SUPPLEMENTARY EXPERIMENTS FOR THE LOCALIZATION OF HCO-PGP-13 MRNA IN THE PARASITE

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Anatomy of Caenorhabditis elegans hermaphrodite adult

The anatomy of C. elegans has been extensively studied at the electron microscopy level, and its complete cell lineage has been made available (www.wormatlas.org). The hermaphrodite adult C. elegans is about 1mm long and presents an unsegmented, cylindrical shape that becomes narrower on the extremities (Fig. 7A). Similarly to H. contortus presented before, its whole body is covered by a striated cuticle secreted by epithelial cells such as hypodermis and seam cells on the two lateral regions of the worm, over which the alae forms at all stages except L2 – L3. A new cuticle is secreted at each stage with a molt at the end of the previous stage.
Figure 7. Caenorhabditis elegans hermaphrodite adult anatomy. A. Major anatomical features. The cuticle and nervous systems are not shown for better visibility of other organs. Two of the four quadrants of body wall muscles are represented. B. Zoom on the head of the nematode for simplified representation of the nervous system: the nerve ring and only the amphidial neurons of the head are shown for better visibility, as well as the start of the dorsal and ventral nerve cords that run along the entire body. C. Cross-section through the mid-body region of the C. elegans hermaphrodite (location marked with a dotted line in B.) showing the intestine and gonad at the centre of the pseudocoelomic cavity. D. Cross-section through the pharyngeal region of the C. elegans hermaphrodite (location marked with a dotted line in A.) showing the four muscle quadrants surrounded by the epidermis and cuticle. Only the nerve processes of the amphids on the two sides of the pharynx and the six labial nerves are shown for simplification. DC = Dorsal cord, VNC = ventral nerve cord, CANL = left CAN neuron with unknown function, LDSC = left dorsal sublateral cord, RVSC = right ventral sublateral cord, DLLN = dorsal lateral labial nerve, PN = pharyngeal neuron, AN = amphidial neuron. Schematic representation constructed from images found at www.wormatlas.org.

elegans adults.

The somatic nervous system is structured by 282 neurons with cell bodies clustered in ganglia in the tail, and the head where they form the nerve ring around the metacorpus part of the pharynx (Fig. 7B). The 4 main classes of neurons are motorneurons connecting muscle cells, sensory neurons that sense many stimuli such as temperature, chemicals, ambient osmolarity, oxygen level, pH, light and mechanical stimuli, interneurons connecting them and polymodal neurons performing at least two of these three possible functions. Most of these neurons travel longitudinally along the worm from the nerve ring, either towards the tip of the head, or throughout the entire body. These are essentially located within the ventral and dorsal nerve cords, with processes located between the hypodermis and body wall muscles (Fig. 7C) and linked by commissures (Fig. 7B). In contrast, the 20 pharyngeal neurons are located directly among pharyngeal muscles (Fig. 7D).
The digestive system is similar to that of H. contortus, starting with the mouth that opens on the anterior end, followed by the pharynx and intestine structures linked by the pharyngeal-intestinal valve (Fig. 7A). These structures occupy the centre of the worm and open through the anus on the ventral side, just before the tail whip. The intestine is found either on the left or on the right side of the reproductive system of the hermaphrodite, with a switch in the middle. Indeed, the self-fertilization is made possible by the presence of two, bilaterally symmetric, U-shaped somatic gonad arms (oviducts), each followed by a spermatheaca, ending with a central uterus that opens on the ventral side of the midbody with the vulva (Fig. 7A).
The muscle system contains two types of muscles. Body wall muscles, that run along the body (Fig. 7A), are obliquely striated and arranged into four quadrants, two dorsal and two ventral (Fig. 7C and 7D). They receive neuronal input from motor neuron processes located in nerve cords or in the nerve ring to permit locomotion (Fig. 7B, 7C and 7D). Other muscles, found in the pharynx and around the intestine, rectum and vulva, are nonstriated and allow the functions of feeding, defecation and egg laying, respectively.
The excretory system allows osmoregulation and waste disposal. It consists of two canals running along the body on the two lateral sides of the worms (Fig. 7A), linked on the ventral side of the posterior head with an opening close to the nerve ring on this side. Finally, the coelomocyte system is composed of the pseudocoelomic cavity and three pairs of coelomocytes that endocytose fluid from the pseudocoelom, which probably plays the role of a primitive immune system (Fig. 7C and 7D).

