Evolution and ecology of commensal gut protists: recent advances

Abstract

The microbial community colonizing the animal gut includes all domains of life, including eukaryotic microbes. Historically viewed as pathogens, increasing evidence has revealed that many protists are commensal members of the microbiome with diverse ecological functions. This review synthesizes recent advances in our understanding of the ecology and evolution of these organisms, with a focus on phylogenetic diversity, microbial interactions, and genomic signatures of adaptation. New technologies such as single-cell genomics and transcriptomics, long-read sequencing technologies, and co-culture strategies have made these new findings possible, but much remains to be investigated. Further work is needed to understand how these diverse organisms contribute to the gut environment and evolve to colonize animal hosts.

Introduction

The gastrointestinal tract of animals contains a microbial ecosystem comprised of all domains of life, including bacteria, archaea, viruses, and microbial eukaryotes. The microbiota interact with the host and with each other, influencing host phenotypes by breaking down host-indigestible dietary components and remodeling host immunity [1]. Microbes in the gut are constantly evolving in response to pressures from the host and other microbiota. The genomes of gut bacteria bear signatures of recent adaptation to changes in host diet, lifestyle, and species [2–6].

Historically, protists in the human gut have been considered potential pathogens due to their phylogenetic proximity to known disease-causing protists [7]. However, as gut microbes from all domains of life can cause disease in certain contexts, the microbiota should be considered on a continuum from pathogenicity to commensalism [8]. Some well-studied gut protists, including Entamoeba histolytica and Giardia, can cause invasive disease or elicit infection-clearing immune responses, and are therefore generally considered pathogens despite the existence of asymptomatic infections [7,9,10]. Recent work from a variety of sources, including deep metagenomic sequencing and large case-control cohorts, has demonstrated that many prevalent gut protists in humans are benign and sometimes correlated with positive health outcomes [11–15]. In contrast to pathogenic protists, these commensal protists lack evidence of acute pathogenicity, are prevalent in asymptomatic individuals, and have long-term associations with the host [7,12,16]. These findings prompt a re-evaluation of commonly detected gut protists in humans, shifting away from a focus on pathogenicity towards a view as resident members of the gut microbiota [7,17].

This perspective shift, coupled with technological advances in sequencing and studying the microbiota, have resulted in new findings about the ecological niches of gut protists and how they have adapted to different hosts and different microbial assemblages. This review focuses on recent advances in understanding the forces shaping the evolution and ecology of commensal gut protists found across animals, with an emphasis on microbial interactions and genetic signatures of adaptation.

Who’s who: the commensal gut protists

“Protist” does not refer to a monophyletic group and is used here as to refer to singlecelled microbial eukaryotes that are not archaeplastids (plants and green algae) or opisthokonts (fungi and metazoans). Protists colonizing the gastrointestinal tract are phylogenetically diverse, including stramenopiles, ciliates, metamonads, and amoebozoans. Several groups of gut protists that include commensal organisms are summarized here and in Table 1.

Table 1.

Key commensal gut protists.

Organism	Taxonomic Group	Primary habitat	Morphology
Blastocystis spp.	Stramenopile	Human & other vertebrate animal gut	Non-flagellated, round morphology
Opalina spp.	Stramenopile	Amphibian gut	Ciliated, multinucleate
Entodinium spp.	Alveolata	Rumen of ruminant animals	Ciliate
Isotricha spp.	Alveolata	Rumen of ruminant animals	Ciliate
Dientamoeba fragilis	Metamonada (Parabasalia)	Human & other mammal gut	Amoeboid, lacks visible external flagella
Pentatrichomonas hominis	Metamonada (Parabasalia)	Human & other mammal gut	Flagellate
Tritrichomonas muris	Metamonada (Parabasalia)	Mouse gut	Flagellate
Tritrichomonas casperi	Metamonada (Parabasalia)	Mouse gut	Flagellate
Chilomastix mesnili	Metamonada (Retortamonadida)	Human and non-human primate gut	Flagellate
Retortamonas intestinalis	Metamonada (Retortamonadida)	Human and non-human primate gut	Flagellate
Enteromonas hominis	Metamonada (Diplomonadida)	Human and non-human primate gut	Flagellate
Entamoeba spp.	Amoebozoa (Archamoeba)	Human & other animal gut (commensal, except for Entamoeba histolytica)	Amoeba
Endolimax nana	Amoebozoa (Archamoeba)	Human & other mammal gut	Amoeba

Stramenopiles: Slopalinata

The most prevalent protist that colonizes the human gastrointestinal tract is Blastocystis, a stramenopile distantly related to oomycetes [12,18]. Blastocystis colonizes most vertebrate animal lineages and some invertebrates [19–21], comprising genetically distinct subgroups that likely represent different species [22]. Subgroups found in mammalian and avian groups are clustered into numbered subtypes based 18S rRNA similarity [23]. Related stramenopile protists such as the Proteromonas and Opalina genera also primarily colonize the gastrointestinal tracts of reptiles and amphibians and have not been reported in primates [18,24,25]. In the human gut, Blastocystis has been linked to alterations in the gut microbiota alongside positive health status [11,12,15,16].

