The first Parasite Microbiome Project (PMP) Workshop (January 9–14, 2019, Clearwater,
Florida, United States) hosted researchers from across continents and disciplines
to lay the foundation of the PMP consortium. The PMP vision is to catalyze scientific
discourse and explorations through a systems approach, toward an integrated understanding
of the microbiota of parasites and their impact on health and disease. The participants
identified knowledge gaps and grand challenges in the field of host–parasite–microbe
interactions summarized here. The PMP will provide an interactive centralized platform
and resource for transdisciplinary collaboration to propel the field of parasitology
forward by disentangling complex interactions between parasites and hosts, their respective
microbiota, and microbial communities in the parasite’s direct environment (Fig 1).
10.1371/journal.ppat.1008028.g001
Fig 1
The complex nature and interrelations of host–parasite–microbe interactions are illustrated
using a Matryoshka (Russian nesting) doll metaphor.
The PMP aims to elucidate nested interactions between a given host and parasites (e.g.,
helminths and protists) that are themselves hosts to their own symbionts and parasites
(e.g., viruses and bacteria). Artwork by Meredith Brindley (http://meredithbrindley.com/).
PMP, Parasite Microbiome Project.
Parasitism is a successful lifestyle that has evolved in virtually every clade of
multicellular organisms [1–3] and protists [4, 5]. Parasitology seeks to develop the
means to prevent, limit, or cure infections by parasites for the benefit of humans,
agriculture, and wildlife and to understand how parasitism and parasitic disease impact
not only the host but also host communities and ecosystem health. This is a challenging
task, considering the diversity and complex nature of host–parasite interactions.
Parasitic organisms harbor a rich tapestry of traits associated with survival and
must navigate the host immune response to reproduce and be effectively transmitted
to the next host.
An improved understanding of underlying molecular mechanisms and evolutionary patterns
that explain interindividual, temporal, and geographic variation in the outcomes of
parasitic infections is much needed [6–9]. There is an increasing recognition of the
potential for host- and parasite-associated microbiota―endo- and/or ectosymbiotic
archaea, bacteria, viruses, and micro-eukaryotes―to influence and shape host–parasite
interactions [10]. In the past few years, the concept of individuality has given way
to that of “holobiont” with the recognition that each organism is a composite of organisms
[11–14] (Fig 1, Box 1). Yet we have limited insight into the nature and importance
of these interactions for parasite ecology and evolution [15, 16], and not a single
parasite species has its entire microbiome fully characterized.
Box 1. Key microbiome and holobiont concepts applied to parasitology
Direct environment: environment of the parasite at the time of sampling (host-associated
and free-living stages).
Parasite-associated microbiome: collection of the genomes of the microbiota (viruses,
bacteria, archaea, and micro-eukaryotes) that are either chromosomally integrated
or episomal, intracellular, or attached to the surface of the parasite.
Host-associated microbiome: collection of the genomes of the microbiota that are associated
with the host, either in the direct environment of the parasite, or in a distant tissue
or anatomic compartment of the host.
Environmental microbiome: collection of the genomes of the microbiota that are present
in the direct environment of free-living (encysted or mobile) life stages of parasites.
Holobiont: a unit of biological organization composed of a host and its microbiota,
inclusive of transient and persistent microbes.
Hologenome: the complete genetic content of an organism’s genome, including nuclear
and organellar genomes, and its microbiome.
The PMP
The PMP envisions a holistic understanding of host–parasite–microbe interactions by
fostering global transdisciplinary explorations of the microbiomes of parasites and
their direct environment (Fig 2; Box 1) [17]. The PMP will be enabled by new and existing
tools, technologies, and standards developed for microbiomes and tailored for analyses
of host–parasite–microbe interactions. Areas of focus will include (1) development
of relevant standards for metadata collection and curation, (2) methods development
for processing of parasite-associated microbes, (3) multi-omics technologies, and
(4) tailoring of analytical tools for parasite-associated microbiome research. Methods
and data will be shared freely with the scientific community and public using open
data standards. Establishing and optimizing these methods will initially require a
“test” collection of well-characterized parasite isolates/models and a comprehensive
identification of parasite-associated microbes. The PMP will establish a workflow
and centralized platform to maximize parasite sampling efforts and facilitate parasite
microbiome research for the community at large (Fig 3; Table 1).