A model nematode in the laboratory

Many other advantages are found in C. elegans for in vitro and in vivo studies. Its short life cycle and high number of eggs produced by a single adult makes it possible to quickly generate genetically identical progeny by self-fertilization of the hermaphrodite, which can, for example, be used for generation of resistance lineages to increasing doses of a drug. On the other hand, isolation, maintenance or spreading of mutations across strains can be done with male mating. Mutations can also be easily obtained by homology recombination, with many deletion strains for various genes made available on the Caenorhabditis Genetics Center (http://cbs.umn.edu/cgc/home), and gene rescue with the homolog from another species is routinely performed by microinjection.
Plus, C. elegans is transparent throughout its life cycle, which simplifies the record of visible phenotypic evolutions. Moreover, it easily feeds on Escherichia coli bacteria on agar plates or in liquid cultures. Its genome being short, about 100 millions of base pairs found across 5 autosomes and 1 X chromosome, C. elegans was the first animal to have its entire genome totally sequenced, in 1998. This, added to its stereotypical development and simple body plan composed of about 1000 somatic cells, made it a model of choice in many fields of life sciences such as genomics, embryogenesis, cell biology, neurosciences and aging. Its large palette of behavior also allows the study of many complex processes such as locomotion, feeding, mating, egg laying, memory, and sensory responses to various stimuli like touch, temperature and chemicals.
However, the difference in the free-living versus parasitic life cycles of C. elegans and H. contortus, respectively, must require the expression of different genes between these two species. Especially from L3 to adult stage, the H.contortus transcription changes for life within a host, e.g. by increasing peptidases production for blood-feeding activity (Schwarz et al., 2013), while C.elegans stays free and feeds from its environment. Nevertheless, the common pharmacology found across species of anti-parasitic drugs commonly used has allowed, over many years, the successful study and application of findings in C. elegans to various parasites (for a review: Holden-Dye and Walker, 2007).

ANTHELMINTICS AND MECHANISMS OF RESISTANCE

In order to reduce haemonchosis and its deleterious impact on livestock, efficient drugs against H. contortus have been sought. However, due to the selection of resistant parasites by each specific anthelmintic (AH) class, there is an ongoing demand for new AHs that overcome existing resistance (Holden-Dye and Walker, 2007). The main classes of anthelmintics efficient against roundworms, including H. contortus, are presented below, ordered by date of their discovery. Some anthelmintics initially used against other pathogens are also listed, because they later became a possibility for combination with commonly used parasiticides to regain control after resistance had arisen.

Main classes of anthelmintic drugs

Imidazothiazoles, also called tetrahydropyrimidines, are the oldest anti-parasitic agents used to treat cattle. They are agonists of the acetylcholine receptors (AChR) present at the surface of muscle cells. Tetramisole and pyrantel were the first two AHs of this family to be described in 1970 (Aceves et al., 1970; Aubry et al., 1970). These drugs cause prolonged activation of the AChR at neuromuscular junctions leading to sustained contraction of the somatic muscle, which results in paralysis of nematodes without causing their death. Pharmacological studies in Ascaris suum have shown that different AChR subtypes exist: N-AChR, B-AChR and L-AChR, each having various subunit compositions and being preferentially activated by nicotine, bephenium and levamisole respectively (Qian et al., 2006). C. elegans L-AChR is composed of five subunits and three proteins are essential to its function, allowing its assembly and targeting to the membrane (Boulin et al., 2008). It is also activated by pyrantel, though to a lower extent than levamisole, and while nicotine cannot activate this receptor it is a potent allosteric inhibitor.

