View metadata, citation and similar papers at core.ac.uk brought to you by CORE MINI REVIEW ARTICLE published: 11 July 2011 provided by PubMed Central doi: 10.3389/fmicb.2011.00153 Microbial eukaryotes in the human microbiome: ecology, evolution, and future directions

Laura Wegener Parfrey1,William A. Walters2 and Rob Knight1,3*

1 Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO, USA 2 Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO, USA 3 Howard Hughes Medical Institute, University of Colorado, Boulder, CO, USA

Edited by: High throughput sequencing technology has opened a window into the vast communities Peter J. Turnbaugh, Harvard of bacteria that live on and in humans, demonstrating tremendous variability, and that they University, USA play a large role in health and disease. The eukaryotic component of the human gut micro- Reviewed by: Alain Stintzi, Ottawa Institute of biome remains relatively unexplored with these methods, but turning these tools toward Systems Biology, Canada microbial eukaryotes in the gut will likely yield myriad insights into disease as well as the Jacques Ravel, University of ecological and evolutionary principles that govern the gut microbiota. Microbial eukary- Maryland School of Medicine, USA otes are common inhabitants of the human gut worldwide and parasitic taxa are a major *Correspondence: source of morbidity and mortality, especially in developing countries, though there are Rob Knight, Department of Chemistry and Biochemistry, University of also taxa that cause no harm or are beneficial. While the role microbial eukaryotes play in Colorado, UCB 215, Boulder, CO healthy individuals is much less clear, there are likely many complex interactions between 80309, USA. the bacterial, archaeal, and eukaryotic microbiota that influence human health. Integrat- e-mail: [email protected] ing eukaryotic microbes into a broad view of microbiome function requires an integrated ecological approach rather than one focused on specific, disease-causing taxa. Moving forward, we expect broad surveys of the eukaryotic microbiota and associated bacteria from geographically and socioeconomically diverse populations to paint a more complete picture of the human gut microbiome in health and disease.

Keywords: intestinal protozoa, host-associated communities, eukaryotic diversity

INTRODUCTION is not phylogenetically meaningful because microbial and macro- Microbial eukaryotes are an important component of the human scopic organisms intermingle in the eukaryotic tree of life (Adl gut microbiome. Eukaryotes that reside in the human gut are dis- et al., 2005; Parfrey et al., 2010). Similarly, terms such as protozoa, tributed across the eukaryotic tree (Figure 1) and their relationship amebae, flagellates, and algae do not have phylogenetic meaning with the human host varies from parasitic to opportunistic to but are useful as morphological descriptors. commensal to mutualistic. For the purposes of this review, we are focusing on the microbial eukaryotes and are not discussing APPROACHES TO STUDYING HOST-ASSOCIATED MICROBIAL metazoan parasites,which are thoroughly covered elsewhere (Stoll, EUKARYOTES 1947; Kassai, 1999; Muller, 2002; Bogitsh et al., 2005). We are also Microbial eukaryotes in the human gut have been studied pri- focusing on the gut microbiome as many eukaryotic microbes are marily from a parasitological point of view and are generally found there, and it is the best-characterized human body site in considered to negatively impact human health. The methods typ- terms of the bacterial communities. ically used focus on elucidating the presence of specific parasitic Eukaryotes are one of the three domains of life and are defined taxa, traditionally with culture and microscopy-based approaches by the presence of nuclei. Animals, plants, and fungi are the most (Bogitsh et al., 2005; Church et al., 2010) and more recently with visible clades of eukaryotes, but these are just three of the 70+ targeted molecular analyses such as PCR based assays (Stensvold lineages (Patterson, 1999), most of which are microbial. In recent et al., 2011). Broad surveys of the eukaryotic diversity in the gut decades, perspectives on the organization of eukaryotic diversity microbiota have only been explored on a limited basis (March- have shifted away from a Five Kingdom view emphasizing plants, esi, 2010), but are likely to provide new insights into the role of animals, and fungi (Whittaker, 1969; Margulis et al., 1990). Molec- microbial eukaryotes in the gut by providing data on the whole ular systematics and evolutionary studies clearly demonstrate that community, including uncultured organisms and potential inter- the bulk of the eukaryotic tree of life is microbial (Adl et al., 2005; actions among taxa. Fewer than 1% of microbes are culturable, Keeling et al., 2005; Parfrey et al., 2010), and eukaryotes are now and culture independent (CI) studies of microbial communities divided into a small number of higher-level clades, although the have revealed abundant insights into the nature and function structure of the eukaryotic tree is still stabilizing (Adl et al., 2005; of bacterial and archaeal communities in diverse environments Burki et al., 2010; Parfrey et al., 2010). Microbial eukaryotes are (Pace, 1997), including the human microbiome (Marchesi, 2010; sometimes colloquially referred to as protists, although this term Robinson et al., 2010). While studies on eukaryotes lag behind