Alveolata: Ciliates

Ciliates are abundant in ruminant animals, and can comprise up to 50% of the total microbial abundance in the rumen [26]. Because of their high relative abundance and presence in agriculturally important animals, rumen ciliates are among the most well-studied commensal protists. Ciliates play varied roles in the rumen including predators of bacteria, metabolic partners of methanogens, and biomass degraders [27]. Ciliates are rare in primates; the sole ciliate known to infect humans, Balantidium coli, can cause severe gastrointestinal disease [28].

Metamonada: Parabasalia

The parabasalids are an ancient and diverse group that contains parasites, commensals, and some free-living lineages [29]. Some parabasalid symbionts of insects participate in processes like lignocellulose digestion, enabling termites to digest wood [30]. Parabasalid protists that colonize the human gastrointestinal tract include Dientamoeba fragilis, which has been linked with gastroenteritis but may be incidental [13], and Pentatrichomonas hominis [29]. The discovery of Tritrichomonads colonizing laboratory mice has led to mechanistic understanding of how these protists interact with the host immune system and with other microbiota [31–33].

Metamonada: Retortamonadidae and Diplomonadida

Like Parabasalia, both Retoramonads and Diplomonads contain parasites, commensals, and some free-living lineages. The retortamonadids Chilomastix mesnii and Retortamonas intestinalis and the diplomonadid Enteromonas hominis are commensals in humans, though they are relatively rare in industrialized populations [34,35]. Classifications based on the 18S rRNA sequence has found host-specific genotypes for Retortamonas and Chilomastix [36,37]. Little is known about how these organisms interact with other microbes, and they lack genome sequences. The diplomonadid Giardia lamblia is a common human gut parasite that attaches to the epithelial wall of the small intestine and, when symptomatic, causes diarrheal disease and nutrient malabsorption. While the contexts that control when Giardia causes disease remain unclear, evidence suggests that microbial interactions play a role in pathogenicity [38]. Giardia has been linked to alterations in the gut microbiome both in the context of disease models and in the context of asymptomatic carriage [39,40]

Archamoeba: Amoebozoans

The amoebozoan Entamoeba histolytica is pathogenic; while many cases are asymptomatic, E. histolytica can invade host tissue and cause serious disease in other body sites [41]. Close relatives of E. histolytica are non-pathogenic and are found as commensals in the guts of humans and other animals, including Entamoeba dispar, Endolimax nana, and others [42]. As for Giardia, interactions between Entamoeba histolytica and other gut microbes may contribute to its disease-causing phenotype [43]. Gut amoebozoans consume bacteria through phagocytosis, which may modulate the gut microbial ecosystem [44].

Genomic signatures of adaptation to the gut environment

Genome sequencing of eukaryotic pathogens, parasites, and symbionts has revealed common genomic features of these organisms, including adaptations to an anaerobic environment, genome streamlining, and expansions of transposable elements. Some of these features are not adaptive and reflect a loss of constraint on pathways essential in free-living eukaryotes, such as nutrient synthesis [45]. For example, the pathogenic protists Entameoba histolytica and Giardia lamblia have lost de novo purine and pyrimidine synthesis pathways and depend on the host for these essential compounds [46,47]. Other genomic changes in gut protists, like major changes to mitochondria and loss of aerobic respiration, reflect the shared need to adapt to an anaerobic environment. One source of lineage-specific innovation that is shared across host-associated eukaryotes is horizontal gene transfer (HGT) from bacteria [48]. Recent advances in sequencing eukaryotic gut symbionts have revealed that niche-specific carbohydrate-active enzyme (CAZyme) genes are frequently acquired via HGT in diverse protist groups.

The genomes of rumen ciliates are challenging to sequence for multiple reasons (see Box 1). To overcome this, multiple recent studies have used single-cell genome and transcriptome sequencing, which have found a high number of horizontally transferred plant-active CAZymes. This was initially observed from individual gene [49] and expressed sequence tag studies in ciliates [50] and from comparative studies, including one that assembled a macronuclear genome for the cultured ciliate Entodinium caudatum [51] and a comparative single-celled transcriptome analysis of three uncultured rumen ciliates [52]. Recent work that generated single-cell genomes for 52 different genomes for 22 morphospecies of rumen ciliates also found high numbers of plant cell wall active CAZymes. In particular, two genera of rumen ciliates have as many CAZymes as the Neocallimastigomycota anaerobic rumen fungi, which degrade plant matter in cellulosome nano-machines analogous to bacterial celluosomes [53–55]. Further work using metagenome-assembled genomes and metaproteomics found in vivo support for rumen ciliates participating in carbohydrate fermentation in the rumen [56].