10.1371/journal.ppat.1008028.g002
Fig 2
Examples conveying context-dependent usage of the microbiome and holobiont concepts
in parasitology.
(A) Procercoid stage of the cestode Schistocephalus solidus in the body cavity of
a cyclopoid copepod. The Pam may be collected from procercoids. The De is the body
cavity of the copepod. The Ham may be collected from the gut or other host tissues
whereas the Em may be collected from the water. (B) Oocyst of Toxoplasma gondii that
sporulated upon excretion with cat feces. The Pam may be collected from purified oocysts.
Distinction between the Ham, De, and Em is difficult. (C) Trypanosoma sp. among red
blood cells. The Em is not represented. The intracellular microbes potentially present
within red blood cells may be considered Ham whereas microbes within the plasma can
be considered in the De of the parasite. Image credits: M. Hahn; L. Knoll and J. P.
Dubey; J. Lukeš. De, direct environment; Em, environmental microbiome; Ham, host-associated
microbiome; Pam, parasite-associated microbiome.
10.1371/journal.ppat.1008028.g003
Fig 3
Proposed workflow for processing PMP samples from parasites and host tissues.
PMP, Parasite Microbiome Project.
10.1371/journal.ppat.1008028.t001
Table 1
Methods to tackle the grand challenges of parasite microbiome research.
Method
Challenge and/or proposed approach
Reference
Sample collection
• Metadata collection
Must be complete and standardized; collect adhering to MIxS environmental package
for parasite-associated samples
[18–20]
• Environmental parasite microbiota
Need to fractionate samples to distinguish parasite-associated microbiome from direct
environment microbiome; freezing and/or preservation in ethanol or RNAlater depending
on downstream processing
• Laboratory parasite microbiota
Need growth conditions, in vitro animal model systems, e.g., tissue, organoids, cell
lines
[21, 22]
Molecular characterization
• Metagenomic DNA sequencing
Capture whole community including prokaryotes, micro-eukaryotes, and abundant or actively
replicating viruses
• Amplicon DNA sequencing
Group-specific taxonomic profiling of key groups
• Viral community sequencing
Viral purification (viral metagenomes) or sequencing of vSAGs
[20, 23–25]
• Parasite genome sequencing
Need to supplement reference genomic databases and identify role of host genotype
in shaping the interactions of resident microbes
[26, 27]
• Transcriptomics, cDNA metagenomics
Detection of RNA viruses
[28]
• Metabolomics
Mass spectrometry (LC-MS/MS, GC-MS)
• Microscopy for spatial organization
FISH and microscopy to identify localization of microbes on/inside parasite and in
relation to each other; microscopy of living parasites to reveal temporal patterns
[29, 30]
Data analysis
• Data mining
Search existing sequence archives and parasite sequencing projects for parasite microbiomes
• Reference databases
Build upon existing databases (e.g., EuPathDB)
https://eupathdb.org
• Genome assembly
Need to assemble microbial genomes from metagenomes in context of host and parasite
genomic DNA; also assemble parasite genomes
• Metagenomic taxonomic and functional analysis
Taxonomic composition using nucleotide composition (e.g., Kraken, Nonpareil) and marker
genes (e.g., MetaPhlAn) and species-specific fuctional composition using nucleotide
and protein databases (e.g., HUMAnN2)
[31–34]
• Amplicon analysis
Database curation and exact-sequence methods
[35–39]
• Multi-omics analysis
Compare profiles of taxa, genes, metabolites across multi-omics methods
Data sharing
• Protocols
Protocols for sample collection, processing, and analysis; share on protocol-sharing
service (e.g., Protocols.io)
https://protocols.io
• Code
Processing and analysis code; share on GitHub repository and permanent archive (e.g.,
Zenodo)
https://github.com, https://zenodo.org, http://gensc.org
• Study metadata
Study title, description, design, points of contact, and publication DOI; share on
GitHub repository and permanent archive (e.g., Zenodo)
• Sample metadata
MIxS-compliant metadata (see above); share on GitHub repository and permanent archive
(e.g., Zenodo)
• Raw data
All raw data after collection; deposit in EBI, GenBank, and other data archives
https://www.ebi.ac.uk
Abbreviations: DOI, digital object identifier; EBI, European bioinformatics institute;
FISH, fluorescence in situ hybridization; GC-MS, gas chromatography-mass spectrometry;
LC-MS/MS, liquid chromatography-tandem mass spectrometry; MIxS, Minimal Information
about any Sequence; vSAG, viral single amplified genome
A primary advantage of a centralized platform like the PMP is the collation of large
aggregates of associated metadata that can be harnessed to uncover, and eventually
understand, patterns of microbial diversity and ecology [40, 35]. Therefore, detailed