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those of bacteria, CI approaches in environmental microbiology Human-associated microbial eukaryotes found in the gut are have also revealed hidden diversity and ecological interactions the focus of this article; however, eukaryotes are also part of the within eukaryotes (Caron et al., 2009; Steele et al., 2011). microbial community in other locations of the human micro- In recent years barcoded high throughput sequencing of marker biome. There is a low-diversity fungal community associated with genes like small subunit ribosomal DNA (Hamady et al., 2008) human skin dominated by the genus Malassezia (Paulino et al., coupled with analytical tools to assess patterns in the resulting data 2006). Fungi are also members of the oral microbiota; CI stud- (Caporaso et al., 2010) have made it possible to assess the compo- ies of western populations reveal diverse fungal lineages (Ghan- sition of bacterial communities and compare communities across noum et al., 2010). The mouth can harbor microbial eukaryotes many samples. These approaches have successfully been applied that are closely related to taxa found in the gut such as Enta- to bacteria and archaea, and have revolutionized our understand- moeba gingivalis and Trichomonas tenax in individuals with poor ing of the human microbiome (Costello et al., 2009; Marchesi, oral hygiene (Bogitsh et al., 2005). Parasites that cause malaria 2010; Robinson et al., 2010). Comparing eukaryotic communities (Plasmodium) and sleeping sickness (Trypanosoma brucei) reside across a diverse collection of individuals from different geographic in the liver and bloodstream, and other taxa cause problems regions, economic status, and disease states are the next steps in when in brain tissue (e.g., Encephalitozoon, Toxoplasma, Naegleria; characterizing the human gut microbiome. Bogitsh et al., 2005). There is growing body of theoretical and experimental liter- ature on co-infection within the infectious disease community CURRENT SURVEYS OF THE EUKARYOTIC COMPONENT OF that stresses the importance of considering the action of disease THE HUMAN MICROBIOME agents within the context of other microbial players (e.g., Cox, Comprehensive study of the eukaryotic component of the human 2001; Graham, 2008; Lafferty, 2010; Telfer et al., 2010). This may microbiome is just beginning and lags far behind our understand- provide a framework for understanding the broader community ing of the bacterial communities. Initial CI surveys that were done context of the gut microbiome. Taking advantage of analytical with low-throughput methods of community finger printing and tools such as co-occurrence networks, which detect positive or clone library sequencing are intriguing and suggest that there are negative correlations among taxa (Steele et al., 2011), will also numerous parallels between the bacterial and eukaryotic compo- be crucial in detecting important interactions among community nents of the microbiome. Fungi and Blastocystis are the dominant members. Together, these approaches will provide a baseline of (and in many cases the only) eukaryotes in the gut microbiome expected microbial eukaryotes in healthy individuals and yield of healthy individuals (Nam et al., 2008; Ott et al., 2008; Scan- a better understanding of community changes associated with lan and Marchesi, 2008). The diversity of microbial eukaryotes disease states. within an individual is low, with fewer than 10 phylotypes recov- ered per individual (Ott et al., 2008; Scanlan and Marchesi, 2008). ROLES OF HOST-ASSOCIATED EUKARYOTES: PATHOGEN, The limited data available thus far suggests that eukaryotic com- COMMENSAL, BENEFICIAL munities are stable across time and unique to individuals (Scanlan Understanding the prevalence and distribution of microbial and Marchesi, 2008). The apparent diversity will likely rise as more eukaryotes in the human gut has large consequences for human individuals from diverse populations are sampled with next gener- health. This is especially true in the developing world, where ation sequencing technologies by that enable rare taxa within the microbial parasites represent a large source of morbidity and microbiota to be detected. mortality (Kaplan et al., 2000; Haque, 2007). Major gastrointesti- nal pathogens include Entamoeba histolytica (amoebiasis), which EVOLUTIONARY CONTEXT infects millions of people and results in 40,000–100,000 deaths The evolutionary patterns of microbial diversity within the ver- annually, and Giardia intestinalis (giardiasis), which is the most tebrate gut are similar across eukaryotes, bacteria, and archaea. prevalent intestinal parasite (Haque, 2007). While intestinal pro- Across all taxa only a small number of lineages have adapted to the tistan parasites are often considered a tropical malady, they are gut environments, but those lineages are successful in colonizing actually broadly distributed across the globe, and their prevalence a wide variety of hosts. Reduced diversity at deep phylogenetic within a population is often linked to poor sanitation of human levels (e.g., the Bacteroidetes and Firmicutes divisions of bacte- waste (Bogitsh et al., 2005; Pritt and Clark, 2008). ria) is a hallmark of host-associated bacteria and archaea (Ley While the focus is generally on pathogens (in terms of study et al., 2006). This pattern is also evident in eukaryotes, where a effort, genome sequencing, etc.), most microbial eukaryotes that small subset of eukaryotic lineages have host-associated repre- reside in the gut do not cause harm, being either beneficial or sentatives (Figure 1). Within the bacteria, the lineages present in commensal. Some eukaryotic microbes are considered probiotics, the gut have flourished and produced extensive species and strain in particular the yeast Saccharomyces boulardii was originally iso- level diversity (Ley et al., 2006). The genetic diversity of microbial lated to combat Cholera,and it now marketed as a cure for diarrhea eukaryotes at shallow phylogenetic levels is lower than observed (McFarland and Bernasconi, 1993). Other eukaryotes have been for bacteria, although there is still extensive strain (or species) level shown to be beneficial in certain context, for example the hypoth- variation in isolates from different individuals (Parkar et al., 2010; esized reduction in asthma and allergies in conjunction with Stensvold et al., 2011). The second major trend is that the taxa hookworm infection (Falcone and Pritchard, 2005). Many other that have adapted to host-associated environments are broadly eukaryotic microbes are present in the gut but do no harm (they are distributed as parasites and commensals of many different ani- commensals), including Pentatrichomonas and Entamoeba dispar mal hosts. For example, commensal flagellate genera Chilomastix (Bogitsh et al., 2005). and Pentatrichomonas are found both in the bovine rumen and in

