Keywords

20.1 Introduction

The term “parasite”, though fairly easy to define in biological terms, presents a major problem for chapters such as this as it covers a wide variety of single- and multi-celled organisms which cover a large phylogenetic range. This is illustrated by Table 20.1, which describes the variety of species that specifically parasitise humans and other mammals. This diversity of phylogeny is matched by an equal diversity in biology and life-cycles, all of which combine to make the task of producing an authoritative chapter on TRP channels across parasitology a major challenge. However, this task can be approached via the realization that many parasites are related to well-studied model organisms, and the dramatic increase in genome sequence information means that it is possible to compare the members of the TRP family encoded by parasites with those of their model cousins and perhaps even deduce what some of the physiological roles of the channels might be. For example, several of the parasites in Table 20.1 are nematodes, and the genome of the model nematode Caenorhabditis elegans was not only the first to be sequenced of any metazoan [1], it has also been superbly annotated.

Table 20.1 Some parasites infecting humans
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There is no denying, though, that there have been hardly any investigations into the functions of TRP channels in parasites. This is a pity, and a lacuna in our knowledge that should be addressed. TRP channels present attractive drug targets [2], especially as many existing anti-parasitic compounds, especially anthelmintics, act on other ion channels [3–6], including ligand-gated ion channels and the SLO-1 potassium channels, and an improved pipeline of new drugs is always needed [7–9]. In addition, the involvement of the TRP channels in so many sensory processes implies that they will have roles in mediating the many host:parasite interactions that are essential for the completion of sometimes very complex life-cycles. Understanding these roles could dramatically improve our understanding of the molecular and cellular mechanisms that underpin the adaptations necessary for successful parasitism to evolve.

This hugely diverse grouping of parasites possesses an equal diverse collection of life-cycles. These life-cycles may be direct, involving only a single host, or indirect, requiring both a definitive host, in which sexual reproduction (if it occurs) takes place, and an intermediate host, which allows for the parasites to be dispersed and in which other developmental stages may take place. As an example, for malaria the human is a definitive host and the mosquito the intermediate host, and there are many different developmental stages within both. Even in those parasites with direct life cycles, many stages of development may take place outside the host and in the environment, for example hookworm eggs are shed into environment, then hatch and undergo several moults before reinfecting their mammalian host. A full description of the events within the various hosts is well beyond the scope of this chapter, as all parasites go through multiple developmental stages and have to find their specific niche within the host body. Many migrate around; the intestinal nematode Ascaris lumbricoides migrates from gut to liver to lungs and back to the gut during the various stages of its development within infected humans.

This requirement for replication within an animal host has severely limited the development of tools to study parasites. Some parasitic protozoans can be cultured in the laboratory and for these advanced molecular genetic tools such as gene knock-outs have been developed [10–13]. However, no such culture methods or tools are readily available for the parasitic helminths, and this has had a severely limiting effect on research on these organisms. For the nematodes and many ectoparasites, good model organisms (C. elegans and Drosophila melanogaster, respectively) are available for comparison and, at the genomic level, these comparisons have been made facile by the amount of sequence information that is now available. A considerable amount of excellent science has been carried out on the TRP channels of these model organisms ( [14–17]), but the biological relevance of these studies to parasitic species remains to be determined. Studies on other nematode gene families show that the composition of these families, and possibly their functions, differ greatly between C. elegans and parasitic species [18, 19] and so simple extrapolation may be misleading.

The biological diversity of parasites demands that this chapter be sub-divided, and we have chosen to do this along the lines of Table 20.1. Separate sections will discuss endoparasites, those organisms that invade their hosts and live inside them (which include the protozoan parasites and the helminthes, or “worms”) and the ectoparasites, organisms that live on the outside of the host, but are dependent on them for food – many are blood feeders.

20.2 Endoparasites

20.2.1 Protozoa

A very diverse group of single-celled organisms has evolved to parasitise humans and other animals. The apicomplexans, which include the Plasmodium species that cause malaria, arguably the most important infectious disease on the planet [20], are an exclusively parasitic grouping that possess a unique organelle, the apicoplast, a non-photosynthetic plastid [21], and an apical complex required for entry into host cells [22]. The kinetoplastids, which cause sleeping sickness and leishmaniasis, are a group of flagellates defined by the presence of a kinetoplast, a granule within the mitochondrion that contains DNA [23]. Both of these groups are normally transmitted via an invertebrate vector, such as a mosquito or biting fly. The metamonads are anaerobic flagellates, most of which are symbiotic with their hosts, but some of which are parasitic. A few species of amoeba are also parasitic.

Two TRPML-like genes, lmmlA (LmjF07.0910) and lmmlB (LmjF26.0990), have been described from Leishmania major, a kinetoplastid [24]. Though lmmlA is constitutively expressed throughout the life-cycle, lmmlB expression was reported to be up-regulated in amastigotes, the form of the parasite found in the vertebrate host [24]. A simple search of TrTrypDB (http://tritrypdb.org/tritrypdb/) revealed that both of these genes are conserved throughout the kinetoplastids, with similar sequences being present in the other Leishmania and Trypanosoma species. There are no published studies on the functions of these channels.

