The mitochondrial genome and a 60-kb nuclear DNA segment from Naegleria fowleri, the causative agent of primary amoebic meningoencephalitis

Abstract

Naegleria fowleri is a unicellular eukaryote causing primary amoebic meningoencephalitis, a neuropathic disease killing 99% of those infected, usually within 7-14 days. Naegleria fowleri is found globally in regions including the US and Australia. The genome of the related nonpathogenic species Naegleria gruberi has been sequenced, but the genetic basis for N. fowleri pathogenicity is unclear. To generate such insight, we sequenced and assembled the mitochondrial genome and a 60-kb segment of nuclear genome from N. fowleri. The mitochondrial genome is highly similar to its counterpart in N. gruberi in gene complement and organization, while distinct lack of synteny is observed for the nuclear segments. Even in this short (60-kb) segment, we identified examples of potential factors for pathogenesis, including ten novel N. fowleri-specific genes. We also identified a homolog of cathepsin B; proteases proposed to be involved in the pathogenesis of diverse eukaryotic pathogens, including N. fowleri. Finally, we demonstrate a likely case of horizontal gene transfer between N. fowleri and two unrelated amoebae, one of which causes granulomatous amoebic encephalitis. This initial look into the N. fowleri nuclear genome has revealed several examples of potential pathogenesis factors, improving our understanding of a neglected pathogen of increasing global importance.

Fig. 1–2

PCR confirmation of assembly. 1. Eleven ~1000 bp amplicons from across the 60-kb segment in the nuclear genome were successfully recovered by PCR amplification. M, ladder. 2. Diagram of where the amplicons were located across the contig.

Fig. 3

Gene content of the Naegleria fowleri and Naegleria gruberi mitochondrial genomes in comparison with other eukaryotes. Genes are included in the classes in the following way, as in (Burger et al. 2003). Reduction and oxidative phosphorylation (horizontal bars): atp1, atp3, atp4, atp6, atp8, atp9; cob; cox1–3; nad1–4, nad4L, nad6–11, sdh2–4. rRNAs (solid black): rnl, rns, rrn5. tRNAs (diagonal dots): trnA-Y. Ribosomal proteins and EF-Tu (solid dark grey): rps1–4, rps7, rps8, rps10–14, rps19; rpl1, rpl5, rpl6, rpl10, rpl11, rpl14, rpl16, rpl18–20, rpl27, rpl31, rpl32, rpl34, rpl36; tufA. Protein import and maturation (solid white): secY, ymf16, tatC, yejR (ccmF), yejU (ccmC), yejV (ccmB), yejW (ccmA); cox11. RNA maturation (solid light grey): rnpB. Transcription (black squares): rpoA-D. Other (hatched): ORFs of unknown function. Data for all organisms except N. fowleri was retrieved from NCBI. In Dictyostelium discoideum, cox1 and cox2 are encoded as a single ORF, but are counted as two genes here. Abbreviations: N. fowleri=Naegleria fowleri; R. americana=Reclinomonas americana; M. jakobiformis=Malawimonas jakobiformis; R. salina=Rhodomonas salina; H. sapiens=Homo sapiens; S. cerevisiae=Saccharomyces cerevisiae; D. discoideum= Dictyostelium discoideum; M. polymorpha=Marchantia polymorpha; P. falciparum=Plasmodium falciparum.

Fig. 4

Circular map of the Naegleria fowleri mitochondrial genome. Genes encoding proteins are annotated based on BLAST results, and genes encoding RNA are annotated based on tRNA and rRNA scanning software predictions (see Materials and Methods section). For the full name of each gene, see Table S2. Black arrows with gene names on the outside of the map represent genes on one strand, and white arrows with gene names on the inside of the map represent genes on the alternate strand. The N. fowleri mitochondrial genome is identical in gene content and organization to the mitochondrial genome of Naegleria gruberi.

Fig. 5

Comparative collinearity analysis of the predicted Naegleria fowleri ORFs on the 60-kb nuclear segment and their homologues in the Naegleria gruberi nuclear genome. Black right arrows indicate ORFs on one strand, while white left arrows indicate ORFs on the other strand. (Top) Genes on the N. fowleri segment and the organization of homologues on individual scaffolds. (Bottom) The N. fowleri segment is in the middle, with the arrangements of homologous genes on N. gruberi scaffolds containing two or more genes above and below. Annotations of N. fowleri genes are based on BLAST results (see Materials and Methods), while those for N. gruberi are the GenBank protein accession for the homologue of each N. fowleri ORF. Scale bars are given for each map at 100,000 bp, with the exception of the N. fowleri segment, for which the scale bar indicates 10,000 bp. There is a distinct lack of collinearity between the two Naegleria species at this region of the N. fowleri genome.

Fig. 6

Phylogeny of cathepsin family homologues. This phylogeny is a composite of MrBayes, PhyML, and RAxML results shown on the best Bayesian topology, arbitrarily rooted between the cathepsin B and L clades. The relevant nodes are marked using dots that represent Bayesian posterior probability/PhyML bootstrap/RAxML bootstrap values better than the following thresholds: 1.00/95/95 (black), 0.95/75/75 (grey), and 0.80/50/50 (white). The N. fowleri sequence is boxed. It is found within the cathepsin B clade, supporting its identity as part of the cathepsin B family.