Transcriptome analyses reveal protein and domain families that delineate stage-related development in the economically important parasitic nematodes, Ostertagia ostertagi and Cooperia oncophora

Studies on the free-living nematode Caenorhabditis elegans have provided a wealth of information on metazoan biology and development. However, being a member of the Nematoda has periodically engendered erroneous assumptions that C. elegans is a measurable representative of other nematodes within this phylum. More recent studies on the genomes and transcriptomes of other nematodes have demonstrated the extensive diversity within this group and the need to look more closely at individual genera to begin addressing questions related to nematode parasitism and host-parasite relationships.

Cooperia oncophora and Ostertagia ostertagi are two parasitic nematodes of the order Strongylida that belong to the same phylogenetic clade as C. elegans[1]. Both species are parasites of bovids in more temperate regions of the world. The diseases caused by these nematodes are among the most costly to the cattle industry where hundreds of millions of dollars are lost each year in lower productivity and higher management expenses. Treatment of cattle infected with these strongylid nematodes commonly involves anthelmintic drugs; however, similar to what has been observed in many microorganisms, drug resistance has become a significant problem within this group of parasites [2]. In spite of their economic impact, a dearth of information is available on their molecular biology.

Parasites of the genera Cooperia and Ostertagia as well as other Strongylida exhibit similar life cycles that begin with fertilized eggs being passed in the host feces. Like C. elegans, the first three larval stages (L1, L2, and L3) are considered free-living because they are environmentally-exposed but with no host dependency. The infective L3 has a protective sheath (L3sh) that allows for movement on pasture while protecting the parasite from ecological pressures. Upon ingestion, however, the nematodes become host-dependent i.e. parasitic; the L3 exsheath (L3ex), develop to the fourth larval stage (L4) and continue development to adults in the abomasum (Ostertagia) or the intestines (Cooperia). Despite their biological similarities, infection by O. ostertagi does not confer strong immunity against reinfection except in cattle which have been infected for extended periods of time [3]. Cattle which have been infected by C. oncophora, on the other hand, attain resistant to reinfection more readily [4]. Furthermore, even though cattle are often found simultaneously-infected with both species, anthelmintic resistance has only been documented in Cooperia spp.

Deciphering the underlying biological differences between these two similar organisms may open the path for more holistic hypotheses on host-parasite relationships, host immunity, and the development of drug resistance. Detailed and comparative explorations of their transcriptomes and genomes would not only provide insights into fundamental biological processes, but underpin the discovery of new treatments and control methods that may be broadly applicable to other less similar nematodes. Although limited transcriptomic information is available for two developmental stages of O. ostertagi, [5] this falls woefully short of representing the entire life cycle and providing insights into what differentiates the free-living and parasitic stages. Currently, no transcriptomic data are publicly available for C. oncophora. Analysis of transcriptome data and their comparison with genomic data is well known to provide useful information about an organism [5–7]. This approach has led to studies on identifying new drug targets (e.g. [8–10]), understanding nematode biology [11], and detecting parasite protein-specific indels and evaluating their importance in parasitism and evolution [12], to name a few.

The present study has generated extensive, stage-related information on the transcriptomes of C. oncophora and O. ostertagi. The comprehensive comparative transcriptomic analysis of stages representing the entire life cycles of these animals established gene expression patterns which characterize and delineate among each of the stages investigated. In addition, transcripts which are unique to free-living or parasitic stages have also been discovered. The resources and results in this study provided molecular insights that improve our understanding of parasite biology and development, and identified differential transcripts among stages/sexes. In broader terms, these analyses will be extremely useful for annotating their upcoming genomes [13] and could enable the development of new methods to control infections by these parasites.

