1) Metabolites: 1,3BPGA, 1,3-bisphosphoglycerate; 2OG, 2-oxoglutarate; Ace-R: acetate/acetyl-CoA, c, cytochrome c; CoQ, Coenzyme-Q, ubiquinone; DHAP, dihydroxyacetone phosphate; e-, electrons; FBP, fructose 1,6-bisphosphate; G3P, glycerol-3-phosphate; GAP, glyceraldehyde-3-phosphate; Glu, glutamate; Gln, glutamine; OAA, oxaloacetate, PEP, phosphoenolpyruvate. (2) Enzymes (in red): ACS, AMP-forming acetyl-CoA synthetase; DHOD, dihydroorotate dehydrogenase; GLS, glutamine synthetase; GPDH, glycerol-3-phosphate dehydrogenase (FAD- or NAD-dependent enzymes); LDH, lactate dehydrogenase; MQO, malate:quinone oxidoreductase; NDH2, type II NADH:quinone oxidoreductase; OGDH, oxoglutarate dehydrogenase; PEPC, PEP carboxylase; PEPCK, PEP carboxykinase; PDH, pyruvate dehydrogenase; PK, pyruvate kinase; SDH, succinate dehydrogenase; TH, transhydrogenase; III, complex III of the respiratory chain; IV, complex IV of the respiratory chain; V, F₀F₁-ATP synthase.