Draft genome of the marine Entamoeba species reveals reduction in the gene family repertoire associated with pathogenicity and lateral gene transfer for adaptation to the marine environment

Draft genome of the marine Entamoeba species reveals reduction in the gene family repertoire associated with pathogenicity and lateral gene transfer for adaptation to the marine environment | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Draft genome of the marine Entamoeba species reveals reduction in the gene family repertoire associated with pathogenicity and lateral gene transfer for adaptation to the marine environment View ORCID Profile Tetsuro Kawano-Sugaya , Shinji Izumiyama , View ORCID Profile Tomoyoshi Nozaki doi: https://doi.org/10.1101/2025.02.12.637563 Tetsuro Kawano-Sugaya 1 Graduate School of Medicine, The University of Tokyo , Bunkyo, Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tetsuro Kawano-Sugaya Shinji Izumiyama 2 Department of Parasitology, National Institute of Infectious Diseases , Shinjuku, Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tomoyoshi Nozaki 1 Graduate School of Medicine, The University of Tokyo , Bunkyo, Tokyo, Japan Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Tomoyoshi Nozaki For correspondence: nozaki{at}m.u-tokyo.ac.jp Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Entamoeba is the amoebozoan parasite commonly found in the intestines of animals. E. marina is the first exception isolated from marine sediments, possibly adapting from animal intestines to the sea. However, the evolutionary process of E. marina remains uncertain due to the lack of a genome sequence. Here, we present the de novo genome and transcriptome of E. marina using Oxford Nanopore MinION and Illumina HiSeq/MiSeq. The genome of E. marina is approximately 37.5 Mbp in length and consisted of 202 contigs, which is the second longest followed by E. invadens . E. marina showed significant reduction in the major virulence-associated gene families, including cysteine proteases, lysosomal enzyme transporters, and surface galactose/N-acetylglucosamine-specific lectins, suggesting diversification, more specifically reduction of pathogenicity-related genes. Genome and RNA-seq analyses also indicated genes either conserved throughout eukaryotes or laterally transferred from prokaryotes, and potentially responsible for salt tolerance. Our study provides insights into the mechanism underlying the lifestyle changes in the evolution of parasitic eukaryotes. Introduction Acclimation to environmental changes is one the major evolutionary driving factors ( Lamichhaney et al. 2015 ). Entamoeba is the genus that belong to the super group Amoebozoa and composed of mostly parasitic organisms that reside in a broad range of environments including the intestine of vertebrates of fish ( E. chiangraiensis ; Jinatham et al. 2019 ), reptilian ( E. invadens ; Meerovitch 1958 ), and non-vertebrates such as cockroaches ( Kawano et al. 2017 ), as well as the oral cavity of humans ( E. gingivalis ; Smith and Barrett 1915 ). There are also a few free-living exceptions of Entamoeba that inhabit sewage water ( E. moshkovskii Tshalaia 1941; see Scaglia et al. 1983 ) and marine sediments ( E. marina ; Shiratori and Ishida 2016 ). Previous phylogenetic studies indicated that E. marina diverged together with the intestinal Entamoeba species at the base of the Entamoeba clade ( Shiratori and Ishida 2016 ; Kawano et al. 2017 ). This phylogenetic evidence is consistent with the premise that E. marina originally inhabited the intestines of animals, and then has subsequently adapted to the marine environment. However, it remains elusive whether E. marina is parasitic with the host remaining unidentified or it represents another free-living Entamoeba sibling. The genomes of various parasitic and free-living Entamoeba have been sequenced ( Loftus et al. 2005 ; Ehrenkaufer et al. 2013 ; Tanaka et al. 2019 ; Kawano-Sugaya et al. 2020 ; Nakada-Tsukui et al. 2018 ). The genome information of Entamoeba species that live in the conditions clearly divergent from other Entamoeba, such as E. marina, should indicate key genes or pathways necessary for the adaptation to such environments, i.e., sea water or sediments, or maybe a free-living lifestyle. Here, we describe the first draft genome assembly of the E. marina strain SRT209 ( Shiratori and Ishida 2016 ), which consists of approximately 37.5 Mbp