BOX 1: Technological advances in sequencing gut protists genomes and transcriptomes.

Sequencing gut protist genomes can be challenging for many reasons, including difficulties in culturing and genomic features that make assembly difficult. Many species of gut protists are difficult or impossible to grow in culture [18,29]. Single-cell genomics and transcriptomics has made it possible to sequence these microbes. Single-cell genomics and transcriptomics can be performed using manual micromanipulation techniques to extract single cells from a sample and amplify the genome or transcriptome [25,52,59]. Flow cytometry methods have been demonstrated to be effective both for single-cell genomes and for increasing protist representation in the metagenomic sequenced sample [75,76].

Many gut protists can be grown in culture alongside other microbes but cannot be grown axenically. Cultures where protists have been enriched via antibiotic treatment or density-based centrifugation can be sequenced and assembled using metagenomic sequencing techniques [77]. Some protists can be grown for short periods in the absence of bacteria, but this period can be sufficient to obtain enough material for sequencing [78].

Other challenges in sequencing commensal protist genomes arises from characteristics of the genomes themselves. Transposable element expansion is frequently seen in parabasalids, which makes genome assembly difficult [79]. Long-read sequencing from Nanopore and PacBio technologies has significantly improved genome assemblies for transposon-rich parabasalids [33,65,80]. Ciliates are particularly challenging as they have multinucleate cells with alternate subgenomes, small chromosomes arising from programmed chromosomal fragmentation, and bacterial endosymbionts [81–83]. Bacterial endosymbionts from protist genomes can be addressed with metagenomic sequencing techniques and by techniques to identify bacterial specific contigs, such as excluding contigs from a final assembly that do not have evidence of polyadenylated gene expression.

The Parabasalia contain many gut symbionts, including termite gut symbionts that participate in lignocellulose digestion, facilitating the wood-eating lifestyle of these insects [57,58]. Termite gut parabasalids are difficult to culture, but single-cell sequencing has enabled genomic investigation into these organisms. Comparative single-cell transcriptomics together with enzymatic assays from the gut of the termite Coptotermes formosanus identified a division-of-labor mechanism for lignocellulose degradation between four different parabasalids species based on differential distribution of CAZyme families [59]. One species contained chitindegrading genes horizontally transferred from bacteria, which may play roles in metabolism of dead skin shed by the host or in preventing infection by entomopathogenic fungi.

The Blastocystis genus of protists are the most prevalent eukaryotic microbes in the human gut and are widespread in other vertebrates. Despite its prevalent nature, how Blastocystis contributes to the gut environment metabolically remains unclear. Recent work sequenced genomes from Blastocystis from herbivorous tortoises, finding that these lineages contained more CAZymes and plant-carbohydrate degrading genes than Blastocystis isolated from humans [60]. Several of these gene families arose from horizontal gene transfer from bacteria to the protist, and in some cases from bacteria that colonize the gut. The contribution of Blastocystis to the breakdown of dietary fiber in the gut remains uncharacterized, but it is likely that these horizontally transferred genes reflect a niche for the protist in breaking down plant fibers in herbivores.

Functional validation of ecological niches of gut protists

Understanding the biology of commensal gut protists requires understanding how they interact with other microbes. In free-living environmental microbial communities, protists have important roles as bacterial predators, which shapes the structure of the bacterial community and can contribute to bacterial evolution [61]. Recent advances have established multiple different niches for protists in the gut microbiome, including as predators of bacteria that shape the bacterial population, carbohydrate fermenters that contribute to host energy balance, and as both metabolic partners to and antagonists of bacterial and archaeal microbiota [27].

Ciliates are abundant in the rumen of ruminant animals and comprise up to 50% of the overall microbial biomass [26]. Protists can be removed through defaunation without negative host impact, though this process substantially alters the microbial community composition and methane production. The precise impact of the protist on the rumen bacterial community remains unclear, but proposed roles have included bacterial predation, metabolic partners providing hydrogen to methanogens, and plant fiber decomposition [27].