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Foraminifera SAR: Polycystina Acantharea Haplosporidia, Rhizaria Plasmodiophora Euglyphida Cercomonadida Thaumatomonads Phaeodarea Desmothoracids, Chlorarachniophytes

Diatoms Brown algae Stramenopiles Blastocystis

Dinoflagellates Cryptosporidium Alveolates Apicomplexa Balantidium Ciliates

Cryptomonads Haptophytes Red algae

Green algae (including plants) Glaucocystophytes Euglenozoa Excavata

Heterolobosea

Jakobids Trichomonas, Malawimonas Dientamoeba Parabasalids Preaxostyla Enteromonas Fornicata Chilomastix Giardia Entamoeba Centroheliozoa Archamoeba Tubulinea Amoebozoa Acanthamoebidae Eumycetozoa

Flabellinea

Ascaris Apusomonads Animals Opisthokonts Choanoflagellates Mesomycetozoa Candida Pneumocystis Enterocytozoon Fungi Microsporidia Nucleariidae

FIGURE 1 | Distribution of host-associated taxa across a broadly represent lineages found in invertebrates (some red lineages are also present sampled eukaryotic tree of life. Red branches represent lineages found in in invertebrates). Yellow taxa are non-gut parasites of humans. Tree is based the vertebrate gut with human-associated genera listed. Orange branches on Parfrey et al. (2010).

