Mining the Haemonchus contortus genome for new anthelmintic targets Salma Siddiqui Forrestor Laboratory

What is Haemonchus contortus? Parasitic nematode Infects sheep, goat and cattle around the world Common name: barber pole worm

Haemonchus contortus Live in stomach of the host Feeds on blood Host falls ill Primary sign - bottle jaw Weakness, weight loss and death ges/intparasite/internal.htm

Life Cycle of Haemonchus contortus Male and female mate, eggs get deposited into external environment Develop from 1 st stage larvae to infective 3 rd stage larvae Ingestion of 3 rd stage larvae causes cattle to become infected with Haemonchus contortus

Drugs for Treatment? Three leading antiparasitic drugs Ivermectin, fipronil and imidacloprid All 3 target ligand-gated ion channels (Raymond, D., Sattelle, D., 2002) Most successful – Ivermectin Targets glutamate- gated chloride channels

What are Ligand-gated Chloride Channels? 5 subunits form a pore in a cell membrane Pore opens with binding of a ligand (a molecule that binds to a receptor, ie. Glutamate) Pore regulates Cl - flow into cell

PlasmaMembrane Extracellular Intracellular Cl - Ivermectin Glutamate Cl - Paralysis CellularInhibition Glutamate-gated Chloride Channel Function

Ivermectin Resistance Ivermectin overused Resistance in parasites Need new drug targets discovered Ligand-gated chloride channels may be the best drug targets

Objective To identify new ligand-gated chloride channel genes in Haemonchus contortus

Why Ligand-gated Chloride Channels (LGCC’S)? Parasitic nematodes are invertebrates Invertebrate LGCC’s very different from mammal LGCC’s Drugs targeting invertebrate LGCC’s may not target mammalian LGCC’s Also, research on invertebrate LGCC’s can give important information on primitive nervous systems.

How was this done? Used the complete C. elegans genome to identify ligand-gated chloride channels Wormbase search glycine-gated chloride channel gaba-gated chloride channel glutamate-gated chloride channel Built a library of C. elegans LGCC gene sequences Used list to blast C. elegans genome for additional ligand-gated chloride channels

What is “BLAST’ing”? “basic local alignment search tool” Using a sequence to search for a similar sequence in a particular organism or groups of organisms In this way more LGCC’s can be found

Blasting! Identified 58 LGCC’s from C. elegans These C. elegans sequences were used to search the partial H. contortus genome available from the Sanger Institute 152 sequences obtained Haemonchus contortus genome not completely sequenced - unorganized Labeled with accession number only

Organizing Sequences To get more information on H. contortus sequences sequences blasted against all organisms sequences blasted against human genome The H. contortus results were compared to the most similar results to obtain a better idea of what type of LGCC they may encode.

Organizing Sequences Organized sequences into a table identifying the most similar sequences in C. elegans and in Humans. Page No.H. ContortusTop C. Elegans MatchTop Human Match Accession No.(% homology) 11haem-16n21.p1kgaba/glycine receptor (96%)DNA polymerase protein (62%) 12haem-26m17.p1kgaba/glycine receptor (69%)glycine receptor (69%) 13haem-31j01.p1kgaba receptor (57%)gaba receptor (53%)

What Now? Choose sequences that will be examined further. This will include: Isolating entire H. Contortus sequence Functional studies to confirm identity of chosen sequences Ie. what ligand opens the channel the sequence encodes New LGCC’s may be used for screening of new drugs

Conclusion Used C. elegans LGCC genes to identify possible H. contortus LGCC genes H. contortus gene sequences were blasted against other organisms To get further information on the possible identity of each of the H. contortus sequences Few gene sequences will be further examined to see if they can be used as new drug targets

Schistosoma mansoni Very little is known about chloride channels from this parasite. Same strategy used to identify LGCC’s in S. mansoni 109 sequences identified so far

Questions?