and encodes 10,771 proteins with 46% being hypothetical proteins. We found a reduction in the repertoire of virulence-associated gene families in E. histolytica, as well as diversification of genes related to vesicular transport and oxidative stress management. Furthermore, we identified a pair of genes, one conserved throughout eukaryotes and the other laterally transferred, that are potentially responsible for salt tolerance in E. marina . Taken together, a newly disclosed E. marina genome supports the premise that a combination of such genetic alterations plays a role for adaptation to diverse living environment and/or for transition from the parasitic to free-living lifestyle. Materials and Methods Organism and culture Entamoeba marina strain SRT209 was kindly provided by Dr. Seiki Kobayashi (National Institute of Infectious Diseases) and cultivated using protocols modified from a previous publication ( Shiratori and Ishida 2016 ). Briefly, the cells were cultured under monoxenic conditions with Shewanella sp. in 10% YIMDHA-S broth ( Suzuki et al. 2008 ) diluted with artificial seawater (Cat. 395-01343; Nihon Pharmaceutical, Japan) at 25.5°C and passaged weekly. Short read genome sequencing A total of 10 8 trophozoites grown in 15 mL polystyrene non-treated culture tubes (CLS430157; Corning, AZ, USA) in the late logarithmic phase were harvested by centrifugation at 400 × g for 5 min. Total genomic DNA was isolated and purified using the QIAGEN Blood & Cell Culture DNA Kit with Genomic-tip 100/G (Qiagen, Germany). The purity and quantity of DNA samples were estimated using a NanoDrop 1000 (Thermo Fisher Scientific, MA, USA) and Qubit dsDNA HS assay kit (Thermo Fisher Scientific), respectively. Purified DNA was used to construct 8 kb and 20 kb long jump distance mate- pair genomic libraries and sequenced using the HiSeq 2000 platform (100MP; Illumina, CA, USA) by Eurofins Genomics (Tokyo, Japan). Finally, the 8 kb mate-pair sequencing produced a total of 115.2 million reads (10.2 Gbp), and the 20 kb mate-pair sequencing produced a total of 61.1 million reads (5.5 Gbp). Long read genome sequencing Trophozoites were grown in 50 tubes (15 mL polystyrene non-treated culture tubes) and harvested by centrifugation at 400 × g for 5 min. High molecular weight DNA was isolated and purified using the NucleoBond HMW DNA Kit (Macherey-Nagel, Düren, Germany). The purity and quantity of the DNA samples were assessed using a NanoDrop 1000 (Thermo Fisher Scientific) and a Qubit dsDNA HS assay kit (Thermo Fisher Scientific), respectively. The purified DNA was used to prepare the library using a Rapid Sequencing Kit (SQK- RAD004; Oxford Nanopore Technologies, Oxford, UK). Sequencing was performed on the MinION platform using R9.4.1 Flow Cells (Oxford Nanopore Technologies) using MinKNOW 3.6.5. Base calling was performed using Guppy 3.2.10 (Oxford Nanopore Technologies). Finally, 435,782 reads (7.3 Gbp) were obtained. RNA-seq Total RNA was isolated from log-phase trophozoites of E. marina SRT209 grown in 15 mL polystyrene non-treated culture tubes using TRIzol reagent. RNA (8.18 ng) was used to prepare the RNA-seq library using the ScriptSeq v2 RNA-seq Library Preparation Kit (Epicentre, WI, USA) following the manufacturer’s protocol. Sequencing was performed at the Pathogen Genomics Center (National Institute of Infectious Diseases, Tokyo, Japan) on the MiSeq platform (300PE; Illumina), yielding a total of 13.9 million reads (5.6 Gbp). The reads were trimmed using fastp v0.22.0 ( Chen et al. 2018 ) with the following options (-q 30 - w 2). Read mapping was performed using HISAT2 with --max-intronlen 3000 ( Kim et al. 2019 ). After removing reads corresponding to the rRNA region (see Supplementary Data) using intersectBed in BEDTools ( Quinlan and Hall 2010 ), the transcripts per million (TPM) values were calculated using the TPMCalculator ( Vera Alvarez et al. 2019 ). De novo assembly The long reads from the MinION were trimmed using Nanofilt v2.8.0 ( De Coster et al. 2018 ) with the following options (-q 8 --headcrop 50 -l 5000). The trimmed reads were then assembled using minimap2 v2.24 ( Li 2018 ) and miniasm v0.3 ( Li 2016 ). The assembly fasta file was generated using the awk command (awk ’/^S/{print ">"$2"\n"$3}’ | fold). The two contigs with too high (>50%) GC content compared to the Entamoeba genome out of 66 contigs were manually discarded as contamination from the co-cultured