metadata associated with each study and sample are absolutely critical to maximize
the utility of each. To facilitate future research opportunities, the PMP will encourage
tracking of metadata connected to both the sample and its processing and the deposition
of host and parasite vouchers into museum collections to allow future analysis opportunities
when new techniques and hypotheses arise [41, 42]. Any additional tissues and extracted
biomolecules should also be maintained in dedicated (cryo-) collections. We will adapt
practices and lessons learned by the Earth Microbiome Project (EMP) [35, 43], e.g.,
preparation of multiple (homogenous) aliquots of the samples to be studied [44], in
a manner that is best suited for the PMP. By providing tested methods and developing
standards for parasite microbiome research, the PMP foresees the following:
Elevating the integral role of the microbiome in host–parasite ecology and evolution
(in a dynamic environment) to promote solution-oriented research in parasitic disease
management.
Steering the larger microbiome community towards analysis of the micro-eukaryotic
component of the microbiome.
Building an inclusive and transdisciplinary PMP community that catalyzes analysis
of natural and model parasite systems.
Becoming a community hub that coordinates discussions fostering collaborative research
to address current and future grand challenges.
Grand challenges
We encourage the scientific community to join the PMP in addressing grand challenges
in the field of host–parasite–microbe interactions and designing creative experiments
in a diversity of systems to explore the areas outlined next.
Identifying core and transient parasite-associated microbes
Parasite-associated microbiomes remain largely unknown, in part due to the inherent
difficulties of studying the parasites themselves, e.g., challenging or nonexistent
in vitro cultivation systems, complex life cycles, ethical considerations, obligate
host environments that are difficult to simulate in experimental models, and national
borders. Another specific challenge for investigating microbial communities associated
with parasites is the necessity to isolate the parasite from the background sampled
material and from host-associated microbes. The microbiota within a parasite can be
divided into core microbes (intrinsic to the parasite or at least to a specific parasitic
life stage) and transient microbes (temporarily acquired by the parasite from its
direct environment). Comparative analyses between the parasite microbiome, the microbiome
of the corresponding infected host, and a control noninfected host from the same environment
will be needed to rule out potential microbial contaminants from the direct environment.
Parasite holobiont research will need to ascertain whether microbes are vertically
transmitted from parasitic parent to offspring, horizontally transmitted between parasites
coinfecting the same host, or transmitted between the parasite and its direct environment
(the host or the external environment of the free-living stages). The objective will
be to discern to what extent the parasite-associated microbiota is determined by the
parasite (maintained across developmental growth, reproduction, and dispersal), by
the composition of the microbiota in its direct environment, or by abiotic factors.
This objective could be examined, for example, by comparing the microbiomes of parasites
(1) infecting multiple host species (for generalist parasites or parasites with complex
life cycles), (2) at different life stages, (3) isolated from spatially and temporally
separated populations or populations with different diets, or (4) coinfecting the
same host.
Understanding the roles of parasites in microbe evolution and host–microbe interactions
Parasite prevalence in a population, route(s) of parasite transmission, and interdependence
between the microbe and its parasitic host will drive the evolution of the microbe’s
modes of transmission. These modes are of particular interest because they will drive
microbial virulence both for the parasite and its host [45]. Parasites may influence
the composition of the microbiota of their hosts by diverse means. For example, parasites
may (1) be vectors or reservoirs of microbes; (2) exert pressure on the host during
infection, leading to the evolution of defensive microbes; (3) compete with host microbiota
for nutrients or provide metabolic and genetic reservoirs to support the growth and
survival of other host microbial species; (4) modify the host environment, e.g., pH,
to the benefit of other microbes; and/or (5) induce an immune response by the host
that, in turn, impacts the host’s microbiome.
