Chapter Three - The Biochemistry of Haemonchus contortus and Other Parasitic Nematodes

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Abstract

Different life cycle stages of Haemonchus contortus adapt to different ecosystems. This adaptation is accompanied by alterations in gene transcription and expression associated with the energy, amino acid, nitrogen, lipid and/or nucleic acid metabolism of the respective stages. For example, the aerobic metabolism of larvae depends on an efficient citric acid cycle, whereas the anaerobic metabolism of adults requires glycolysis, resulting in the production of volatile fatty acids, such as acetic acid and propionic acid. There are only few anthelmintics targeting nematode energy metabolism. In addition, H. contortus has reduced pathways for amino acid metabolism, polyamine metabolism and nitrogen excretion pathways. Moreover, nucleic acid metabolism comprising purine and pyrimidine salvage pathways as well as lipid metabolism are reduced. In addition, nematodes possess a particular composition of their cuticle. Energy production of adult worms is mainly linked to egg production and complex regulation of the neuromuscular system in both females and males. In this context, microtubules consisting of α- and β-tubulin heterodimers play a crucial role in the presynaptic vesicle transport. Due to the significant distinction of its quarternary structure in nematodes in comparison to other organisms, β-tubulin was identified as a major target for benzimidazoles used for anthelmintic treatment. Concerning the function of the neuromuscular system, acetylcholine, a ligand of the nicotinic acetylcholine receptor (nAChR), is the major excitatory neurotransmitter in H. contortus. In contrast, glutamate-gated chloride channels, calcium- and voltage-dependent potassium channels as well as γ-aminobutyric acid (GABA)A and its receptors act as inhibitory neurotransmitters and thus opponents to nAChR. For example, the calcium- and voltage-dependent potassium channel SLO-1 is an important target of emodepside, which is involved in the sensitive regulation of activatory and inhibitory receptors of the nervous system. Most of the modern anthelmintics target these different neuromuscular receptors. The mechanisms of resistance to anthelmintics, either specific or non-specific, are associated with changes in the molecular targets of the drugs, changes in metabolism of the drug (inactivation, removal or prevention of its activation) and/or increased efflux systems. The biochemical and molecular analyses of key developmental, metabolic and structural process of H. contortus still require substantial efforts. The nAChR, glutamate-gated chloride channel and calcium- and voltage-dependent potassium channel SLO-1 have long been known as being essential for nematode survival. Therefore, future research should be intensified to fully resolve the three-dimensional structures of these receptors, as has already been started for glutamate-gated chloride channel. With this knowledge, it should be possible to design new anthelmintics, which possess improved binding capacities to corresponding receptors.

Introduction

Haemonchus contortus establishes and lives in different ecosystems. The development, migration and establishment of this parasite are accompanied by adaptions to diverse macro- and micro-environments. These adaptions are characterized by differential gene transcription and expression patterns that have significant influences on the energy, amino acid, nitrogen, lipid and nucleic acid metabolism of the respective stages. As an example, the aerobic metabolism of larvae depends on an efficient tricarboxylic acid (TCA) cycle, whereas the anaerobic metabolism of adults is reliant predominantly on glycolysis (Kapur and Sood, 1987). As a common energy-saving feature for parasites, H. contortus has considerably reduced pathways for amino acid, nucleic acid as well as lipid metabolism, thus being reliant on source materials from their hosts (Köhler, 2006).

The energy acquired via anaerobic carbohydrate degradation in adult worms is mainly used for egg production in female worms and the complex regulation of the neuromuscular system in both females and males (Köhler, 2006). A well-functioning neuromuscular system relies on the controlled and accurate presynaptic release of neurotransmitters via vesicles and their interaction with postsynaptic receptors (Dallière et al., 2015).

Microtubules play an important role in axonal vesicle transport. They consist of α- and β-tubulin dimers. Due to the significant distinction of its quarternary structure in nematodes, in comparison with other organisms, β-tubulin was identified as a major target for anthelmintic therapy (Harder, 2002). In addition, the chemical signal represented by the major excitatory neurotransmitter acetylcholine is translated into an electrical signal via its postsynaptic receptors, the nAChRs. Glutamate-gated chloride channels, calcium- and voltage-dependent potassium channels as well as γ-aminobutyric acid (GABA)A and its receptors act as opponents via their inhibitory effects on the neuromuscular signal transmission (Dallière et al., 2015). In this context, the SLO-1 receptor, a voltage-gated and calcium-dependent potassium channel, a target of emodepside, is involved in the regulation of activatory and inhibitory receptors of the nervous system (Dallière et al., 2015). The composition of this receptor is highly complex and specific to nematodes, such as H. contortus, and its specificity makes it an attractive drug target for a number of anthelmintics.

As there are serious resistance problems in parasitic nematode populations against many modern anthelmintics, it is of great importance to know and understand the biochemical mechanisms of resistance to these anthelmintics to prevent resistance. The resistance mechanisms are multifactorial and relate to a number of alterations, including (1) changes in the molecular target of a drug, resulting in a loss of interaction with the drug target; (2) changes in metabolism that inactivate or remove a drug, or prevent its activation; (3) changes in the distribution of a drug, preventing it from reaching its target or increasing its efflux; (4) amplification of target genes to circumvent drug action; and (5) compensation of the molecular target via an expression of closely related proteins which are not sensitive to the drug. The purpose of the present chapter is to review salient information on the biochemistry of H. contortus and other parasitic nematodes as a foundation for future anthelmintic discovery and drug resistance research.