There are no other reports of TRP channels from any of these parasites, so we undertook a simple bioinformatics search of some of the available genome sequences, using the membrane-spanning domains of the yeast trp sequence [25] and of C. elegans CUP-5 (a TRPML [26]) as probes. This search revealed sequences in Trichomonas vaginalis (a metamonad) with low levels of identity (~22%) to both the yeast trp and C. elegans CUP-5, and a similar sequence (24% identity to CUP-5) in Cryptosporidium parva (an apicomplexan). The C. parvum protein (cdg6_1510) possesses membrane-spanning regions and is conserved in C. hominis and C. muris. The T. vaginalis “hits” were less convincing, and probably do not represent true channel proteins. Though none of these proteins, including those from the kinetoplastids, has yet been shown to form a genuine ion channel, these early data do suggest that that TRP-like channels might be present in a wide variety of protozoan parasites. We found no evidence of any sequence similar to other sub-families of TRPs, except for TRPML, in these organisms, nor did we find any evidence of TRP-like channels in Plasmodium falciparum, Toxoplasma gondii, Neospora caninum or Giardia lamblia. This may be because these organisms do not have any such genes, or it might be that they are just too divergent to be easily detected – further work is clearly needed to clarify this. Apicomplexans possess most of the other components for regulation of intracellular [Ca2+] found in other eukaryotes, though P. falciparum and C. parva apparently lack voltage-operated Ca2+ channels or PMCA [27]. Given the role of TRPML channels in intracellular events in yeast [25] and higher eukaryotes [28, 29], we would hypothesise that any such channels in protozoan parasites will be involved in the intracellular trafficking and membrane fusion events that take place in these organisms [30, 31]. One possible example of such an event is the fusion of acidocalcisomes, a dense acidic organelle, to the contractile vacuole of T. cruzi and related organisms that is part of the osmoregulation process [32]. Additionally, entry into and replication within host cells by the kinetoplastids is intimately associated with lysosomal-like organelles and membrane fusion events [33, 34]. These events are regulated by TRPML in uninfected cells [28] and it is possible that the related proteins encoded by these parasites function as part of the process by which they control this compartment in their hosts.

20.2.2 Helminths

20.2.2.1 Nematodes

Nematodes, or roundworms, represent about 80% of all animal species. Many of these species are parasitic, causing chronic infections and debilitating, though rarely fatal, disease. A couple of parasitic genome sequences have been published [35, 36] in addition to that of the “model worm”, C. elegans [1], and a considerable amount of sequence information is available, primarily from the groups at Washington University, St Louis, USA (http://www.nematode.net) [37] and the Sanger Centre, Cambridge, U.K. (http://www.sanger.ac.uk/Projects/Helminths/). Many of the C. elegans TRP genes have been extensively studied and reviewed [17]; some of this information is summarized in Table 20.2. However, phylogenetic analysis suggest that C. elegans may not be typical of all nematodes [38], and previous analyses have revealed big differences between the ion channel genes of C. elegans and some parasitic species [19]. We searched the annotated genes of the parasitic nematode Brugia malayi [35] and the plant parasite Meloidogyne incognita [36] together with the unannotated sequence of Trichinella spiralis (http://genome.wustl.edu/genomes/view/trichinella_spiralis/) for TRP channel sequences; the results are summarized in Table 20.2. Compared to C. elegans, it seems that all three parasite species possess fewer such genes, as observed previously for the ligand-gated ion channels [19]; this may not reflect any physiological simplicity of the parasites, but rather the number of gene duplications that have occurred during C. elegans evolution, though considerably more comparative genomic analysis is required before any firm conclusions can be drawn. We also searched the B. malayi genome (http://blast.jcvi.org/er-blast/index.cgi?project=bma1) for TRPM-, TRPA- and TRPP-related sequences in case these had not been annotated; we found some evidence of TRPM-like sequences but not for TRPA or TRPP. Many of the TRP channels are involved in sensory processes, in nematodes as in other organisms, and the loss of these channels may reflect a reduction in the complexity of sensory inputs encountered by the animal parasites as they inhabit the homeostatically maintained environments of their vertebrate and, in the case of B. malayi, invertebrate hosts. The T. spiralis and M. incognita genomes contain members of most of the TRP channel sub-families, but not TRPA, nor TRPP; M. incognita seems to have more TRPM channels than the animal parasites. The TRPP channels of C. elegans are required in males for successful mating and responses to hermaphrodite pheromones [39, 40]; their absence from the parasitic species may reflect differences in mating behavior between the species. This hypothesis may be supported by the absence of an obvious spe-41 from B. malayi and the divergent form of this gene seen in T. spiralis since this gene is also involved in mating [41]. The TRPV channels of C. elegans function as heteromers [42], and the reduction in TRPV gene number in the parasites may reflect an expression of homomeric channels in these species.

