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

www.smo.uhi.ac.uk/~sm067635/boiteagan.jpg

http://ag.ansc.purdue.edu/sheep/ansc442/Semprojs/images/intparasite/internal.htm

http://cal.vet.upenn.edu/merial/Introduction/images/bottle%20jaw.jpg

Life Cycle of Haemonchus contortus

Male and female mate, eggs get deposited into external environment

Develop from 1st stage larvae to infective 3rd stage larvae

Ingestion of 3rd 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

PlasmaPlasmaMembraneMembrane

ExtracellularExtracellular

IntracellularIntracellular

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-Cl-

IvermectinGlutamate

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-

Cl-Cl-

ParalysisParalysisCellular Cellular InhibitionInhibition

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. Contortus Top C. Elegans Match Top Human Match

  Accession No. (% homology) (% homology)

11 haem-16n21.p1k gaba/glycine receptor (96%) DNA polymerase protein (62%)

12 haem-26m17.p1k gaba/glycine receptor (69%) glycine receptor (69%)

13 haem-31j01.p1k gaba 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?