Transporter gene acquisition and innovation in the evolution of Microsporidia intracellular parasites

Abstract

The acquisition of genes by horizontal transfer can impart entirely new biological functions and provide an important route to major evolutionary innovation. Here we have used ancient gene reconstruction and functional assays to investigate the impact of a single horizontally transferred nucleotide transporter into the common ancestor of the Microsporidia, a major radiation of intracellular parasites of animals and humans. We show that this transporter provided early microsporidians with the ability to steal host ATP and to become energy parasites. Gene duplication enabled the diversification of nucleotide transporter function to transport new substrates, including GTP and NAD⁺, and to evolve the proton-energized net import of nucleotides for nucleic acid biosynthesis, growth and replication. These innovations have allowed the loss of pathways for mitochondrial and cytosolic energy generation and nucleotide biosynthesis that are otherwise essential for free-living eukaryotes, resulting in the highly unusual and reduced cells and genomes of contemporary Microsporidia.

Fig. 1

Ancestral reconstruction and functional characterisation of nucleotide transporters. a Schematic representation showing the position of the nodes in the NTT phylogenetic tree for which ancestral sequences (AncNTT_Roz/Mic and AncNTT_Mic) were inferred and functionally characterised. We obtained point estimates of the ancestral NTT protein sequences by selecting the amino acid with the highest posterior probability at each site in the alignment (Supplementary Data 1 and 2). b Inferred secondary structure and membrane topology of one contemporary (ThNTT4) and the two ancestral sequences, predicted using HMMTOP. c Truncated western blot of fractionated E. coli expressing different NTTs detected using an anti-HIS antibody. Ancestral gene = AncNTT_Roz/Mic, E. cuniculi = Ec, R. allomycis = Ra. Total = sonicated bacteria, Inclusions = 20,000 g pellet, Membranes = 150,000 g pellet, Cytosol = supernatant after 150,000 × g spin. Complete blots are shown in Supplementary Figure 4. d Kinetics of [³²P]-ATP uptake by ancestral NTTs expressed in E. coli; pET16b = empty vector control. e Substrate saturation curve for the uptake of [³²P]-ATP in the presence of increasing concentrations of unlabelled ATP. Data is fitted to a Michaelis–Menten equation to determine K_m (µM) and V_max (pmol/min/mg) by iteration. f Competitive substrate inhibition against [³²P]-ATP uptake. Competitors were at 50,000× excess over the radio-labelled ATP. Data points represent residual radioactivity within the bacteria after subtraction of the empty vector control. g Nucleotide uptake of [³²P]-labelled pyrimidine (dTTP and UTP) or purine (ATP and GTP) nucleotides or NAD⁺ by the ancestral NTTs. h Effect of the protonophore CCCP on [³²P]-nucleotide uptake by the two ancestral proteins and PamNTT5 of Protochlamydia. Significant difference (*) to the control was only seen for PamNTT5 (p < 0.05; one-way ANOVA). i Back-exchange assay whereby [³²P]-ATP-loaded E. coli expressing AncNTT_Roz/Mic were incubated in the presence or absence (=Buffer) of unlabelled ATP. Data shows residual intracellular label in harvested E. coli cells. All data points represent means ± SD of at least three independent experiments

Fig. 2

Phylogeny and ATP transport by Microsporidia and Rozella NTTs. a NTT phylogeny for the Microsporidia/R. allomycis clade of endoparasitic fungi inferred under the CAT+GTR model in PhyloBayes. The tree is the posterior consensus tree inferred under the CAT+GTR model, in which all relationships with posterior support <0.5 were collapsed. Branch lengths are proportional to the expected number of substitutions per site. Scale bar = 0.5 changes per site. A single-ancestral acquisition of a bacterial NTT gene is inferred in the common ancestor of Microsporidia and Rozella followed by independent gene duplications and family expansion during the radiation of Microsporidia. The NTTs that were functionally characterised in this study are indicated, including the two ancestral NTTs shown at the base of the tree. Support values are Bayesian posterior probabilities. b Uptake of [³²P]-ATP by E. coli cells expressing NTTs for 30 min. All NTT gene and species names are given. ATP uptakes for all NTTs were significantly different (p < 0.05, one-way ANOVA) to the pET16b control. c Kinetics of [³²P]-ATP uptake in E. coli expressing NTTs from E. bieneusi (EbNTT1–4) or R. allomycis (RaNTT1). d Substrate saturation curves for [³²P]-ATP to determine K_m and V_max for NTTs from T. hominis (ThNTT1–4), E. bieneusi (EbNTT1–4), and R. allomycis (RaNTT1). Curves were fitted to the Michaelis–Menten equation and the K_m and V_max were calculated by iteration. All data points represent means ± SD of at least three independent experiments