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the human gut (Williams and Coleman, 1992), and E. histolytica eukaryotic tree (Orange branches in Figure 1), and several novel and E. invadens are parasitic in humans and snakes, respectively lineages have been recently discovered (e.g., Hertel et al., 2002). (Barnard and Upton, l994). This suggests that relatively few taxa This pattern likely stems from two factors:“invertebrates” are a far have adapted to host-associated environments, but these taxa are more diverse category phylogenetically than are vertebrates, and successful in a variety of hosts and have diversified at shallow invertebrate-associated environments span a far broader range of phylogenetic levels. conditions. The common patterns of diversity in the gut microbiome seen across the three domains of life are likely driven by the fact that THE ECOLOGICAL CONTEXT OF THE GUT MICROBIOME host-associated environments, and especially the gut, are unique The species composition and general patterns of diversity found in microbial habitats that are difficult to colonize because the sta- the intestinal microbiota are best understood from an ecological ble, warm, low-oxygen, and eutrophic conditions represent an perspective (reviewed in Ley et al., 2006; Robinson et al., 2010). extreme environment (Ley et al., 2008). Eukaryotes that live in Eukaryotes in the gut are not acting in isolation but are part of the gut are anaerobic or microaerophilic, and in most cases have a complex community of microorganisms that includes bacteria, highly reduced mitochondria (Hjort et al., 2010). Global analysis archaea, and viruses that interact through predation (Wildschutte of bacterial communities demonstrate that host-associated com- et al., 2004), competition (Graham, 2008; Lafferty, 2010), and munities are quite distinct from their free-living counterparts (Ley mutualism (Radek, 1999). The microbiota are also interacting and et al., 2008). While global patterns of eukaryotic diversity have responding to the host immune system. Thus, it is important to yet not been examined quantitatively, it is likely that similar pat- determine changes in the bacterial and archaeal communities that terns to bacteria will emerge, as most gut taxa are known only correspond to the presence or absence of eukaryotic microbes. from host-associated environments and are found across a range Next-generation sequencing of the total microbiota across diverse of hosts (Kreier and Baker, 1987). However, extensive CI studies samples will enable this type of investigation. Placing eukaryotic of eukaryotes may reveal additional hidden diversity either within microbes within the ecological context of the microbiome may be known lineages or in lineages not previously found within the important for determining factors that influence pathogenicity of microbiome. those that reside in the human gut. An ecological perspective will be particularly useful in understanding asymptomatic infections, EUKARYOTIC COMMUNITIES IN OTHER HOSTS opportunistic infections, and complex phenotypes such as IBS and The eukaryotic microbiota has been extensively studied in animals malnutrition. where eukaryotes play a more central role, such as ruminants and The range of the host response to many individual microbial termites, and these studies may provide expectations for the role eukaryotes varies greatly from asymptomatic to causing morbid- of eukaryotes in the human microbiome. Culture independent ity or mortality. For example, many people infected with known studies have shown that mice harbor fungal communities that are parasites, such as E. histolytica and G. intestinalis, are asympto- much more diverse than those found in humans (Scupham et al., matic (Prado et al., 2005; Pritt and Clark, 2008). In the case of 2006; Marchesi, 2010), and animals that break down cellulose may E. histolytica, only 10% of infections are invasive and result in generally harbor higher eukaryotic diversity. Eukaryotes make up amoebic dysentery, but these cases often lead to death (Pritt and the bulk of microbiota residing in the rumen and hindgut of many Clark, 2008). Conversely, a number of microbial eukaryotes that herbivores. Yet, even here the high-level phylogenetic diversity is are normally commensal or absent from the human microbiota limited: for example, the multitude of rumen ciliates all belong to a can occasionally cause disease when they become abundant or single order, the Litostomatea (Williams and Coleman, 1992). The infect immunocompromised people. Many factors underlie the human commensal Balantidium also belongs to this order. One differential manifestations of pathology, including host immune of the best-characterized examples of mutualism occurs between response, prior exposure to the pathogen, host genetics, and nutri- symbiotic flagellates that reside in the hindgut of basal termites tional status, and co-infection with multiple parasites (e.g., Pritt and the wood-eating cockroach Cryptocercus and break down cel- and Clark, 2008). Differences in the bacterial and archaeal com- lulose (Radek, 1999). These insects rely on the parabasalid and munities may be a further factor determining whether or not oxymonad symbionts to break down cellulose and release energy, an individual manifests symptoms when their intestinal tract is and these flagellates can constitute 15–30% of the body weight of colonized by protistan parasites. Building a large database of co- the termite (Radek, 1999). Going a step further, parabasalids of the occurring eukaryotic and bacterial communities will allow one to genus Trichonympha that inhabit Cryptocercus are themselves inti- test the hypothesis that changes in the bacterial community are mately associated with bacterial symbionts (Carpenter et al.,2009). responsible for the variance in the pathogenicity of eukaryotic There are also complex endosymbioses between rumen ciliates, parasites. methanogenic Archaea (related to Methanobrevibacter smithii) Opportunistic parasites are a significant source of morbid- and proteobacteria (Irbis and Ushida, 2004). ity in immune compromised patients, although these same taxa Examining the eukaryotic microbiota associated with diverse may be present in healthy people without apparent consequence. animals has the potential to reveal new lineages of eukaryotes These emerging opportunistic pathogens include Cryptosporidium as several clades are known only from the microbiome of spe- parvum, Pneumocystis carnii, and microsporidia, and all generally cific taxa. For example opalinids, a clade of stramenopiles, are cause severe diarrhea (Kaplan et al., 2000). This shift in path- only found in the hindgut of amphibians and hypermastigid ogenicity between healthy and immunocompromised individuals parabasalids in the hindguts of termites and cockroaches. Parasites might result from changes in the microbial community in addition of invertebrate animals are more broadly distributed across the to changes in the immune system. These parasitic infections may