bacteria Shewanella sp. To correct misassemblies, the 64 contigs were divided into a total of 564 contigs using Tigmint v1.2.9 ( Jackman et al. 2018 ) with subsequent options (tigmint-long span=auto G=37295306 dist=auto). The consensus sequence was called by Racon v1.5.0 ( Vaser et al. 2017 ) for five times. The polishing step was performed using Pilon v1.24 ( Walker et al. 2014 ) with the following options (-Xmx40G --tracks --verbose) using the short reads generated from Illumina mate-pair sequencings and trimmed by fastp. Final assembly had a total length of 37,460,168 bp consisted of 202 contigs. Assembly statistics were calculated using QUAST v5.0.0 ( Mikheenko et al. 2018 ). The scripts used in this study are provided in the supplementary data. Gene prediction and annotation We detected repeat regions in the assembled E. marina genome using RepeatModeler v2.0.1 ( Flynn et al. 2020 ) and soft-masked them using RepeatMasker v4.1.1 (Smit et al. 2013; http://www.repeatmasker.org ). Gene prediction was performed by BRAKER2 ( Brůna et al. 2021 ), providing OrthoDB v10 ( Kriventseva et al. 2019 ) as a protein database. Predictions with supports based on OrthoDB were used for the subsequent process. The remainder of the prediction was additionally filtered with RNA-seq data. The RNA-seq reads from MiSeq were assembled by Trinity v2.15.1 ( Grabherr et al. 2011 ). The reads were remapped to the RNA-seq assembly by HISAT2 ( Kim et al. 2019 ). The reads mapped on RNA-seq were recovered from bam file and then mapped to the genome assembly using HISAT2 with -- max-intronlen option. The annotation of related species Entamoeba histolytica HM-1:IMSS provided in AmoebaDB release 63 (2022-12-06; Aurrecoechea et al. 2011 ) was transferred to E. marina genome using pipelines in GeMoMa 1.9 ( Keilwagen et al. 2019 ). Annotations from GeMoMa and BRAKER2 were merged by GffCompare v0.12.6 ( Pertea and Pertea 2020 ) using the annotation from GeMoMa as a reference. As a result, a total of 10,771 genes were identified. They were compared to the UniProt Reference Clusters (UniRef90; Suzek et al. 2015 ) in EnTAP v0.10.8 ( Hart et al. 2020 ). Finally, total a total of 9,363 genes (including 4,954 hypothetical or uncharacterized genes) were annotated (Supplementary Data Table S1). Statistics for genes were calculated by AGAT ( https://www.doi.org/10.5281/zenodo.3552717 ). Phylogenetic analysis Sequences in Entamoeba species were retrieved from AmoebaDB release 63 using BLASTp (threshold: 1e -5 ). They were aligned using the mafft-linsi command in MAFFT v7.520 ( Katoh and Standley 2013 ). TrimAl v1.2 ( Capella-Gutiérrez et al. 2009 ) automatically selected well- aligned positions using the -automated 1 option. Phylogenetic analyses were performed using FastTree 2.1.10 with the -lg option ( Price et al. 2010 ). The results were visualized using FigTree v1.4.4 ( https://github.com/rambaut/figtree ). Identification of Rab GTPases in E. marina When we used BLASTp to identify Rab GTPases in the E. marina genome using the criteria from a previous study ( Saito-Nakano et al. 2005 ), it gave innegligible false positives from other Ras superfamilies due to GTPase domains. Thus, we conducted RPS-BLAST ( Altschul et al. 1997 ) searches using proteome in E. marina as queries and conserved domains database (CDD) as a database to evaluate if Rab GTPases from E. marina under analysis most likely contain Rab GTPase conserved domains (PSSM-ID: 206640, 206653, 206654, 206655, 206656, 133267, 206657, 206658, 206659, 206660, 206661, 206688, 206689, 133306, 206692, 206693, 206694, 133310, 133311, 206695, 206696, 133314, 133315, 206697, 206698, 133318, 133319, 206699, 133321, 133322, 133323, 133324, 133326, 206700, and 206701; Table S4). We defined Rab GTPases based on top-hits under the e-value 1e -10 . Results and Discussion E. marina possesses the second largest genome in Entamoeba, but its gene repertoire resembles other parasitic Entamoeba The draft genome of E. marina is the second largest among available Entamoeba genomes, with a total length of 37,460,168 bp, and consisted of 202 contigs. This places it second in size, following E. invadens (40.9 Mbp), among the sequenced Entamoeba species including E. histolytica , E. dispar , E. moshkovskii , and E. invadens ( Fig. 1A ). The basic genomic characteristics of E. marina , including GC content and gene number, fall within the range observed in other Entamoeba