Genomic Characterization of Neoparamoeba pemaquidensis (Amoebozoa) and Its Kinetoplastid Endosymbiont

ABSTRACT We have performed a genomic characterization of a kinetoplastid protist living within the amoebozoan Neoparamoeba pemaquidensis. The genome of this “Ichthyobodo-related organism” was found to be unexpectedly large, with at least 11 chromosomes between 1.0 and 3.5 Mbp and a total genome size of at least 25 Mbp.

Kinetoplastids are an important group of eukaryotic microbes named by virtue of their shared possession of a conspicuous mass of DNA-the "kinetoplast"-inside the mitochondrion (21,31). They are remarkable for their bizarre suite of biochemical features (e.g., spliced leader [SL] trans splicing and mitochondrial RNA editing) (1,5,21) and are best known as pathogens: the so-called "tritryps" are responsible for mass mortality and morbidity in humans and other animals (1,14,31). The kinetoplastids also contain a variety of other parasites, such as the fish pathogen Cryptobia, as well as many free-living groups (e.g., Bodo) (31). Phylogenetic studies suggest that within kinetoplastids, parasitic lineages have evolved from free-living ones on at least four occasions (31). Despite many genomics-enabled advances for the tritryp pathogens (e.g., see references 2, 14, and 18), a comprehensive evolutionary framework for understanding the biology of these important pathogens is currently lacking.
The kinetoplastid endosymbiont of Neoparamoeba pemaquidensis, an amoebozoan that causes disease in fish (e.g., Atlantic salmon) and invertebrates such as lobster (20,27), represents an apparent example of "recent" adaptation to intracellularity. We refer to this enigmatic endosymbiont as the "Ichthyobodo-related organism" (IRO) based on 18S ribosomal DNA (rDNA) analyses showing its close affinity to Ichthyobodo (6,(10)(11)(12). In this study we characterized genes from N. pemaquidensis and its kinetoplastid endosymbiont and carried out the first investigation of the chromosomes of both organisms using pulsed-field gel electrophoresis (PFGE) and Southern blotting.
N. pemaquidensis strains CCAP 1560/4 and 1560/5 and Neoparamoeba branchiphila AFSM3/II were cultured as described previously (13). Using standard transmission electron microscopy (TEM) and 4Ј,6-diamidino-2-phenylindole (DAPI) staining protocols, N. pemaquidensis strain 1560/4 was mostly found to possess 1 (and only occasionally 2) oval or round IRO per cell, each 4 to 6 m in diameter. The IRO was typically very closely associated with the host cell nucleus, with their surfaces often appearing to be in direct contact ( Fig. 1A and C). This is consistent with previous reports based on studies of multiple strains of Neoparamoeba (e.g., see references 10, 12, 13, and 17). Within the IRO, a single mitochondrion was found to occupy more than half of the endosymbiont cell volume, with its distinctive kinetoplast DNA (kDNA) appearing as a complex fibrillar structure in TEM micrographs ( Fig. 1A and B). The close proximity of the IRO to the amoeba host nucleus was also apparent under DAPI staining (Fig. 1C). The IRO nucleus stained weakly with DAPI relative to the amoeba nucleus, suggesting that the host and endosymbiont nuclei differ greatly in terms of their DNA content (Fig. 1C).
In order to gain insight into the evolutionary origins of Neoparamoeba and the IRO, we amplified six protein genes from both the host and its endosymbiont using PCR and/or reverse transcriptase PCR (RT-PCR) ( ; primer sequences are available upon request). PCR products were cloned into suitable vectors, and several clones per amplicon were sequenced on a CEQ8000 capillary DNA sequencer (Beckman Coulter, Inc., Fullerton, CA). Protein alignments were analyzed individually (data not shown) and as a single concatenate. Trees inferred from the concatenated data set showed the IRO branching robustly with the tritryps Leishmania and Trypanosoma, to the exclusion of two other euglenozoans, Diplonema and Euglena (Fig. 2). We also amplified and sequenced the SL RNA gene from the IRO genome and confirmed the presence of an SL mini-exon addition to IRO alpha-and beta-tubulin mRNAs by RT-PCR and 5Ј rapid amplification of cDNA ends (RACE)-PCR (data not shown). Additional RACE-PCR experiments showed that the host-derived alpha-tubulin mRNA does not undergo mini-exon addition. These results suggest that SL trans splicing takes place in the kinetoplastid endosymbiont but not in its host. No spliceosomal introns were found in any of the IRO protein genes sequenced herein, which are among the first to be determined, so it remains to be seen whether both cis and trans splicing occurs in these organisms, as has been shown for euglenoids and other kinetoplastids (3).
