The genome of Naegleria lovaniensis, the basis for a comparative approach to unravel pathogenicity factors of the human pathogenic amoeba N. fowleri

Abstract

Background: Members of the genus Naegleria are free-living eukaryotes with the capability to transform from the amoeboid form into resting cysts or moving flagellates in response to environmental conditions. More than 40 species have been characterized, but only Naegleria fowleri (N. fowleri) is known as a human pathogen causing primary amoebic meningoencephalitis (PAM), a fast progressing and mostly fatal disease of the central nervous system. Several studies report an involvement of phospholipases and other molecular factors, but the mechanisms involved in pathogenesis are still poorly understood. To gain a better understanding of the relationships within the genus of Naegleria and to investigate pathogenicity factors of N. fowleri, we characterized the genome of its closest non-pathogenic relative N. lovaniensis.

Results: To gain insights into the taxonomy of Naegleria, we sequenced the genome of N. lovaniensis using long read sequencing technology. The assembly of the data resulted in a 30 Mb genome including the circular mitochondrial sequence. Unravelling the phylogenetic relationship using OrthoMCL protein clustering and maximum likelihood methods confirms the close relationship of N. lovaniensis and N. fowleri. To achieve an overview of the diversity of Naegleria proteins and to assess characteristics of the human pathogen N. fowleri, OrthoMCL protein clustering including data of N. fowleri, N. lovaniensis and N. gruberi was performed. GO enrichment analysis shows an association of N. fowleri specific proteins to the GO terms "Membrane" and "Protein Secretion."

Conclusion: In this study, we characterize the hitherto unknown genome of N. lovaniensis. With the description of the 30 Mb genome, a further piece is added to reveal the complex taxonomic relationship of Naegleria. Further, the whole genome sequencing data confirms the hypothesis of the close relationship between N. fowleri and N. lovaniensis. Therefore, the genome of N. lovaniensis provides the basis for further comparative approaches on the molecular and genomic level to unravel pathogenicity factors of its closest human pathogenic relative N. fowleri and possible treatment options for the rare but mostly fatal primary meningoencephalitis.

Keywords: Comparative genomics; Genome de novo assembly; Naegleria fowleri; Naegleria lovaniensis; PacBio sequencing; Primary amoebic meningoencephalitis.

Fig. 1

Workflow Genome de novo Assembly. To achieve a complete as possible assembly of the N. lovaniensis genome, PacBio data were assembled using FALCON quality and completeness assessment using BUSCO. Repetitive sequences were identified and masked by RepeatModeler. In a further step, non-coding RNAs were identified using INFERNAL and proteins were predicted by an ab-initio method applying BRAKER1 including RNAseq data. Functional annotation was performed using Blast2GO pipeline. To gain an overview of Naegleria species, a genome similarity network as well as a phylogenetic tree was constructed. Additionally, Naegleria proteins were clustered using OrthoMCL

Fig. 2

Overview of gene annotation of the N. lovaniensis genome. Of total 15,195 ab initio predicted proteins, 8749 (57.6%) proteins mapped to at least one GO term using Blast2GO; 4678 (30.8%) only have a BLAST hit, while 1768 (11.6%) show no similarity to known proteins or domains

Fig. 3

Genome Similarity Network of unicellular eukaryotic species. To gain insights of the phylogenetic relationship of Naegleria, a BLAST based Genome Network was constructed including N. lovaniensis and N. fowleri predicted protein as well as Uniprot proteomes of T. brucei, T. cruzi, A. castelanii, and E. histolytica. Nodes in the graph represent the organisms and the edges, which are inversely proportional to the number of shared gene families, are the measurement of similarity between nodes. Additional file 2 shows the number of shared gene families between species

Fig. 4

Maximum likelihood tree of Naegleria species. Based on maximum likelihood and bootstrapping using RAxML a species tree was constructed to achieve a comprehensive overview of the relatedness of the species within the genus of Naegleria. Phylogenetic distances were estimated based on 100 single copy orthologs defined by OrthoMCL clustering. Beside Naegleria species, the more distantly related protists T. brucei and T. cruzi were chosen as taxonomic outgroups

Fig. 5

OrthoMCL Clustering of Naegleria proteins. To gain an overview of the diversity of the Naegleria protein repertoire, predicted protein of N. lovaniensis and N. fowleri as well as protein sequences of N. gruberi retrieved form Uniprot were clustered using OrthoMCL