species ( Table 1 ). The E. marina genome encodes 10,771 proteins, of which 4,954 are annotated as hypothetical proteins. Download figure Open in new tab Figure 1. Similarities among the genomes of E. marina and other Entamoeba ( A ) The flow diagram for genome analysis of E. marina . ( B ) Codon usage in Entamoeba genomes. The y-axis represents the frequency (%) of each codon. ( C ) Venn diagram showing the inclusion relation of the orthogroups in Entamoeba . View this table: View inline View popup Table 1. Basic features of the genome assemblies of five Entamoeba species Statistics for genome assemblies of Entamoeba . The data for E. dispar , E. histolytica HM-1:IMSS, E. moshkovskii , and E. invadens were retrieved from AmoebaDB.org. The data for E. histolytica HM-1:IMSS Clone 6 2001 was from our previous study ( Kawano-Sugaya et al. 2020 ). A notable feature of the E. marina genome is its high repeat contents, with repeat elements accounting for 36.3% of the genome, as detected by RepeatModeler and RepeatMasker. This repeat-rich nature of the E. marina genome is similar to E. histolytica HM-1:IMSS Clone 6 2001 (37.6%; Kawano-Sugaya et al. 2020 ). Furthermore, the codon usage and overall repertoire of E. marina are comparable to those of other Entamoeba species ( Fig. 1B ; Table S1). Orthologous clusters identified by OrthoVenn2 ( Xu et al. 2019 ) revealed 4,100 conserved clusters across five Entamoeba species, with 373 clusters unique to E. marina ( Fig. 1C ; Table S3). These findings suggest that, despite its large genome, E. marina maintains a gene repertoire similar to other parasitic Entamoeba species, indicating that its evolutionary divergence may not be strongly associated with the expansion of virulence- related gene families. Reduction in the repertoire of virulence-associated gene families in E. marina We investigated key virulence-associated genes in E. marina , including cysteine proteases (CP), cysteine protease-binding protein family (CPBFs; Marumo et al. 2014 ), intrinsic inhibitor of CPs (ICP; Sato et al. 2006 ), and Rab small GTPases, using BLAST ( Altschul et al. 1990 ). CPs play critical roles in the pathogenesis of E. histolytica , such as cell killing, phagocytosis, trogocytosis, destruction of extracellular matrix and tissues ( Irmer et al. 2009 ; Ralston et al. 2014 ), and encystation ( Ebert et al. 2008 ), which is necessary for human-to- human transmission. Our BLAST search revealed that approximately half of the CP family A members present in other human-infecting Entamoeba species are absent in E. marina ( Fig. 2A ; CP-A1, A2, A4, A7, A9, A11, and A12; labeled in white). On the other hand, E. marina possesses a species-specific subfamily of CP family A genes (cyan; Fig. 2A ), suggesting that these unique CPs may have specialized biological roles in E. marina . Download figure Open in new tab Figure 2. Half-loss of virulence-associated genes in E. marina The maximum likelihood tree of virulence-associated gene families in the five Entamoeba including ( A ) cysteine protease family A genes, ( B ) cysteine protease-binding protein families, ( C ) lectin heavy subunits, ( D ) lectin light subunits, and ( E ) lectin intermediate subunits. The bootstrap values are shown on each branch. The absence or presence of subgroups in E. marina are shown in white or black, respectively. Intracellular traffic and secretion of lysosomal enzymes, including CPs, are regulated by specific transporters such as CPBF1 ( Marumo et al. 2014 ; Nakada-Tsukui et al. 2020 ) and inhibitors like ICPs (inhibitors of cysteine proteases). The repertoire of CPBPs in E. marina was markedly different from that of other Entamoeba species. Specifically, six of the eleven CPBFs (CPBF3, 4, 5, 7, 8, and 11) were absent in E. marina , while the other CPBFs (CPBF1, 2, 6, 9, and 10) were conserved ( Fig. 2B ; labelled in white). Among them, CPBF1 is the only CP carrier (receptor) among the CPBF members, responsible for transporting all family A CPs from the endoplasmic reticulum to lysosomes ( Marumo et al. 2014 ). ICPs are intrinsic proteins that bind CPs, inhibit CP activities, and prevent their secretion ( Sato et al. 2006 ). In terms of ICPs, E. marina retained two ICPs (QTN25_002404 and QTN25_005631), based on a BLASTp search against ICP1 of E. histolytica (EAL47869.1), showing 35.9% and 25.7% identities, respectively. These values are comparable to those observed between E. histolytica