Consideration of the Neoparamoeba host-and IRO-derived sequences revealed interesting differences in base composition. The GϩC content of the IRO genes was 51.0 to 65.5% for the protein coding genes sequenced in our study, and the previously sequenced ribosomal intergenic spacer regions (ITS1 and -2) showed 68.1% GϩC when considered together. In stark contrast, sequences derived from the Neoparamoeba host were 49.8 to 55.2% GϩC in protein-coding genes and were much lower overall in GϩC content, especially in noncoding regions, such as the internal transcribed spacer (ITS) (21.5%). Although low GϩC content is often seen in endosymbiont and In addition to changes in base composition, adaptation to a life of intracellularity often leads to a reduction in genome size. This phenomenon is commonly seen in plastids and mitochondria (15,32), bacterial endosymbionts, such as Buchnera (25,26,30), and eukaryotic intracellular pathogens, such as microsporidia (7,8,35). A strong signal for coevolution of Neoparamoeba-IRO pairs is clear from analyses of rDNA sequences (6,11), and given that Neoparamoeba is invariably found with at least one IRO residing within it, the relationship between the two is very likely obligate (6). How big is the IRO nuclear genome, and where does this enigmatic organism reside along the continuum between transient endosymbiont and organelle? To address this question, we used pulsed-field gel electrophoresis (PFGE) to investigate the structure of the host and endosymbiont genomes of N. pemaquidensis. PFGE plugs were prepared using the general procedure described by Tanifuji et al. (34), with N. pemaquidensis prey bacteria being removed by filtration with a 3-m pore polycarbonate membrane (Sterlitech, Kent, WA) prior to plug formation. A strong "smear" of similarly sized chromosomes in the range of ϳ0.45 to 1 Mbp was apparent under a variety of different PFGE conditions and ethidium bromide stainings and with different PFGE plug concentrations (Fig. 3 A and B). In order to differentially target host and IRO chromosomes, nonradioactive Southern hybridization analyses were performed using digoxigenin (DIG)-labeled probes against the predicted telomeric repeat of the N. pemaquidensis host [(TTTAGGG) n ], the host rDNA ITS, and the SL RNA gene of the IRO. Southern hybridizations using host ITS and telomere probes detected strong signals between 0.45 and 1.2 Mbp, a pattern that was also seen with a host 28S rDNA probe (data not shown). These results suggest that the host genome contains many chromosomes in the 0.45-to 1.2-Mbp range.
Chromosomes of the N. pemaquidensis IRO were detected in Southern hybridization analyses using an SL RNA gene probe. Euglenoids and dinoflagellates are known to possess SL RNA genes in high copy number (2,18,28,36), and our results suggest that the same is true for the N. pemaquidensis IRO. To our surprise, hybridizations using the SL RNA gene probe against membranes derived from multiple independent PFGE runs and using various plug cell concentrations revealed a total of 11 distinct hybridizing bands between ϳ1.0 and 3.5 Mbp; particularly strong signals were detected at ϳ1.0, 2.4, and 3.5 Mbp (Fig. 3). Taking into account the fact that the chromosomal band migration distance was found to vary depending on PFGE plug cell density (see Fig. 3B; this is often the case in PFGE), these 11 bands total ϳ25 Mbp. We consider this to be a tentative absolute lower bound for the size of the N. pemaquidensis IRO nuclear genome. This estimate must be considered in the context of several important caveats, including (i) the likelihood of comigrating chromosomes in our PFGE analyses, (ii) interchromosomal variation in the SL RNA gene copy number (and thus variation in hybridization intensity), and (iii) the possibility that not all IRO chromosomes contain an SL RNA gene.
Across the breadth of eukaryotic diversity, nuclear genome size varies tremendously (16), but even free-living organisms, such as the red-alga Cyanidioschyzon merolae and the green VOL. 10, 201110, NOTE 1145 alga Ostreococcus tauri, can have very "small" genomes (16.5 Mbp and 12.6 Mbp, respectively) (9,22). Trypanosomatid haploid genomes are in the range of 35 to 55 Mbp (14), and based on the preliminary data presented herein, it is possible that the IRO genome is similar in size. Our base composition analyses are also significant. A reduction in GϩC content is also a hallmark feature of many (but not all) reduced genomes (15,23,24,26,29,32), but our newly determined IRO protein gene sequences exhibit a GϩC content between 55% and 65%, similar to that seen in other kinetoplastid taxa, including the free-living bodonid Bodo saltans (19). In fact, the rDNA ITS of the N. pemaquidensis IRO is 68.1% GϩC over 188 bp, more GϩC rich than the ITS of the prokinetoplastid Ichthyobodo necator (40.8% GϩC) and the metakinetoplastids Bodo caudatus (49.2% GϩC), Trypanosoma brucei, and Leishmania major (33.6 to 49.0%) (2,4,18). In sum, while the N. pemaquidensis IRO nuclear genome is very likely not "large," there is currently no evidence to suggest that it has undergone significant reductive evolution in response to what is clearly a life of obligate intracellularity. In concert with investigation of the Neoparamoeba nuclear genome, determining the sequence of the IRO genome will provide a framework for better understanding Neoparamoeba pathogenesis and the nature of this interesting host-endosymbiont relationship. Nucleotide sequence accession numbers. Genes of Neoparamoeba and the IRO, encoding the following proteins, were sequenced: alpha-tubulin, beta-tubulin, elongation factor 2, heat shock protein 90, an RNA polymerase I subunit, and an RNA polymerase II subunit; GenBank accession numbers are JF262536 to -42, JF262544 to -53, JF419568 to -72, FJ706693 to -98, and JF706718. Sequence of the SL RNA gene from the IRO genome has been deposited in GenBank under accession numbers JF262543 and JF441171.