The extent to which the host microbiome is determined by its parasites can be investigated
by comparing the microbiome of individuals infected by different parasitic species
and/or strains [46]. When treatments are available, they can be used to determine
whether the host microbiome returns to its original state after removal of the parasite.
In addition, characterizing the underlying mechanisms will be necessary to determine
whether the parasite directly or indirectly interacts with the host microbiome and
whether this is beneficial to the parasite or a side effect of the infection. Furthermore,
by serving as vectors or reservoirs of microbes, parasites could alter the evolution
of microbes by providing opportunities for host switching or novel microbe–microbe
interactions that may lead to genetic exchanges. In order to gain an evolutionary
perspective on host–parasite–microbe interactions, evolutionary studies encompassing
microbes across host and parasite species are necessary to identify patterns of cospeciation
and speciation following host shifts.
Understanding the functional role of microbes in parasite fitness and host diseases
Parasites and associated microbes can be viewed as a community of organisms that experience
different selection pressures, despite the high potential for interdependence. Microbes
can be either beneficial (mutualistic) or antagonistic (parasitic or with fitness
conflicts) to the parasite. The nature of the interaction would lead to radically
different effects of the microbes on the evolution of the holobiont and the host–parasite
interaction. Similarly, microbes associated with the host may be beneficial for the
parasite, as a result of selection for cooperation, or they may be detrimental due
to the competition for nutrients and/or space. The nature of parasite–microbe interactions
may have a critical effect on the parasite’s fitness and host disease. For example,
viral symbionts of parasitic protists can divert host responses toward antiviral immunity,
which is inefficient in clearing the eukaryotic infection and may aid the parasite
survival [21].
Understanding the impact of microbes on the fitness of hosts and parasites is of relevance
to epidemiological studies and is expected to provide new opportunities for therapeutic
interventions. The inherent complexity of the study of host–parasite–microbe interactions
necessitates the application of methods from the field of community genetics, wherein
it is acceptable that the gene that governs a given phenotype resides in the genome
of another species and is dependent on the environment [47]. Here, the environment
of the host and parasite is the microbiome, and its impact on the evolution of the
system can be tested by measuring parameters of the host and parasite fitness in the
presence of different microbes. Alternatively, host–microbe interactions can be tested
by considering the host as the environment.
Identifying patterns and processes of host–parasite–microbe coevolution
Interindividual variations in the outcome of a parasitic infection resulting from
variations in host susceptibility, parasite virulence, and host–parasite compatibility
can be better understood in the context of the geographic mosaic of coevolution [48].
Microbes also show geographic variation, and they can participate in coevolution by
shifting selection pressures away from or towards either the host or the parasite
[49–51]. With appropriate experimental systems, geographic variations affecting the
role of microbes in host–parasite interactions can be assessed by using a complete
cross-experimental design, in which hosts from different localities are infected with
parasites from their corresponding localities in the presence of either microbes isolated
from the same localities or microbes from different test localities. Identification
of temporal variations in selection pressures on microbes involved in host–parasite
interactions would require time-shift experiments, wherein the microbes that have
evolved with the host and parasite are transferred back to an ancestral host and parasite.
Finally, when possible, experimental evolution of parasites and hosts in the presence
or absence of the identified microbes can been used to test the effect of specific
microbes on the evolution of the system and to identify mechanisms involved in parasite–microbe
interaction.
Moving forward
The PMP consortium proposes a two-phase development, analogous to the Human Microbiome
Project (HMP) [52]. Phase one will compile information on previously characterized
parasite-associated microbes and parasite–microbe interactions (already partially
reviewed in [15–16, 53]), mine genomic and transcriptomic databases to detect microbial
sequences, and characterize the complete microbiome of a set of parasites representing
diverse taxa and environments. A main focus during this phase will be on preparing
a website and developing and sharing best practices, methods, and standards for effective
sample management and integration of data. The PMP, in collaboration with the Genomic
Standards Consortium (GSC; gensc.org), has initiated the development of a new parasite-associated
package to be added to the Minimal Information about any Sequence (MIxS) standard
[18]. This package will facilitate the collection, standardization, reporting, and
integrated analyses of metadata to capture the parasite microbiome contextual information
describing the host, environment, sample and sequencing data. We anticipate the MIxS-PMP
to be available by the end of 2019.