ICP1 and its E. invadens orthologs (XP_004185788.1 and XP_004256329.1) (48.5% and 27.5%). Notably, CPBF6 and CPBF8 regulate the transport of lysosomal hydrolases involved in glycoprotein and carbohydrate degradation in E. histolytica ( Furukawa et al. 2012 ; Furukawa et al. 2013 ). In E. histolytica, it was demonstrated that CPBF6 binds to α-amylase and γ-amylase while CPBF8 binds to lysozymes and β-hexaminidase. In E. marina, only CPBF6, is retained, while CPBF8 is absent. This absence is particularly interesting because CPBF8 is also missing in E. invadens , suggesting that CPBF8 was likely acquired during the divergence of the common ancestor of E. histolytica, E. dispar, and E. moshkovskii from the lineage leading to E. marina and E. invadens . A similar pattern of the absence of isotypes in E. marina may apply to CPBF3 and CPBF4, as the clade containing E. marina and E. invadens is positioned at the base in the phylogenetic tree relative to the CPBF3/4 clade. In contrast, CPBF5 and CPBF11 are absent in E. marina, but are retained in E. invadens , suggesting lineage-specific expansions of certain CPBF isotypes after the divergence of E. marina from other Entamoeba species. Note that it was demonstrated that CPBF3 and CPBF4 likely makes heterodimeric complex and the ligands of CPBF3, 4, 5, and 11 remain undermined. Adherence to and cytolysis of the intestinal epithelia are hallmarks of E. histolytica pathogenesis. While adherence, but not cytolysis, is conserved across Entamoeba species, including commensal, non-invasive siblings, E. histolytica uses the surface galactose/N- acetylgalactosamine inhibitable lectin as a receptor for adhesion to the host epithelium. The Gal/GalNAc lelcting of E. histolytica consists of three subunits, heavy (Hgl), intermediate (Igl), and light subunits (Lgl), whose encoding genes are conserved across the Entamoeba genus ( Frederick and Petri 2005 ). Hgl is a transmembrane protein with the amino-terminal carbohydrate-recognition domain (LecA), responsible for binding to specific carbohydrate epitopes on the human target cells, while Lgl is GPI-anchored and forms a disulfide bond with Hgl. Igl, also GPI anchored, plays a role in adherence ( Petri et al. 2006 ; Min et al. 2016 ; Kato and Tachibana 2022 ). Phylogenetic analysis of Hgl and Lgl across E. marina and four other Entamoeba revealed similar topologies ( Fig. 2C-D ). Both Hgl and Lgl trees consist of two major clades: the first clade contains the core cognate members from each Entamoeba species (cyan), while the second clade includes expanded members (magenta). This pattern suggests an early gene duplication event in the common ancestor, followed by the additional gene duplication leading to species-specific subclades (magenta). In contrast, Igl showed no diversification across Entamoeba species, with the exception of E. invadens , which has 11 isoforms ( Fig. 3E ; magenta). Download figure Open in new tab Figure 3. The genes potentially related to the adaptation to the marine environment ( A ) The sequence alignment of arginases from E. marina and other Entamoeba . ( B ) The maximum likelihood tree of arginases from E. marina . The orthologous arginase in Entamoeba is shown in cyan, whereas the E. marina -specific arginase is shown in magenta. Lineage-specific diversification of major gene families in E. marina The diversification and expansion of gene families through duplication has been well documented in various gene families including those encoding Bacteroides surface protein A (BspA) family ( Lorenzi et al. 2010 ), avrRpt2-induced gene 1 (AIG1) family ( Nakada-Tsukui et al. 2018 ), and Rab GTPases ( Saito-Nakano et al. 2005 ). These gene families are known to be associated with the pathogenicity of Entamoeba . Our analysis revealed that E. marina lack the AIG1 family, a feature shared with E. invadens and E. moshkovskii . In contrast, the BspA family is highly diversified in E. marina , which encodes a total of 449 BspA proteins, approximately four times more than the 121 BspA proteins found in E. histolytica . (Table S1). Rab small GTPases, conserved across eukaryotes, regulate directional vesicular traffic between cellular compartments in a GTP- or GDP-dependent manner. The genomes of Entamoeba species contain a large number of Rab GTPases, indicating extensive diversification of this protein family in the genus (Table S5). The number of Rab GTPases (111-142 genes per