Benzimidazoles

Benzimidazoles (BZ) were the first class of broad-spectrum anthelmintics established with the discovery of thiabendazole in 1961 (Gordon, 1961). They remained the mainly used anti-parasitic agents until the 1980s. The success of these AHs, that also comprise albendazole, febendazole, mebendazole and oxfendazole, is mainly due to their selective toxicity for helminths (Lacey, 1990). These drugs prevent microtubule polymerization by binding to β-tubulin (Lacey, 1988). Capping of the associating end of the microtubule, which constantly dissociates on the other extremity, results in its depolymerization (Lacey, 1990). The disintegration of the microtubule matrix, first observed in Ascaris suum, impairs many critical cellular processes such as cell division and transport, causing cell death, and eventually leading to the death of the parasite (Borgers and De Nollin, 1975). The variability in efficacy of each drug in vivo was correlated to their affinity for β-tubulin, except for oxfendazole and albendazole sulfone (Lubega and Prichard, 1991). Triclabendazole, on the other hand, is not effective against nematodes and cestodes, but controls all larval and adult stages of the parasitic trematode Fasciola hepatica (Boray et al., 1983).

Salicylanilides and Cyclodepsi-peptides

Salicylanilides represent a wide range of compounds initially developed as antifungal agents (Kraushaar, 1954). Among this class of anthelmintics, closantel and rafoxanide are mostly used, which are highly efficient against the adult stage of the trematode F. hepatica and blood sucking nematodes such as H. contortus (Swan et al., 1999; Van Den Bossche et al., 1979). Their molecular mode of action, however, is not completely elucidated. By uncoupling oxidative phosphorylation in the cell mitochondria, they disturb ATP production critical for energy metabolism, but this could also be due to initial impairment of glycolysis (Fairweather and Boray, 1999). The consequence is spastic paralysis of the parasite that dies from starvation after detachment. The first compound of the cyclodepsi-peptides anthelmintic class, discovered in 1992, was PF1022A, a natural product of the fungus Mycelia sterilia that grows on the leaves of Camellia japonica (Sasaki et al., 1992). Emodepside (EMD) is a derivative of this compound and is licensed for treating roundworms and hookworms in cats. PF1022A and EMD are also known to be efficient against H. contortus strains resistant to IVM, BZ and LEV (Harder et al., 2005). The mode of action of EMD has been extensively studied and starts with the activation of a presynaptic latrotophilin receptor. This induces a complex signaling cascade that leads to a flaccid paralysis of pharyngeal and somatic muscles in nematodes.

Macrocyclic lactones

Since Ivermectin (IVM), the first registered macrocyclic lactone (ML) anthelmintic, was introduced on the market in 1980, other ML showing the same type of activity have been extensively developed. Abamectin, eprinomectin, doramectin and selamectin belong to the class of avermectins and moxidectin (MOX) and mylbemycin oxime are of the mylbemycin class (Haber et al., 1991). These two sub-families all share a macrocyclic lactone nucleus, but the mylbemycins lack the sugar group(s) present at the C13 of the macrocyclic lactone ring in avermectins, thus being more lipophilic (Fig. 8).