To investigate the impact of ciliates on the rumen microbiota, recent work used an in vitro culture system using freshly sampled rumen fluid, culturing size-separated fractions of rumen protists together with rumen prokaryotic microbes [62]. Each size-fractionated protist group corresponded to different genera of ciliates, and the co-cultures were tracked for changes in prokaryotic species composition and metabolite production. Only the protist co-culture fraction containing Isotricha ciliates produced higher levels of methane; Isotricha has especially active hydrogen-producing hydrogenosomes, which is used by methanogens for methane production [27]. All protist-bacterial co-cultures maintained higher bacterial diversity than protist-free cultures. Specifically, protist-containing cultures were more likely to contain phylogenetically related taxa than the protist-free cultures, suggesting that the presence of the protist discouraged strain interference. While the mechanisms remain unclear, this work provides evidence from real rumen microbial communities that the presence of ciliates maintains bacterial diversity, possibly through reducing bacterial competitive exclusion. Commensal protist presence has been linked in many animals, including humans, to higher levels of bacterial diversity [15,16,63], and this study demonstrates a causal role for protists in maintaining this diversity.

Microbes in the gut metabolize host-inaccessible dietary as well as host derived compounds. This includes dietary fiber, much of which is not directly metabolized by animal hosts, host mucin, a highly glycosylated protein that lines the intestinal wall of the large intestine, along with many other dietary-, host-, and microbial- derived compounds [64]. Recent work using parabasalid protists of mice, Tritrichomonas musculis and Tritrichomonas casperi, found that these protists can participate in carbohydrate metabolizing pathways characterized in gut bacteria to digest both dietary fiber and host mucin glycans [65]. Tritrichomonas musculus can metabolize dietary fiber and host mucin glycans, though in vivo prefers dietary fiber and can outcompete fiber-digesting bacterial species. However, under fiber limiting conditions, the protist is outcompeted by mucin-digesting bacteria. Tritrichomonas casperi preferentially digests host mucin glycans in vivo, and can exclude the mucin-consuming bacteria it competes with for this ecological niche. These protists are closely related, but can co-colonize without clonal interference.

These studies have advanced our understanding of the ecological roles of gut protists, but data remain scarce for commensal protists that colonize humans. Blastocystis in humans is linked to altered gut microbiota, with increases in the methanogenic archaea Methanobrevibacter smithii, increases in butyrate-producing Roseburia bacteria, and decreases in some pro-inflammatory bacteria such as Ruminoccocus gnavus and Eggerthella lenta [12,16]. However, little is known about interactions between Blastocystis and the other microbiota. Less is known about Dientamoeba fragilis and the gut microbiome, but several reports have indicated changes in the bacterial population when this protist is present [66,67].

Open questions and opportunities

Many open questions and opportunities remain for understanding the evolution and ecology of gut commensal protists. Many species have not been cultured, and many lack genomes or transcriptomes necessary to make inferences about their metabolism and evolution. A fruitful new direction includes combining genomic data with experimentation. Pairing genomic data with in vivo experimentation revealed that although Tritrichmonas musculus has the metabolic pathways to digest both dietary fiber and host mucin, it preferentially consumes dietary fiber in vivo [65]. Single cell RNA-seq of this species demonstrated expression of meiotic genes in specific cell types, suggesting a potential sexual reproductive cycle that occurs inside the host [33].

Work studying protist-microbiota interactions is vital to understanding the ecology of protists, as microbial interactions are fundamental to the composition of the gut microbiome. While animal models to study microbial effects are powerful, they are difficult to establish for protists that do not colonize laboratory mice and sometimes lack relevance to the host species. In vitro experimentation with individual microbes or microbial communities is a fruitful direction, especially as the reduced complexity of these systems can build predictions that can be directly tested in animal models.

Much remains to be learned about the role of host-derived and microbiota-derived compounds in shaping the ecology and functional impact of protists on the gut microbiome. Host-derived compounds such as bile acids [68], microbiota-derived compounds such as short chain fatty acids [69], and host immune factors like secretory IgA [70] are critical factors contributing to bacterial ecology in the gut, and it is likely that these factors influence commensal protist ecology as well. Bacteriophage shape the ecological dynamics of all microbial ecosystems, and studies from free-living systems have found interactions between bacteriophage and bacterivorous protists in shaping microbial ecosystems [71]. How phage dynamics may impact commensal protists in the gut microbiome is not yet understood.

Substantially more work is needed to understand many elements of gut protist function. Many large gene families in protists with signatures of rapid evolution remain uncharacterized, including the large and diverse families of adhesin-like genes present in parabasalids, amoebozoans, and stramenopiles [45,72,73]. Recent work in bacteria described a family of adhesins that plays a host species-specific role in adhering gut bacteria to the epithelial wall of the Drosophila gut [74], and it may be the case that some of these uncharacterized gene families contribute to adaptation to different hosts and to different microbiota. Future work in these areas is likely to shed light on many of the remaining mysteries of these organisms.

Figure 1.

Types of trans-kingdom microbial interactions in the gut and genetic signatures of adaptation. A) Examples of protist-microbiota interactions in the gut. B) Horizontal gene transfer of carbohydrate-active enzymes has enabled protists to degrade the same carbon sources as gut bacteria.