species) exceeds that in multicellular eukaryotes, including humans (∼70), suggesting that Rab GTPases play a complex role in regulating membrane traffic in Entamoeba ( Saito-Nakano et al. 2005 ). Notably, many Rabs (e.g., RabA, RabB, Rab5, Rab7A, Rab7B, Rab7D, Rab8A, Rab11A, Rab11B, Rab11D, Rab21, Rab35) are involved in key virulence and transmission-related processes such as phagocytosis, trogocytosis, and encystation ( Saito-Nakano et al. 2004 ; Welter et al. 2005 ; Mitra et al. 2007 ; Hernandes-Alejandro et al. 2013 ; Emmanuel et al. 2015 ; Verma and Datta 2017 ; Saito-Nakano et al. 2021 ). Among the five Entamoeba species analyzed, E. marina has the highest number of Rab small GTPases, encoding a total of 160 proteins for 76 subfamilies (e.g., Rab7) (Table S5), based on RPS-BLAST search. Many gene duplications were identified specifically in E. marina , contributing to the expansion of the Rab GTPase family. Of 76 subfamilies, twenty include multiplicated isotypes, including Rab7D, Rab7H, Rab7I, Rab11C, RabA, RabC1, RabC7, RabD1, RabF5, RabK3, RabM1, RabM3, RabP2, RabX6, RabX11, RabX14, RabX15, RabX23b, RabX33 and RabX33b. TLDc [Tre2/Bub2/Cdc16 (TBC), Lysin motif (LysM), Domain catalytic] domain- containing proteins are another gene family that shows extensive diversification in Entamoeba . The E. marina genome encodes a total of 246 TLDc domain-containing proteins (Table S1), which is 2.1- to 5.7-fold more than those in other parasitic Entamoeba species (43-62 in E. histolytica , 50 in E. dispar , 50 in E. nuttalli , and 116 in E. invadens ), but less than the number in the free-living E. moshkovskii (499). Proteins containing a TLDc domain are known to provide oxidative resistance by reducing reactive oxygen species ( Finelli et al. 2016 ) and to interact with proton-pumping V-ATPase ( Eaton et al. 2021 ). Genes potentially related to lifestyle conversion in E. marina In an effort to identify genes responsible for lifestyle conversion to the marine environment, we hypothesized that the genes gained by E. marina via lateral gene transfer (LGT) might have played significant role in this process. We investigated potential LGT genes that are exclusively present in E. marina but absent or nearly absent in other Entamoeba species. According to annotations from EnTAP ( Hart et al. 2020 ), we identified a total of 95 bacterial- origin genes gained by E. marina via LGT. Of these, eighty two were also shared by at least one other Entamoeba species, while thirteen genes were unique to E. marina, making them strong candidates for LGT-derived genes involved in marine adaption ( Table 2 ). View this table: View inline View popup Table 2. E. marina specific LGT genes Sequence identifier, length, TPM, hit in UniRef90, EggNOGTaxScope, and EggNOGDescription for the 13 genes that E. marina specifically acquired via lateral gene transfer. Our RNA-seq analysis confirmed that among 13 LGT genes, several – such as genes encoding one arginase (QTN25_003979) and four cyclase-like (QTN25_004667, QTN25_006567, QTN25_007270, and QTN25_008921) were expressed at levels exceeding the median value of all transcripts (TPM > 7.101; Table S6). In addition to the LGT-acquired arginase, E. marina also contains a gene encoding a conserved arginase that is found in all Entamoeba species. Interestingly, neither the E. marina- specific LGT-derived arginase nor the conserved Entamoeba arginase contain an organelle-targeting transit peptide, unlike mitochondria-targeted arginase-2 in humans. Alignment of amino acid sequences revealed a highly conserved PROSITE pattern (D-A-H-X-D) across all Entamoeba arginases ( Fig. 3A ; cyan), and residues involved in ligand interactions are also well conserved ( Fig. 3A ; grey diamond), consistent with previous studies ( Malik et al. 2019 ). In contrast, residues involved in oligomerization ( Malik et al. 2019 ) were not conserved in the E. marina- specific arginase, suggesting a potential lack of oligomerization or differences in the oligomerization state ( Fig. 3A ; triangle). Phylogenetic analysis indicated that the evolutionary origins of the E. marina - specific LGT-derived arginase and the conserved Entamoeba arginase were clearly distinct ( Fig. 3B ; magenta and blue rectangle). The close phylogenetic relationship between of E. marina- specific arginase and arginase from Psittacicella melopsittaci , a Gram-negative bacterium in γ−proteobacteria, indicates this