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 nucle...

Global Kinetoplastea phylogeny inferred from a large-scale multigene alignment including parasitic species for better understanding transitions from a free-living to a parasitic lifestyle

“…1). Prior to this study, the phylogenetic position of Prokinetoplastida has been assessed only by single-gene alignments including kinetoplastid species sampled from all five orders (Callahan et al, 2002;Moreira et al, 2004;Simpson et al, 2004;Breglia et al, 2007;Hirose et al, 2012) or by four-gene and 11-gene alignments with restricted taxon samplings (Tanifuji et al, 2011;Cenci et al, 2016). Thus, the current study provides the first phylogenomic support for the earliest-branching status of Prokinetoplastida in Kinetoplastea (Fig.…”

The unconventional kinetoplastid kinetochore: from discovery toward functional understanding

“…Furthermore, at least some KKT proteins are present in an early diverging Prokinetoplastina, Perkinsela sp. [213], suggesting that KKT proteins are likely a conserved feature of all kinetoplastids (my unpublished data).…”

Diversity and Evolution of Paramoeba spp. and their Kinetoplastid Endosymbionts

“…; Tanifuji et al. ). These facts point towards a stable, obligatory relationship between the two organisms, albeit one in which the benefits to one or both them are unknown.…”

“…and its host (Tanifuji et al. ). This is indicative of a relatively recent adaptation to intracellularity, the study of which has the potential to shed light on the early stages of reductive evolution and the transition from eukaryotic endosymbiont to organelle.…”

“…PARAMOEBA/NEOPARAMOEBA species are small lobose amoebae with dactylopodiate pseudopodia, best known as pathogens of various fish and invertebrates in marine and estuary environments around the world. They are of significant economic and ecological interest because of their association with Amoebic Gill Disease (AGD) in various fishes, paramoebiasis in blue crabs and lobster, and wasting disease in green sea urchins (Caraguel et al 2007;Dykov a et al 2005;Feehan et al 2013;Fiala and Dykova 2003;Lee et al 2006;Mouton et al 2014;Sprague et al 1969;Tanifuji et al 2011;Young et al 2007Young et al , 2008. Despite the ongoing interest in the study of Paramoeba/ Neoparamoeba spp., the pathobiology of the organism is still poorly understood.…”