The gastrointestinal parasites studied here exhibit numerous biological similarities. They begin their lives as eggs that are passed in the feces from the host. They remain as free-living organisms up to and including the L3sh at which time they are ingested by the host, ex-sheath and then continue their development as parasitic organisms within the host. Examination of transcripts in both species revealed that 68.8% in C. oncophora and 73.0% in O. ostertagi have sequence homologues in the other species examined in this study (Figure 1) and that ~60% of strongylid genes have homologs in C. elegans[7, 18]. While we have identified few peptides (0.2% and 0.3%, respectively) that share homology only to non-Strongylida species (Additional file 1), mainly Ascaris. suum and Brugia malayi, these are likely homologous peptides not yet identified in other Stongylida species because of the incomplete nature of their genome sequences. Our study showed similar results in that BLAST searches identified homologous sequences in 55.1% of C. oncophora and 57.9% of O. ostertagi polypeptides when compared with free-living nematodes. The slightly higher percentages observed in this study can be attributed to the better coverage of the Cooperia and Ostertagia transcriptomes using pyrosequencing relative to the coverage obtained from conventional EST libraries in previous investigations (e.g. [5]). Because of differences in the environments and living requirements between the free-living and parasitic stages, it is expected that some pathways and enzymes will be unique to these two phases of development and coincide with the requirements and challenges imposed by the different environments. Comparisons of domains and pathways present in the free-living stages to those in the parasitic stages revealed many of these differences.

Given the similarities between C. oncophora and O. ostertagi, it was not unexpected that there would be significant overlap in the domains found in up-regulated peptides in the various stages. For example, among the 20 most abundant domains in all stages, ten were identical in both organisms. The domains that were prevalent in the free-living vs. parasitic stages may provide clues to the lifestyles and environments in which these organisms live. In the free-living stages, domains previously implicated in growth and development tended to dominate. In C. oncophora three different chromo domains (IPR000953, IPR016197, and IPR023780) and the MADF domain (IPR006578) were enriched (Figure 4, Additional file 17). Chromo domains are often found in association with heterochromatin protein 1 (HP1) which functions in germline and vulval development in C. elegans[19]. The MADF domain is a transcription factor in Drosophila that activates genes necessary for development [20]. Chromo domains and MADF domains were found in proteins that predominate in the egg as would be expected (Additional file 18). Interestingly, the chromo domain (IPR000953) and MADF domain (IPR006578) were also found elevated in adult O. ostertagi. Two domains identified as basic leucine zippers [21] (IPR004827 and IPR011700) were up-regulated in the free-living stages of O. ostertagi.

As the organisms transition to L1, the domain prevalence shifts as well. In C. oncophora, the most prevalent domain was EF-hand-like domain (IPR011992). This domain tends to be found in calcium binding proteins [22]. In contrast, the most prevalent domain in O. ostertagi was globin (IPR012292). Globin and saposin domains were prevalent in the L2 of both species. Both of these domains were found in secreted peptides of both species. Saposin domains are expressed in all stages of Ancylostoma caninum[23]. While they were not found in enriched peptides in every stage of C. oncophora or O. ostertagi, these domain containing peptides were expressed in all stages.

During the L3sh, the worms both protect themselves from environmental stress as well as prepare for uptake by and development within the host. Among the most prevalent domains in the L3sh were protease inhibitor I8 (IPR002919) and late embryogenesis abundant (LEA) protein (IPR004238) in C. oncophora and O. ostertagi, respectively. Among the multitude of roles played by protease inhibitors, it has been suggested that they are also involved in protecting invading organisms from host molecules, in particular, those derived from the gastrointestinal tract, such as pepsin. In this way gastrointestinal nematodes can safely navigate and survive within the host digestive tract [6]. Late embryogenesis abundant proteins have been shown to play a role in protection from the environment. In Aphelenchus avenae (plant pathogenic roundworm), LEA proteins help protect other proteins from aggregating during times of low water and possibly play a role in preventing desiccation [24].