Modifying drug biotransformation

Increasing the rate of modification of the drug to a non-toxic compound, or reducing the activation of pro-drugs can also alter their efficacy, so that such mechanisms may spread in the population by selection pressure (Cvilink et al., 2009). A biotransformation study performed ex vivo on microsomal fractions of Fasciola hepatica showed that the rate of triclabendazole metabolism into triclabendazole sulphoxide was significantly higher in triclabendazole-resistant flukes compared to susceptible ones (Alvarez et al., 2005). Metabolism of BZs was shown to possibly play a role in their resistance to this class of anthelmintics in various organisms. For example, H. contortus was shown to be more resistant to thiabendazole after glutathione S-transferase (GST) expression was induced by a cambendazole treatment (Kawalek et al., 1984). Plus, another team later found that inhibiting glutathione synthesis led to an increase in thiabendazole sensitivity in H. contortus resistant strains (Kerboeuf and Aycardi, 1999). However, these studies are indirect and the effect observed could be due to other induced mechanisms such as modification of transport.
On the other hand, ML resistance does not appear to be linked to the biotransformation of these molecules (Lespine, 2013). In fact, IVM and MOX were shown to have high chemical stability in sheep ruminal and abomasal content as they are only poorly metabolized by biotransformation enzymes usually detoxifiying xenobiotics (Lifschitz et al., 2005). Plus, the enzymes involved in this process, mainly cytochromes, differ between host species and drugs (Zeng et al., 1998; Zeng et al., 1996; Zeng et al., 1997). 60 to 80% of macrocyclic lactones are then found as the parental form in the plasma of the host (Gonzalez-Canga et al., 2009). Elimination of these compounds is thus thought to be mainly due to their transport.

Modifying drug transport

An increased efflux or decreased influx of the drug can also reduce its action inside the cell. This can, for example, be due to the overexpression of ATP-binding cassette (ABC) transporters from the multidrug resistance (MDR) family. As these transporters are able to expel ML out of mammalian cells, their overexpression in parasites could also be a mechanism of resistance to these drugs (Pouliot et al., 1997).

ABC MDR TRANSPORTERS AND ANTHELMINTICS RESISTANCE

Proteins of the ABC transporters family all share a common structure: they contain at least one nucleotide binding domain (NBD) also called ATP binding cassette (ABC), which gives its name to the protein family (Hyde et al., 1990). To be active, ABC transporters need two such domains to bind to and hydrolyze ATP, thus supplying the protein with energy necessary for transport. ABC transporters are mostly membrane transporters, and contain transmembrane domains (TMDs) most often composed of 6 transmembrane helices (TMs) each. These TMDs are formed by a majority of hydrophobic amino-acids that allow the anchoring of the protein within the leaflets of the plasma membrane. Substrates bind within the funnel-shaped intertwining TMDs. Functional ABC transporters are either full-transporters that contain 2 NBDs and 2 TMDs, forming a typical “tandem” structure, or half-transporters that only contain one TMD and one NBD, and need to homo- or hetero-dimerize to be active (Table 1). Exceptions are soluble ABC transporters that are mainly expressed in the nucleus to act as gene regulators.
In all living kingdoms, ABC transporters contribute to cell homeostasis. In bacteria, ABC transporters can import compounds essential for cell viability and pathogenicity, or export endogenous molecules out of the cell (Table 1) (Davidson et al., 2008; Sarkadi et al., 2006). They can also promote the translocation of lipids from the inner to the outer leaflet of the cell membrane. In eukaryotes, most of ABC pumps extrude molecules from the plasma membrane, leading them to the extracellular compartment, but some ABC transporters can also be found in organelles such as the mitochondria, the endoplasmic reticulum, the peroxisomes, or vacuoles in plant cells. In mammals, seven sub-families of ABC transporters are expressed in various tissues, and are responsible for the transport of a wide variety of compounds, as indicated in Table 1. They are composed of different combinations of NBD and TMD domains (Sarkadi et al., 2006; Szakacs et al., 2006):
The ABCA sub-family contains 13 full transporters, ABCA1 to A13, with the same domain arrangement shown in Table 1. They are all mainly involved in lipid transport in different tissues. The ABCB sub-family contains 11 proteins. Three of them are full transporters (ABCB1, B4 and B11) and display remarkable multispecific properties. The eight other ABCB transporters are half-transporters and are expressed in internal membranes where they handle specific endogenous substrates.
The ABCC family is composed of 13 full transporters. Seven of them, ABCC1 to C3 and C6 to C9, contain an additional TMD of 5 helices at the N-term of the protein (“TMD0” in Table 1), linked to the first TMD1 by the loop “L0” (Bryan et al., 2004; Deeley et al., 2006). ABCC7 harbors a supplementary cytosolic regulatory domain (R) that plays a critical role in the regulation of its function. Indeed, it is a specific ion channel that passively conducts chloride ions in epithelial cells. The R subunit must be phosphorylated in order to facilitate the channel gating (Gadsby and Nairn, 1999). During the transport of Cl-, ATP binding on the NBDs only has a regulatory effect on the ionic conduction and ATP hydrolysis remains very slow. Various mutations in this protein, also called cystic fibrosis conductance regulator (CFTR), make it not functional, so that the ensuing default of Cl- transport causes damages in various tissues of patients suffering from cystic fibrosis. The sulfonylurea receptors SUR1/ABCC8 and SUR2-ABCC9 are not transporters but form the ATP-binding subunit regulating the ATP-dependent potassium channels in pancreatic and heart cells respectively (Bryan et al., 2004). However, they also form the receptors of various compounds acting as blockers and openers of the K+ channel, thereby presenting an unusual capacity of multispecific recognition of various drugs (Bessadok et al., 2011).
All four ABCD members are half-transporters and transport various fatty acids.
The unique ABCE, a regulator of protein synthesis, and the three ABCF proteins believed to play a role in inflammatory processes, lack TMD domains.
All five ABCG transporters are half-proteins (ABCG1 to G4 and G8) with an inverted NBD-TMD arrangement. They form homodimers except for ABCG5 and G8.