The second phase of the project will rely on the development of experimental model
systems that may be employed to prove cause-effect relationships between parasite
virulence, diseases, and microbiome composition, as well as to investigate the underlying
molecular mechanisms and the evolution of host–parasite–microbe interactions. Findings
from initial microbiome characterizations during phase one and previously proposed
experimental model systems [53] will guide the evaluation and selection of systems
most suitable for addressing the scientific grand challenges identified herein.
Given the important role of parasites in ecosystems, human health, and agricultural
management, propelling the field of parasitology in a coordinated way with the PMP
can have an enormous payoff (Table 2). The PMP will necessitate both significant funding
to conduct challenging research as well as engagement from the community to provide
high-quality samples and to share detailed and accurate metadata information. Therefore,
we propose constituting a community of researchers that meet annually for workshops
and symposia. With this opinion article, we invite reader comments to better define
grand challenges and research needs moving forward.
10.1371/journal.ppat.1008028.t002
Table 2
Representative examples of organisms for which uncovering parasite–microbe interactions
is allowing major scientific advances.
It is anticipated that the PMP will advance the field by facilitating similar research
on diverse parasites and uncover patterns of microbial diversity and ecology that
apply across phyla.
Parasite
Microbe(s)
Significance for health, agriculture, and/or the environment
References
Opisthorchis viverrini
Helicobacter pylori and other host gut bacteria
O. viverrini often leads to cholangiocarcinoma. Co-infection with oncogenic bacteria
that are vectored towards the liver by the fluke may contribute to cancer development
[54–56]
Trichomonas vaginalis
TVV 1 through 3
Different clinical isolates of T. vaginalis show variable pathogenicity to the human
host cells dependent on the TVV they carry; TVV released by dying and stressed parasites
can explain why antibiotic therapy fails to prevent the inflammatory sequelae of parasitic
infection
[57]
Trichomonas vaginalis
Host vaginal microbiome
Infection is detrimental to Lactobacillus and favors pathogenic bacteria associated
with bacterial vaginosis
[58]
Leishmania spp.
LRV1
LRV1-infected Leishmania spp. increase severity of human leishmaniasis and lead to
drug treatment failures
[59, 60]
Filarial nematodes
Wolbachia
Antibiotics, such as doxycyline and rifampicin, targeting the Wolbachia endosymbiont
lead to loss of worm viability and fertility in human trials and increase antifilarial
treatment efficacy
[61, 62]
Parasitoid wasps
Polydnaviruses and RNA viruses
Viruses contribute to parasitoid wasps virulence by modulating host immune response,
host behavior, and feeding ability
[63–65]
Ticks
Coxiella-like endosymbiont
Symbiont codiversifies with its parasitic host and provides B vitamins missing from
blood meals, enabling ticks to specialize in hematophagy
[66, 67]
Vibrio shiloi
Symbiotic zoonxanthellae of corals
V. shiloi produces toxins that target symbiotic zooxanthellae of the coral host inhibiting
photosynthesis
[68]
Trichuris spp.
Host gut microbiome
The whipworm ingests bacteria from its direct environment and favors growth of mucolytic
bacteria.Bacterial attachment is required for egg hatching
[69–72]
Digenetic trematodes including species of Nanophyetes, Echinostoma, Fasciola
Neorickettsia species
Endosymbiotic bacteria within cells of the trematode. These symbionts can be transferred
horizontal from the trematode to mammalian host, where they are facultative pathogens
[73, 74]
Pseudocapillaria tormentosa
Zebrafish gut microbiota
Abundance of some bacteria taxa predicts helminth burden and intestinal lesions in
host. Gut microbiome serves as diagnostic for parasite infection.
[75]
Abbreviations: LRV1, Leishmania RNA virus 1; PMP, Parasite Microbiome Project; TVV,
Trichomonas vaginalis virus