During the parasitic stages beginning with the L3ex, it is expected that transcriptional profiles will shift towards host interaction while maintaining those profiles associated with worm development. Zinc finger domains (IPR007087 and IPR015880) which are important in cell differentiation and development [25] were indeed among the most prevalent domains in the L3ex of C. oncophora and in O. ostertagi adults possibly resulting from additional rapid growth as the worms emerge from the gastric glands. In O. ostertagi L3ex, the most prevalent domains found in the greatest number of peptides, were DUF148 and metridin-like ShK toxin. The metridin-like ShK toxin domain (IPR003582) was up-regulated in O. ostertagi parasitic stages and was the most prevalent domain in the L4 stage. Noteworthy is that the metridin-like ShK toxin domain is often found near the C-terminus of C. elegans metallopeptidases. It is suggested that these domains are important in parasitic interactions [26]. CAP domains (IPR014044) were also among the most prevalent domains in C. oncophora L4 and O. ostertagi adults (Figure 4 and Additional files 17 and 18); however, among putatively-secreted peptides, CAP domains were observed in C. oncophora L3sh, L4, and adults, and in O. ostertagi L4. In mammalian species, proteins harboring CAP domains are divided into nine subfamilies which encompass cysteine-rich secretory proteins (CRISPs). Similar CRISP domains (PF00188) were up-regulated in Ostertagia (Additional file 10) and have recently been identified in the Lethenteron japonicum (parasitic lamprey) [27] which secretes a CRISP containing protein from its buccal glands once it has attached to the host. It is believed that this CRISP protein enhances vasodilation and feeding [28]. It should be noted that the concept of “secretory proteins” is defined as a cellular event and not necessarily a function related to parasites secretions. As such, there need not be a direct relationship between CRISP proteins and “extraorganismal” functionality i.e., parasite secretory products. Case in point, in mammals, CRISP proteins are well known to be associated with cell signaling, reproduction, fertilization and the maturation of spermatozoa. As such, it may not be coincidental that in parasites, an abundance of CRISP proteins is associated with the later larval and adult stages of worm development. CRISP domains have been found associated with proteins with immunomodulatory activity [29] and have been studied in few parasitic nematode species including the hookworm A. caninum[30] and the murine strongylid nematode, Heligmosomoides polygyrus[31].

It is well known that proteins such as chymotrypsin, trypsin, and peptidase [32] are involved in the breakdown of proteins into constituent parts. Chymotrypsin (IPR001254) domains were up-regulated in the parasitic stages of C. oncophora and found only in the parasitic stages of O. ostertagi (Figure 4 and Additional file 17 and 18); trypsin-like domains (IPR009003) were up-regulated in C. oncophora, and; peptidase S1/S6 (IPR001254) was one of the most prevalent domains in female C. oncophora (Figure 4 and Additional file 17). Given their abundance in the later stages of development (e.g. L4 and adult), it is possible that proteins associated with these domains collectively play a role in the feeding process [24]. This is supported in part by the observation that these domains are present in nine secreted peptides in C. oncophora and 75 in O. ostertagi. It is possible that a subset of these is not only secreted from the cell but also from the parasite. Given that the adult diets of these parasites vary based upon either abomasal (Ostertagia) or intestinal (Cooperia) contents, these secreted proteases may also participate either in countering the host immune responses (L4) by hydrolyzing antibodies, or in establishment in the host (L3 ex) particularly as it relates to Ostertagia and its need to enter the gastric glands and keep inflammation at bay.

The three C-type lectin domains (IPR001304, IPR016186, and IPR016187) were the most prevalent domains in male C. oncophora (Additional file 17) and were up-regulated as well in O. ostertagi (Additional file 18). As expected, all three of these domains are found in putatively secreted peptides in both species predominantly because evolutionarily, the superfamily of proteins containing C-type lectin domains is comprised of extracellular metazoan proteins with diverse functions. In general, these domains are involved in calcium dependent carbohydrate binding. However, it should also be noted that not all proteins containing C-type lectin domains can actually bind carbohydrates or even Ca²⁺. Indeed, most of the proteins containing this domain and referred to as C-type lectins are not lectins. Nonetheless, those with functionality have been implicated in innate immune responses in invertebrates, and have been linked to proteins involved at the host-parasite interface which may assist in evading the host immune response [33]. As such, differences in the levels of these domains between C. oncophora and O. ostertagi may in part be associated with the observed variation in host immunity as well as distinction in the predilection sites of the respective L4s and adult worms. A closer investigation of sequence similarity to C-type lectins from free-living and parasitic nematodes and an analysis of the locus to which these proteins are eventually translocated might shed light on physiological functionalities as they relate either to sustaining life within the organism or controlling the host-pathogen interface. Some nematode C-type lectins have been linked to the parasite surface i.e., the epicuticle. Among other things, the nematode cuticle is comprised of collagen proteins and these proteins exhibit stage specific expression [16, 34].