Fig. 3

Substrate competition assays and nucleotide uptake assays for Microsporidia and Rozella NTTs. a Substrate competition assays whereby [³²P]-ATP uptake by NTT-expressing E. coli was performed in the presence of 50,000 × excess unlabelled substrate. Th T. hominis, Eb E. bieneusi, Ra R. allomycis. b [³²P]-nucleotide uptake assays with E. coli cells expressing NTT genes. Uptake of NAD⁺ by E. cuniculi NTTs was not significantly different to empty vector controls (p > 0.05, one-way ANOVA). Coloured bars show transport data for the indicated pyrimidine nucleotides (dTTP or UTP), purine nucleotides (ATP or GTP) or NAD⁺. pET empty vector control. c [³²P]-nucleotide uptake by NTT-expressing E. coli in the absence (set to 100% after control was subtracted) or presence of carbonyl cyanide m-chlorophenyl hydrazone (CCCP). Significant difference at p < 0.05 (one-way ANOVA) is shown with *. d Back-exchange assay with [³²P]-ATP-loaded E. coli incubated in the presence or absence of 100 μM unlabelled ATP. Data shows the residual radioactivity following washing of the bacteria. PamNTT5 is a positive control symporter from the bacterium Protochlamydia amoebophila. All data (mean ± SD) is representative of at least three independent experiments

Fig. 4

Protein localisation and transcript abundance for T. hominis NTT transporters over a time course of T. hominis infection. a Immunofluorescence time course (3–96 h) of rabbit kidney (RK-13) cells infected with T. hominis spores using published individual rabbit anti-ThNTT antibodies (red). Rat anti-HSP70 (green) was used to label the mitosomes of intracellular parasites (meronts). The first time point at 3 h is shortly after injection of the T. hominis sporoplasm into the host cell when labelling by antisera to ThNTT1 and ThNTT4 is already apparent. The top DIC image shows the spore bags (arrows) at 96 h with superimposed labelling (red) by antisera to ThNTT1 and ThNTT3 but not by antisera to ThNTT4. Scale bar is 1 µm. b Increase in parasite biomass during T. hominis infection time course measured using cell diameter and cell volume. Diameter of parasite cells (minimum 100 counted) was taken at their widest point, and cell volumes were calculated using Axiovision software. Error bars are standard deviation. N = 3. c RNAseq analysis showing transcript abundance (log₁₀ FPKM (Fragments per kilobase per million mapped reads)) for the four ThNTTs, spore protein (PTP2), a glycolytic enzyme (PGK1) and ribosomal protein L37 throughout the infection time course

Fig. 5

Gene duplication and evolution of Microsporidia NTTs. a Phylogeny of Microsporidia and Rozella NTTs and their transported substrates. ATP transport by the predicted ancestral NTTs AncNTT_Roz/Mic and AncNTT_Mic are shown at the respective nodes. The tree topology suggests that the common ancestor of Microsporidia clade III and IV could already transport both purine nucleotides and potentially NAD⁺ as indicated by the cross-hatched boxes. b A model for T. hominis NTT-mediated acquisition of energy and nucleotides from infected cells. NTTs are located at the parasite plasma membrane and can act as exchangers (ThNTT1-3) or symporters (ThNTT4) enabling energy parasitism or net nucleotide uptake, respectively. The transporters and/or pathways used by T. hominis to acquire pyrimidine nucleotides are currently unknown