Table of contents :

LITERATURE REVIEW
I. HAEMONCHUS CONTORTUS AND CAENORHABDITIS ELEGANS
1. Haemonchus contortus, a parasitic gastro-intestinal nematode
2. Caenorhabditis elegans, a model nematode
II. ANTHELMINTICS AND MECHANISMS OF RESISTANCE
1. Main classes of anthelmintic drugs
2. Mechanisms of resistance to anthelmintics
III. ABC MDR TRANSPORTERS AND ANTHELMINTICS RESISTANCE
1. ABC transporters: structures and functions
2. ABC MDR transporters and resistance to anthelmintics
3. Overcoming ML resistance in parasitic nematodes
HYPOTHESIS AND OBJECTIVES
EXPERIMENTAL WORK
PART I – IN SILICO FUNCTIONAL CHARACTERIZATION OF CEL-PGP-1
A.MANUSCRIPT N°1 IN PREPARATION: MODELING MULTISPECIFIC DRUG RECOGNITION BY CAENORHABDITIS ELEGANS P-GLYCOPROTEIN 1
I. INTRODUCTION
II. COMPUTATIONAL METHODS
1. Structure of Cel-Pgp-1
2. Preparation and conformational analysis of ligands
3. Docking calculations
4. Data analysis
5. Determination of the residues constituting the « hotspots for drug binding »
III. RESULTS
1. Docking experiments on compounds stimulating or not the ATPase activity of Cel-Pgp-1
2. Binding mode of other substrates of mammalian Pgp previously tested on the ATPase activity of Cel-Pgp-1
3. Binding mode of other molecules of interest
IV. DISCUSSION
1. Validation of in silico docking approach on Cel-Pgp-1
2. Further analysis of the in silico/in vitro correlation
3. Molecular properties of the multispecific binding domain of Cel-Pgp-1
B. MANUSCRIPT N°2 P
PARASITOLOGY: DRUGS AND DRUG RESISTANCE: IN SILICO ANALYSIS OF
ANTHELMINTICS BINDING TO CAENORHABDITIS ELEGANS P-GLYCOPROTEIN 1 .
PART II – IDENTIFICATION, LOCALIZATION AND FUNCTIONAL CHARACTERIZATION OF H. CONTORTUS P-GLYCOPROTEIN 
A. HAEMONCHUS CONTORTUS P-GLYCOPROTEIN
I. INTRODUCTION
II. MATERIAL AND METHODS
1. Parasites
2. RNA extraction and reverse transcription.
3. Amplification of Hco-pgp-13 cDNA sequence
4. Prediction of Hco-Pgp-13 protein sequence and phylogenic analysis
5. Construction of 3D models of Hco-Pgp-13 based on Cel-Pgp-1 4F4C PDB structure as template
and in silico docking calculations
6. Cloning and transfection of Hco-pgp-13 gene in Pichia pastoris
7. ATPase activity measurement of Hco-Pgp-13 stimulated by actinomycin D
8. Design of specific antibodies against Hco-Pgp-13
9. Polyacrylamide gel electrophoresis and Western-blot
10. Immunohistochemistry on larvae and adult H. contortus sections
III. RESULTS
1. Amplification and sequencing of Hco-pgp-13 cDNA
2. Translation product amino-acid sequence and topology of amplified Hco-pgp-13
3. Phylogenetic analysis of Hco-Pgp-13
