The long journey of the African trypanosome

Terry PearsonUniversity of Victoria

Doyle Foundation African Trypanosomosis Meeting, 6-7 June 2006, InternationalLivestock Research Institute, Nairobi

Trypanosomes-still a problem

Geographic distribution of tsetse flies, Geographic distribution of tsetse flies, cattle and human sleeping sickness in sub Saharan Africacattle and human sleeping sickness in sub Saharan Africa

Hersey, 2001

• estimated range occupied by tsetse is 9 million km2 of land

• tsetse-transmitted animal trypanosomiasis, Nagana, results in an annual loss of 5 billion dollars due to cattle mortality and loss of agricultural land

• 60 million people are at risk of contracting African sleeping sickness in endemic areas

Trypanosomes are likely toremain as important pathogens

A Brief History

Human sleeping sickness was described by an Arab writer, al Qualquashaudi in the 14th century

Many African cattle-owning tribes knew of the disease in animals for centuries.

In colonial times, the diseases were described in humans and cattle and were spread into theinterior of the continent along exploration routes.

Discovery of trypanosomes:Researcher: Dr. Gabriel Valentin

Professor, Department of AnatomyUniversity of Bern

1841(Funded by The Swiss National Science Foundation)

(time: 1hour: cost: 4 Swiss Francs)

Researcher: Dr. Mike LehaneProfessor, Liverpool School of Tropical Medicine

2006 Funded by taxpayers

(time 8 days: Cost 1200 pounds)

1894

Drawing by Mary Bruce

Drawing by Declan McKeever

(Bloodstream form trypanosome)

First Wave of Research on Tryps

Discovery of trypanosomesLife cyclePathology

Early drugs

Antigenic Variation

Dick LePage 1968-immunologically different surface coats

George Cross 1975-purified Variant Surface Glycoprotein (VSG)

Recombinant DNA Berg/ Gilbert Monoclonal Antibodies Kohler/Milstein2-D gel electrophoresis O’Farrell/AndersonsTrypanosome culture Hirumi

The Second Wave of Research on Trypanosomes

(Mid 1970s)

Rockefeller Foundation“Great Neglected Diseases Program”

WHO“Special Program for Research and Training in Tropical Diseases”

CGIAR-ILRAD

What was the result?-trypanosome culture-VSG structure-mechanisms: antigenic variation-immune perturbations of the mouse-initiation of the study of bovine immune system-trypanosome differentiation-sensitivity of trypanosomes to serum killing-rudimentary diagnostic tests-identification of a few non-VSG molecules-rudimentary attempts at immunization-training of people

Donor fatigueLoss of faith (research administrators, agencies, some researchers) Decentralization of research into small labs

African trypanosomes in human blood

VSG: an immunological and intellectual barrier

Third (slow)Wave of Trypanosome Research

VSG+/Procyclin - Procyclin+/VSG-

Two classes of Procyclins

The “Spiny Norman” model of procyclic trypanosome surface molecules

Bacteriome

Tsetse-Trypanosome Interactions

Identification of proteins from 2-D gels

Excise spot, wash, reduce, alkylate, digest

Run gel, stain,scan

Extract peptides-mass analyze

Database search

1-D and 2-D gels of midguts from Glossina m. morsitans

N-terminalMicrobial HSP-60:(Wigglesworthia)

N-terminal(microbialOMP)

EP midgut protein:

-increased expression in tsetse midgut after antigen challenge

A strategy for interference with trypanosome transmission:

Express anti-tryps/tsetse molecules in Sodalis (transfection in vitro)

Infect tsetse with engineered Sodalis

Sodalis is transferred to offspring via milk gland secretions

Drive engineered Sodalis into tsetse populations

Anti-trypanosome effectors:

-midgut lectins-midgut EP protein

-anti-procyclin mAbs-antimicrobial peptides

Could these be expressed in tsetse?(by paratransgenesis)

Trypanosoma brucei(bloodstream forms)

Untreated BMAP-18 treated

Trypanosoma brucei(procyclic forms)

Leishmania donovani(promastigotes)

Bloodstream form Bloodstream form Trypanosoma Trypanosoma bruceibrucei treated with treated with antimicrobial peptide BMAPantimicrobial peptide BMAP--1818

Flagellar Pocket

-endocytosis-exocytosis-VSG recycling

VSG recycled every 7 minutesVSG translocation 45 minutes(VSG expressway)

Specific targeting of endocytic pathway-single chain Abs or other targeting ligands? Coupled with ant-trypanosomal peptides?

Fourth Wave:Genome and Post genome

Serengeti 1977

Protein Discovery:

-life cycle stages-serum sens vs serum resistant

-species specific antigens-biomarkers for diagnosis

-biomarkers for tsetse interactions-biomarkers for host responses

iTRAQ

Biomarker Validation:

Stable Isotope Standards and Capture by Anti-Peptide Antibodies

(SISCAPA)

Proteins Measured Clinically in Plasma Span > 10 Orders of Magnitude in Abundance

(199 proteins, literature values)

PPI

SISCAPA*: A New Method Combining The Specificity of MS Detection with Sensitivity of

Antibody Capture

* patent pending

*Mass Spectrometric Quantitation of Peptides and Proteins Using Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA). 2004. Anderson, N.L., et al Journal of Proteome Research 3;235-244.

Multiple-Reaction Monitoring (MRM): Specific Mass Spectrometric Assay for Peptides

• MRM – is a 2-stage MS assay capable of absolute analyte specificity with high precision (CV < 10%). – measures selected peptides in a sample digest as quantitative surrogates for the proteins from which they derive– assays for peptides can be designed directly from protein sequence

MRMs are implemented using triple-quadrupole mass spectrometers (QqQMS), very widely used for small molecular assays in plasma (drug metabolites, inborn errors, pesticides)

Assays can be multiplexed (>100 per run)

LC-MS/MS as Universal Antibody

• SISCAPA is effectively a sandwich immunoassay for a target peptide, in which the second antibody is replaced by a mass spectrometer (acting as a generic second antibody applicable to all analytes and with absolute structural specificity).

Target Target

Sandwich immunoassay

SISCAPA assay

Unfractionated x10047 MRM’s

albuminLVNEVTEFAK

hemopexinNFPSPVDAAFR

α1antichymotrypsinEIGELYLPK

α1antichymotrypsinEIGELYLPK

hemopexinNFPSPVDAAFR

AlbuminLVNEVTEFAK

Unfractionated x1

SISCAPA: Ab3 Hx400-fold enrichment

SISCAPA: Ab4 AAC4,400-fold enrichment

SISCAPA: Specific Enrichment of Peptides from a Plasma Digest

Eye of Science/ Photo Researchers

Thanks!

Isabel RoditiPeter ButikoferSerap AksoyRon GoodingMike LehaneJody HaddowLee HainesAngela Jackson

UVIC ProteomicsLeigh Anderson

Gabrielle & Margaret(Doyle Foundation)

If God had amused himself inventing the lilies of the field, He surely knocked His own socks off with the African parasites

Barbara KingsolverThe Poisonwood Bible

mAb 4A2 (teneral G. m. morsitans midgut)

10nm

mAb 4A2 (teneral G. m. morsitans midgut)

10 nm

Could tsetse molecules serve as targets?

Midgut molecules

1. secreted proteins

2. structural proteins