Section snippets

Ecosystems of Haemonchus contortus Life Cycle Stages

The ecosystems in which H. contortus reside vary and change considerably during the parasite's life cycle. Free-living stages, including eggs, first-stage larvae (L1), second-stage larvae (L2), third-stage larvae (L3) and fourth-stage larvae (L4), are confronted with very different physicochemical, environmental conditions, such as pO2, pCO2, pH, osmotic pressure, redox potential and temperature (Köhler, 2006).

Adult nematodes usually have access to abundant water and food resources in the

Gene Expression in Parasitic Life Cycle Stages

Distinct life cycle stages of H. contortus are adapted to their environments, which is reflected in differences in gene transcription among stages (Laing et al., 2013, Schwarz et al., 2013; see also chapter: Haemonchus contortus: Genome Structure, Organization and Comparative Genomics and Understanding Haemonchus contortus Better Through Genomics by Laing et al., 2016; chapter: Ranscriptomics” by Gasser et al., 2016 – this issue). Therefore, significant variation in transcription patterns for

Energy metabolism in larval nematodes

The transition through the egg, L1, L3 and L4 stages of H. contortus is accompanied by considerable alterations in transcription profiles linked to various enzymes. From L1 to L3, the genes of most enzymes are downregulated, including those involved in carbohydrate, lipid and energy metabolism, but many of them are upregulated in the transition from L3 to L4 (Laing et al., 2013). This finding can be explained by the fact that L3 development is arrested, analogous to the dauer larva of

Amino Acid Metabolism

The transition of L4 to adult male H. contortus is accompanied by an increased amino acid metabolism (Laing et al., 2013). Nematodes, like all other organisms, use amino acids for protein synthesis, as precursors for specific biosynthetic pathways and, also, but in a very limited manner, for the production of ATP. Essential amino acids are absorbed from host diet and/or hydrolysed by proteinases or peptidases before they are further degraded in the intestinal lumen of nematodes (Köhler, 2006).

Nucleic Acid Metabolism

Parasitic nematodes, like other eukaryotic parasites, are characterized by substantial cellular multiplication rates associated with high nucleic acid synthesis. One adult female of H. contortus can produce up to 10,000 eggs per day (Veglia, 1916). In comparison, A. lumbricoides can produce 2 × 105 eggs per day (Wehner and Gehring, 1995a, Wehner and Gehring, 1995b).

Lipid Metabolsim

During the transition from L4 to adult male H. contortus, there is a decreased lipid metabolism coupled to an increase in amino acid metabolism (Laing et al., 2013). In nematode eggs, long-chain fatty acids from triacylglycerols are used for the resynthesis of carbohydrates via a functional glyoxylate cycle (Barett, 1981, Köhler, 2006). The presence of this pathway in the developing eggs of some helminths is unique, and is not seen in other animals studied to date (Köhler, 2006).

The lipid

Structure and Biochemical Composition of the Cuticle

The cuticle of parasitic nematodes forms the exoskeleton and consists mainly of cross-linked collagens (Page and Johnstone, 2007); its overlaying surface coat represents the primary interface between the pathogen and the host's immune system (Page et al., 1992). However, nematodes, including H. contortus, also absorb nutrients as well as relatively large amounts of some anthelmintic drugs (eg, levamisole and macrocyclic lactones) through the cuticle, whereas other anthelmintics are absorbed via

Tubulin as a Major Structural Component and Drug Target

Microtubular functions are important for numerous cellular processes, such as cell division, axoplasmic transport, cell movement and cell-to-cell communication. The cytoskeleton is intimately involved in the growth of axons, and microtubuli are involved in axonal transport of compounds (Wehner and Gehring, 1995a, Wehner and Gehring, 1995b). Almost all biosynthetical activities of the neuron can be found in the cell soma, which contains a highly developed endoplasmic reticulum. Via the Golgi

Nervous System in Nematodes

Of all nematodes, the nervous system of C. elegans is the best understood. It contains 302 neurons with 118 neurone classes (Joyner, 2010). Approximately 5000 chemical synapses and 600 electrical synapses (gap junctions) are functional (Bargmann, 2006, Thomas and Lockerly, 1999). More than one-third of the neuronal cells in C. elegans release acetylcholine (ACh), the major excitatory neurotransmitter, causing contraction of the body wall muscle by opening sodium-gated ACh receptors (Holden-Dye

Biochemistry of Drug Resistance

In principle, nematodes can employ a range of different strategies to achieve a state of reduced susceptibility to a particular anthelmintic drug. These strategies include the modification of a drug target (eg, binding site), increased target site numbers (eg, neuronal receptors), increased drug efflux (eg, through transmembrane pumps), increased metabolization (eg, through CYP450) and/or sequestration of the drug (James et al., 2009).

Conclusions

Understanding the biochemistry of nematodes is central to gaining insights into catabolic and anabolic pathways of these worms. Moreover, it helps to better understand nematode–host interactions in the habitats where nematodes reside in the host. In addition, this research field supports the finding of new target sites and thus anthelmintic screening. Unfortunately, there is a paucity of information on biochemical processes in parasitic nematodes in general, and also specifically in H. contortus

Acknowledgement

I would like to thank Prof. Dr Robin Gasser and Prof. Dr Georg von Samson-Himmelstjerna for their great support during writing this manuscript.

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