4. Homology modelling of Hco-Pgp-13 on Cel-Pgp-1
5. In silico docking of actinomycin D on Hco-Pgp-13
6. Expression of Hco-Pgp-13 in Pichia pastoris cells and stimulation of its ATPase activity by
actinomycin D
7. Immunolocalization of Hco-Pgp-13 protein in larvae and adult parasites
III. DISCUSSION
1. The Hco-pgp-13 corrected cDNA sequence encodes a protein matching the topology of an ABC
transporter
2. Hco-Pgp-13 can interact with actinomycin D
3. Hco-Pgp-13 sequence and localization are very close to those of Cel-Pgp-12, Cel-Pgp-13 and Cel-Pgp-14
B. SUPPLEMENTARY EXPERIMENTS FOR THE LOCALIZATION OF HCO-PGP-13 MRNA IN THE PARASITE
I. MATERIAL AND METHODS
1. DNA Probes amplification
2. RNA probes amplification and validation
3. Fluorescence in situ Hybridization
II. RESULTS
1. Probes amplification and validation
2. Fluorescence in situ hybridization
III. DISCUSSION
C. SUPPLEMENTARY EXPERIMENTS FOR THE CHARACTERIZATION OF HCOPGP- 13 FUNCTION
I. MATERIAL AND METHODS
1. Codon optimization and transfection of Hco-pgp-13 in LLCPK1 cells
2. Characterization of mRNA expression
3. Characterization of protein expression
4. Transport assays
5. Cytotoxicity assays
6. ATPase activity assays
7. In silico docking of various molecules on the two Hco-Pgp-13 3D structural models
II. RESULTS AND DISCUSSION
1. Expression of Hco-Pgp-13 in LLCPK1
2. Functional characterization of Hco-Pgp-13 expressed in LLCPK1 cells
3. Passage-dependent expression and function of Hco-Pgp-13 in LLCPK1 cells
4. Functional characterization of Hco-Pgp-13 expressed in Pichia pastoris
5. In silico docking calculations on Hco-Pgp-13 3D structural models
GENERAL DISCUSSION AND PROSPECTS
1. Cel-Pgp-1 is a multidrug transporter with some homologies with mammalian Pgp
2. Cel-Pgp-1 interacts with ML with high affinity
3. Hco-Pgp-13 presents many homologies with Cel-Pgp-1, which makes it a putative ABC multidrug transporter
4. Hco-pgp-13 and Cel-Pgp-1 might also have homologies in their substrate recognition sites with other Pgps
5. The expression of Hco-Pgp-13 in Haemonchus contortus is widely distributed, supporting an important function for this protein
6. Perspectives for future investigation of Hco-Pgp-13 substrate profile
7. Could Hco-Pgp-13 also transport endogenous sterols?
8. Further description of Hco-Pgp-13 molecular properties and implication for other Pgps
9. Investigation of Hco-Pgp-13 importance in the living worm
10. Perspective for fighting ML resistance in the field: design of inhibitors of Pgps which will have been identified as transporters of ML
REFERENCES

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