E. marina- specific arginase gene was acquired from this bacterial lineage ( Fig. 3B ; magenta). In contrast, the other arginases common to Entamoeba did not show close kinship with other organisms ( Fig. 3B ; blue rectangle). These findings suggest that arginases may have undergone frequent replacement events throughout evolution, providing selectable advantages under unique habitat conditions ( Fig. 3B ; green). Furthermore, the E. marina genome encodes four closely related proteins that show similarity to bacterial kynurenine formamidase (UniRef90_A0A7C3CCR2), with e-values ranging from 5.71e -88 to 6.48e -107 (52.8–61.3% similarities and 93.7–94.7% coverages; Table S1). These E. marina proteins also exhibited 53.97–60.08% similarit to cyclase from a Deltaproteobacteria bacterium (MBN2538769.1), with 94–95% coverage and e-values ranging from 4e -91 to 5e -109 as determined by in NCBI BLAST. Kynurenine formamidase catalyzes the hydrolysis of N-formyl-L-kynurenine to L-kynurenine, an intermediate in the kynurenine pathway of tryptophan degradation. L-kynurenine is further transaminated to kynurenic acid by kynurenine transaminase, which is known for its neuroprotective effects and antioxidant activity, scavenging free radicals originating from FeSO 4 ( Lugo-Huitrón et al. 2011 ). RNA-seq data indicate that these L-kynurenine formamidase genes (TPM = 198.5 ∼ 2,738.2; Table 2 ) and two kynurenine transaminase genes (TPM = 98.0 for QTN25_008178 and 70.3 for QTN25_008089; Table S6) are expressed in E. marina , suggesting their involvement in defense against oxidative stresses in trophozoites. The phylogenetic tree indicates that E. marina cyclase-like protein genes were apparently acquired from bacteria by LGT, with distinct bacterial origins compared to other eukaryotic cyclase-like genes ( Fig. 3C ). In addition to its antioxidant properties, kynurenine has been implicated in several other biological functions, including maintenance of NAD + levels, as well as immune tolerance and inflammatory responses and neuroprotective effects. Since the kynurenine pathway ultimately contributes to the synthesis of NAD + , a critical molecule for cellular energy production and redox homeostasis, it is possible that E. marina might use this pathway not only to defend against oxidative stress but also to maintain cellular energy balance under the challenging marine conditions. In addition, well-documented neuroprotective effects of kynurenic acid, a metabolite of kynurenine, including its role in preventing neurodegenerative diseases may be relevant to the survival and functionality of E. marina under stress conditions in its marine habitat. Conclusion Here, we presented a draft genome of ocean-derived E. marina and conducted genome comparisons between E. marina and other Entamoeba species, with aims to elucidate the mechanisms by which E. marina adapted to the marine environment. The E. marina genome largely resembles those of other Entamoeba species, with the majority of genes and gene families being conserved. However, we observed a reduction in the number or a loss of virulence-associated gene families. Notably, the loss of a large fraction of CPs and CPBFs is considered to be associated with lack of virulence to vertebrate and invertebrate hosts. Distinct evolutionary patterns for three subunits for Gal/GalNAc lectins, known to play a pivotal role in recognition of and binding to the target organisms (pray), indicated that the lectins evolved in a way how individual species adapt to different environment. In addition, E. marina has undergone an expansion of certain gene families, including the BspA family, Rab family, and TLD genes, which are involved in the regulation of essential biological processes. On the contrary, repertoire expansion of AIG1 gene family, commonly observed in Entamoeba species that infect primates, such as E. nuttalli and E. dispar , did not occur in E. marina . This observation is consistent with the premise that AIG1 repertoire expansion contribute to virulence and mammalian host specificity. We also identified 13 genes uniquely acquired through LGT in E. marina , including genes encoding kynurerine formamidases and arginase. Taken together, the first draft genome of marine free-living Entamoeba marina demonstrated striking diversifications from other Entamoeba species, which underlie its unique features in marine adaptation and potentially free-living lifestyle