Examination of KEGG categories demonstrated significant associations between life cycle stages and peptides involved in energy metabolism in O. ostertagi where 24 peptides were found in the free-living stages and only four in the parasitic stages (Figure 7). Further analysis of these 24 polypeptides provided clues about environmental adaptation. From the egg stage through L2, the worms are present in the fecal pat. Upon developing to L3sh they become more motile and migrate from the pat to better position themselves for ingestion by the host. Of the 24 peptides involved in energy metabolism in the free-living stages of development, 17 are associated with methane metabolism. As the free-living stages of both species are found in the fecal pat and the fecal pat is a methane rich environment, this is not surprising. Only one of the 24 peptides is up-regulated in the L3sh and classified as an enzyme involved in oxidative phosphorylation rather than methane metabolism. It is possible that this becomes more functional as the worm distances itself from the fecal pat and readies itself for ingestion by the host. It is also interesting to speculate that environmental queues i.e. host GI tract, may down-regulate transcriptional activity of the proteins involved in methane metabolism and in turn induce exsheathment and worm development.

In C. oncophora, the KEGG category “metabolism of cofactors and vitamins” was significantly more abundant in the parasitic stages than in the free-living stages (Figure 7). The specific enzymes involved are associated with pantothenate and CoA biosynthesis [35], and thiamine metabolism. All three peptides were up-regulated only in adult females. Inasmuch as these enzymes were not observed in abundance in fecal eggs, their functions are likely related specifically to females or to egg development in utero.

While many of the transcripts were stage specific, others were expressed in all stages. These constitutively-expressed transcripts are likely involved in core molecular processes used to sustain life, as shown by the domains found within them. This conclusion is also bolstered by the embryonic lethal phenotypes predicted for the majority of the constitutively-expressed transcripts that link to an RNAi phenotype in C. elegans. These transcripts and their encoded proteins should make attractive drug targets provided sufficient variation can be found between parasite and host proteins.

Control of parasitic nematodes is routinely accomplished through anthelminthic drugs. Resistance to these drugs is increasingly becoming a problem especially in livestock hosts. To date, resistance has surfaced to nearly all commercially available drugs [36]. In an effort to better understand this resistance and help combat the higher production costs associated with the lack of efficacy, a detailed study of these parasites at a molecular level was conducted. To this end, we have generated comprehensive data on the transcriptomes of all discernible life cycle stages of these two organisms. The genome sequences for C. oncophora and O. ostertagi have been initiated in an effort to complement and complete this work (http://www.genome.gov/10002154) [13]. The cDNA sequences generated in this study will enable better annotation of these genomes upon completion. In the current study, many differences were revealed between the free-living and parasitic stages of these nematodes when examined at the domain, process and pathway levels. During the free-living stages of development, peptides and pathways involved in growth and development were more prominent. In contrast, peptides, domains and pathways that traditionally function in the degradation of proteins were more prevalent during the parasitic stages. These differences are likely associated with host adaptation and therefore parasitism. Further in-depth examination of the differences in domain prevalence and expression between the free-living and parasitic stages may reveal conservation in genes linked to infection, host recognition, immune response and disease. Equally important is understanding the similarities between evolutionarily related organisms in the hope of detecting biological and molecular threads that link the parasitic stages. In this way, we may better identify targets for the development of new classes of nematocides. Holistic approaches such as this could extend new treatments to human pathogens as well.