Horizontal gene transfer provides insights into the deep evolutionary history and biology of Trichinella

Abstract

Evolution involves temporal changes in the characteristics of a species that are subsequently propagated or rejected through natural selection. In the case of parasites, host switching also plays a prominent role in the evolutionary process. These changes are rooted in genetic variation and gene flow where genes may be deleted, mutated (sequence), duplicated, rearranged and/or translocated and then transmitted through vertical gene transfer. However, the introduction of new genes is not driven only by Mendelian inheritance and mutation but also by the introduction of DNA from outside a lineage in the form of horizontal gene transfer between donor and recipient organisms. Once introduced and integrated into the biology of the recipient, vertical inheritance then perpetuates the newly acquired genetic factor, where further functionality may involve co-option of what has become a pre-existing physiological capacity. Upon sequencing the Trichinella spiralis (Clade I) genome, a cyanate hydratase (cyanase) gene was identified that is common among bacteria, fungi, and plants, but rarely observed among other eukaryotes. The sequence of the Trichinella cyanase gene clusters with those derived from the Kingdom Plantae in contrast to the genes found in some Clade III and IV nematodes that cluster with cyanases of bacterial origin. Phylogenetic analyses suggest that the Trichinella cyanase was acquired during the Devonian period and independently from those of other nematodes. These data may help inform us of the deep evolutionary history and ecological connectivity of early ancestors within the lineage of contemporary Trichinella. Further, in many extant organisms, cyanate detoxification has been largely superseded by energy requirements for metabolism. Thus, deciphering the function of Trichinella cyanase may provide new avenues for treatment and control.

Keywords: Cyanase; Evolution; Horizontal gene transfer; Nurse cell; Trichinella.

Fig. 1

Unrooted maximum likelihood tree of cyanases. Lineages are color coded and include parasitic nematodes, bacteria, fungi, and plants. The tree shows distinction in the placement of Clade I nematodes (sister to plant cyanases) and those from Clades III and IV (cluster with bacterial cyanases). (Reprinted from Zarlenga et al., 2019).

Fig. 2

Unrooted maximum likelihood tree of cyanases. Key lineages are color-coded and include archaea, bacteria, metazoan, fungi, eukaryotic algae, and land plants. Distinctions among the parasitic nematodes are presented. (Reprinted from Mao et al., 2021 with modification).

Fig. 3

Unrooted, maximum likelihood tree of Trichinella cyanases including those from Trichuris spp. and Soboliphyme. Alignment was performed with CLUSTAL (part of the MEGA-11 package). Gaps were removed at the variable N-terminus prior to tree building. Phylogenetic classifications for Trichinella genotypes T6, T8 and T9 remain unresolved. Protein accession numbers and DNA reference numbers (Soboliphyme) for the aligned sequences are as follows: T1 = EFV60696; T2 = OUC42830; T3 = QTG10995; T4A = KRX89740; T4B = ABR10530; T5 = KRX40590; T6 = KRX81296; T7 = KRX22994; T8 = KRZ91604; T9 = KRX64400; T10 = KRZ67954; T11 = ABR10534; Tsu = KHJ46081; Tt = CDW58178; Sb = SBAD_0000658401.

Fig. 4

Alignment of cyanase active sites for species and genotypes of Trichinella. Amino acids designated with “*” indicate key loci for enzyme activity as per Mao et al., 2021. The species and genotype designations are defined in the legend to Fig. 3 as are the accession numbers from which these sequences were obtained and the alignment parameters.

Fig. 5

Genome expression cassette for T. spiralis cyanase. The locations for the cyanase gene and the two arginine N-methyl transferase genes are defined. Transcript read coverage is represented by the colored blocks, and normalized expression levels (RPKM = reads/kilobase of gene length/10⁶ mapped sequence reads) are indicated for the genes of interest.

Fig. 6

Hypothetical sources of cellular cyanate with access to T. spiralis. Potential sources of cyanate are designated by dashed lines as are mechanisms for [A] depleting cellular arginine via nutrient flux into macrophages or [B] liberating macrophage-derived arginase into the cell cytoplasm to control arginine concentration. Mechanisms are based upon a pseudo-urea cycle which has been demonstrated in muscle cells. Enzymes facilitating such a cycle are designated as CPS1 = Carbamoyl Phosphate Synthetase 1; OTC = Ornithine Transcarbamylase; AS = Argininosuccinate Synthetase; AL = Argininosuccinate Lyase; NOS = Nitric Oxide Synthase; ARG = Arginase 1. Pi = inorganic phosphate. The rete is presumed to envelop the entire NC.

Supplementary Fig. S1

Multiple sequence alignment of cyanase genes. Genomic sequence alignments for the cyanase genes appear in order as Trichinella spiralis, Trichinella nativa, Trichuris muris, Trichuris suis, Trichuris trichiura, and Soboliphyme baturini. Alignment was performed with M-COFFEE (part of the T-COFFEE package, version 11.00). Color coding indicates the level of agreement between eight aligners combined for the final alignment (blue = very poor, green = poor, yellow = average, pink = good, red = perfect).

Supplementary Fig. S2

The positions of genes assigned to different Compara families relative to the cyanase gene in species of interest. (A) Compara family membership (identified in IHGC, 2019) among the 10 genes upstream and downstream of cyanase in each of the species of interest. Only families identified in two or more species are shown. Numbers indicate the position of the genes relative to cyanase (0 = cyanase, negative = upstream, positive = downstream). Color coding is used to indicate functions of interest. (B) Positional orientation of genes upstream and downstream of cyanase in species of interest. Color coding corresponds to the colors shown in panel (A), and greyed-out genes indicate that no other genes are present further upstream/downstream on the contig containing the cyanase gene. Plus, signs indicate genes oriented on the same strand as cyanase, and minus signs indicate genes on the opposite strand as cyanase.

Supplementary Fig. S3

The positions of genes annotated with different InterPro domains, relative to the cyanase gene in species of interest. (A) InterPro (IPR) domain annotations (identified in IHGC, 2019) among the 10 genes upstream and downstream of cyanase in each of the species of interest. Only IPR domains identified in two or more species are shown. Numbers indicate the position of the genes relative to cyanase (0 = cyanase, negative = upstream, positive = downstream). Color coding is used to differentiate overall biological functions of interest. (B) Positional orientation of genes upstream and downstream of cyanase in species of interest. Color coding corresponds to the colors shown in panel (A), and greyed-out genes indicate that no other genes are present further upstream / downstream on the contig containing the cyanase gene. Plus, signs indicate genes oriented on the same strand as cyanase, and minus signs indicate genes on the opposite strand as cyanase.

Supplementary Fig. S4

The positions of genes annotated as different KEGG proteins, relative to the cyanase gene in species of interest. (A) KEGG annotations (identified in IHGC, 2019) among the 10 genes upstream and downstream of cyanase in each of the species of interest. Only KEGG annotations identified in two or more species are shown. Numbers indicate the position of the genes relative to cyanase (0 = cyanase, negative = upstream, positive = downstream). Color coding is used to differentiate overall biological functions of interest. (B) Positional orientation of genes upstream and downstream of cyanase in species of interest. Color coding corresponds to the colors shown in panel (A), and greyed-out genes indicate that no other genes are present further upstream/downstream on the contig containing the cyanase gene. Plus, signs indicate genes oriented on the same strand as cyanase, and minus signs indicate genes on the opposite strand as cyanase.