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be similar to Clostridium difficile induced diarrhea, which has been taxa correlate with differences in the bacterial community host shown to result from disruption of the gut microbial community malnourishment. (Walk and Young, 2008). A better understanding of the micro- bial ecology of opportunistic infections may lead to more effective PROSPECTS FOR THE FUTURE treatment options, as it has with C. difficile (Walk and Young, Incorporation of eukaryotes into human microbiome studies is 2008). There are also other organisms that may be present in a just beginning, and will benefit from broad CI surveys of the healthy microbiome community, but become problematic when eukaryotic communities in healthy and diseased individuals. These their numbers increase, including the flagellate Chilomastix (Bog- surveys should encompass diverse populations of healthy and dis- itsh et al., 2005) and the fungus Candida albicans (Schulze and eased individuals, and should exploit within-subject time-series Sonnenborn, 2009). In these cases it may be very important to designs (to control for inter-individual diversity of the micro- look simultaneously at the bacterial and eukaryotic communities biome), and use identical twins (to control for genetic variability). to determine whether a shift in the normal (bacteria dominated) Analyzing these data with emerging computational tools, such as microbiota is correlated with a rise in opportunistic microbial co-occurrence networks, and pipelines for comparing large num- eukaryotes. bers of samples (Caporaso et al., 2010) will enable the patterns Eukaryotes have also been implicated as causative agents of in these data to be elucidated. In-depth sequencing of the meta- diseases such as IBS (Blastocystis), IBD (fungi), and “leaky gut” transcriptome of eukaryotes in the gut is another technique that (Candida; Boorom et al., 2008; Ott et al., 2008; Schulze and Son- will offer a window into the functioning of eukaryotes in the gut nenborn, 2009). These diseases, which have become prevalent in microbiome (Gianoulis et al., 2009). This could be targeted toward western populations, are due to complex changes in the microbial eukaryotes by selecting Poly(A) + RNAs for sequencing, although community. Elucidating community-wide changes, rather than depletion of host transcripts in an unbiased way will be a major presence or absence of specific taxa, will be crucial to under- issue. Genomic surveys of uncultured host-associated eukaryotes standing the cause and potential treatment of in these complex will soon be possible using techniques such as single-cell genomics polymicrobial diseases (Swidsinski et al., 2009). Samples taken and transcriptomics (Heywood et al., 2011; Yoon et al., 2011). from along the gastrointestinal tract may also be important to By connecting single-cell techniques, improved phylogenetic tree determine whether community changes associated with IBS or construction, broad surveys, and the parameters of host pheno- IBD are restricted to the site of disease or are systemic changes in types, these studies have great potential to improve our under- the overall gut community, as has been the observed in bacterial standing of the links between microbial eukaryotes and human communities (Peterson et al., 2008). health, and our understanding of the eukaryotic component of Several eukaryotic parasites are thought to interact with malnu- the tree of life. trition and increase morbidity in parts of the developing world. For example, children infected with Giardia may experience reduced ACKNOWLEDGMENTS growth even when the infection is asymptomatic (Prado et al., Many thanks to Valerie McKenzie and members of the Knight lab 2005). Other eukaryotes that inhabit the gut are only patho- for comments on the manuscript, especially Catherine Lozupone, genic when their host is malnourished or otherwise stressed, as Jesse Zaneveld, Justin Kuczynski, and Daniel McDonald. This in the ciliate Balantidium (Schuster and Ramirez-Avila, 2008). manuscript was improved following comments from two review- These eukaryotes may interact with the bacterial community to ers. Support from the Bill and Melinda Gates Foundation, the impact nutrient cycling and absorption in the gut. Given the patchy Crohn’s and Colitis Foundation of America, the National insti- distribution of these parasitic taxa in human populations it tutes of Health, and the Howard Hughes Medical Institute is should be possible to assess whether the presence of these acknowledged.

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