Table 20.2 TRP channels of nematode parasites compared to those in C. elegans. C. elegans TRP channels from [15]. The Brugia malayi and Meloidogyne incognita databases were searched for predicted proteins annotated as TRP-like, and then the B. malayi, M. incognita and Trichinella spiralis nucleotide sequence databases searched with each of the C. elegans protein sequences, using tblastn. The Accession Numbers are given for specific gene “hits”; where this is not possible (for example where matching sequences were found in genomic sequence that was not part of an annotated predicted gene) this is indicated by the word “present”. Putative B. malayi and M. incognita TRP sequences were reciprocally blasted back against the C. elegans database
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20.2.2.2 Trematodes and Cestodes

The most important trematode, or flatworm, parasites of humans are the schistosomes, or blood flukes [20]. There are three major species of these, S. mansoni, S. japomonicum and S. haematobium: we searched the completed and annotated genome of S. mansoni [43] for TRP channel genes (Table 20.3). We also searched the incomplete and unannotated genome of the tapeworm (cestode) Echinococcus multilocularis (http://www.sanger.ac.uk/Projects/Echinococcus/) for TRP channel sequences. These searches produced similar and quite remarkable results; both organisms possess multiple TRPC and TRPM genes (at least nine in S. mansoni), single TRPA, TRPP and TRPML genes, and apparently no TRPV or TRPN genes. To date, there is no information of the function of any of these channels; there are ESTs for several of the S. mansoni TRP genes, but these seem to be expressed at low levels; Sm_169150 (a TRPC) and Sm_147140 (a TRPM) are the most highly represented, with Sm_147140 having multiple ESTs from adult libraries. In mammals and nematodes, TRPM6 and TRPM7 channels are required for ion homeostasis in the gut [44–46]. Since trematodes and cestodes absorb most of their nutrients, including ions, from the blood or gut contents of the host organisms, it is tempting to speculate that one role of the parasite TRPMs might be in the uptake or release of essential ions. The apparent absence of TRPV channels from these organisms might, as with the parasitic nematodes, reflect the simpler sensory requirements of a parasitic life-style.

Table 20.3 The predicted TRP channel genes of Schistosoma mansoni and Echinicoccus multilocularis. The S. mansoni database was searched for predicted genes annotated to encode a TRP channel (indicated as Smp_XXXXXX), then the S. mansoni and E. multilocularis nucleotide sequences searched using single members of each gene family from C. elegans and D. melanogaster and tblastn
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20.3 Ectoparasites

“Ectoparasites” is a term that makes biological but very little phylogenetic sense. For the purposes of this chapter, we have chosen to focus on a few examples of insect and arachnid parasites, the bugs, lice and ticks. Bugs, such as bedbugs, are closely related to aphids, which could be considered to be ectoparasites of plants; the genome sequence of the pea aphid, Acyrthosiphon pisum, has just been published [47], resulting in an analysis of the ion channel genes, including the TRP channels, present in this organism [48]. We compared the predicted TRP channel genes in A. pisum and D. melanogaster to those annotated from the human head louse, Pediculus humanus, and the tick, Ixodes scapularis (Table 20.4). We also carried out some Blast searches using the Drosophila peptide sequences as probes. The results, which may be incomplete, show a remarkable conservation in gene number between the various insects and ticks, with members of all seven sub-families of TRP channel present except for TRPV in the tick, and TRPP and TRPML in the aphid. This greater conservation of gene number between parasitic and non-parasitic insects, as compared to nematodes, may reflect the more limited adaptations required for ectoparasitism as opposed to endoparasitism. Ticks, which are not insects but arachnids, find their hosts by detecting their temperature and carbon dioxide “footprints” and so might be expected to conserve some of the temperature detecting channels of other arthropods. Insects detect heat via several TRP channels, including the TRPV channels [49], trpA1 [50] and pyrexia [51]; of these we found a clear homologue for only trpA1 (ISCW011428) in I. scapularis. TrpA1 has a role in larval thermotaxis in Drosophila [50] and so might make a good candidate for mediating the same phenomenon in ticks. Head lice move between hosts as adults and seem to have retained a larger number of temperature-sensitive TRP channels to allow them to find a new host at this stage of the life-cycle. The larvae, or nymphs, are not considered to be as infectious, which might explain the apparent loss of the larval thermotaxis gene, trpA1, in the louse.

Table 20.4 The TRP channel genes of ectoparasites compared to those of Drosophila melanogaster. The D. melanogaster gene information is taken from flybase.org. The louse and tick genes were identified by a simple text search of Genbank and by tblastn searches using the Drosophila protein sequences. The aphid genes are taken from [48]
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20.4 Conclusions

An immediate conclusion from this brief survey is that, for invertebrates, far more is known about the TRP channels of model than target organisms. Even from the simple bioinformatics searches that we have carried out, it is clear that the TRP channel genes can vary, especially in the protozoa and helminths, and that these differences between species do warrant further investigation. It may be premature to consider TRPs to be viable drug targets, but they are clearly worth considering and exploring further. We hope to read about such studies in the years to come.