or parasitism to organisms in marine sediments. Conflicts of interest Authors declare that they have no competing interests. Author contribution TN conceptualized and supervised whole study and acquired funds. TKS determined methodology and investigated genome data. SI gave essential advice and technical supports. TKS and TN wrote the manuscript. All authors read and approved the final manuscript. Data availability All sequence data produced in this study were deposited in the Sequence Read Archive (SRA) and GenBank at the National Center for Biotechnology Information (NCBI). The identifiers in the BioProject and BioSample are PRJNA985239 and SAMN35793876, respectively. The SRA accessions for the raw reads after quality trimming were as follows:1) SRR24958723 for long read genome sequence using Oxford Nanopore MinION; 2) SRR24958724 and SRR24958725 for genome sequence with 8kb or 20kb insert using Illumina 100MP sequencings; and 3) SRR24958722 for RNA-seq using Illumina 300PE sequencing. The final genome assembly and annotations were deposited in GenBank (accession number JAUKTS010000000). The all authors confirmed that all supporting data have been provided in the article through the supplementary data files. Supplemental Data Supplemental files | Assembly and annotation data used in this study Table S1. Annotation of E. marina genome from EnTAP Table S2. Codon usage in Entamoeba with frequencies Table S3. E. marina -specific orthologous clusters Table S4. List of entries for Rab conserved domains in CDD Table S5. Rab GTPases in E. marina , E. histolytica , E. dispar , E. moshkovskii , and E. invadens Table S6. TPM for genes of E. marina in RNA-seq Acknowledgements We thank Seiki Kobayashi and Emi Sato-Ebine (National Institute of Infectious Diseases; NIID), Tetsuo Hashimoto (University of Tsukuba), and Kumiko Shibata (The University of Tokyo) for technical assistance on cultivation. We also thank Avik Kumar Mukherjee, Tsuyoshi Sekizuka, and Makoto Kuroda (NIID) for initial analysis of the E. marina genome. This study was supported in part by Grant for Research on Emerging and Re-emerging Infectious Diseases from Japan Agency for Medical Research and Development (AMED, JP22fk0108138 to TN), Grants-in-Aid for Scientific Research (B) (KAKENHI JP21H02723 to TN) from the Japan Society for the Promotion of Science (JSPS), and Grant for Science and Technology Research Partnership for Sustainable Development (SATREPS) from AMED and Japan International Cooperation Agency (JICA) (JP22jm0110022 to TN). Footnotes sugaya{at}tetsu.ro , izmym{at}niid.go.jp References ↵ Altschul SF , Gish W , Miller W , Myers EW , Lipman DJ . 1990 . Basic local alignment search tool . J. Mol. Biol . 215 : 403 – 410 . OpenUrl CrossRef PubMed Web of Science ↵ Altschul SF , Madden TL , Schäffer AA , Zhang J , Zhang Z , Miller W , Lipman DJ . 1997 . Gapped BLAST and PSI-BLAST: a new generation of protein database search programs . Nucleic Acids Res . 25 : 3389 – 3402 . 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Share Draft genome of the marine Entamoeba species reveals reduction in the gene family repertoire associated with pathogenicity and lateral gene transfer for adaptation to the marine environment Tetsuro Kawano-Sugaya , Shinji Izumiyama , Tomoyoshi Nozaki bioRxiv 2025.02.12.637563; doi: https://doi.org/10.1101/2025.02.12.637563 Share This Article: Copy Citation Tools Draft genome of the marine Entamoeba species reveals reduction in the gene family repertoire associated with pathogenicity and lateral gene transfer for adaptation to the marine environment Tetsuro Kawano-Sugaya , Shinji Izumiyama , Tomoyoshi Nozaki bioRxiv 2025.02.12.637563; doi: https://doi.org/10.1101/2025.02.12.637563 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Genomics Subject Areas All Articles Animal Behavior and Cognition (7622) Biochemistry (17648) Bioengineering (13870) Bioinformatics (41880) Biophysics (21423) Cancer Biology (18553) Cell Biology (25458) Clinical Trials (138) Developmental Biology (13364) Ecology (19866) Epidemiology (2067) Evolutionary Biology (24290) Genetics (15589) Genomics (22475) Immunology (17711) Microbiology (40326) Molecular Biology (17145) Neuroscience (88471) Paleontology (666) Pathology (2826) Pharmacology and Toxicology (4815) Physiology (7635) Plant Biology (15114) Scientific Communication and Education (2044) Synthetic Biology (4286) Systems Biology (9815) Zoology (2268)