Identification of Trypanosome Proteins in Plasma from African Sleeping Sickness Patients Infected with T. b. rhodesiense

Brett A Eyford,Terry W Pearson,John C Enyaru,Steven A Carr,Rushdy Ahmad,Joy Sturtevant

doi:10.1371/journal.pone.0071463

Brett A Eyford, Terry W Pearson + Show 4 more

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https://doi.org/10.1371/journal.pone.0071463

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Abstract

Control of human African sleeping sickness, caused by subspecies of the protozoan parasite Trypanosoma brucei, is based on preventing transmission by elimination of the tsetse vector and by active diagnostic screening and treatment of infected patients. To identify trypanosome proteins that have potential as biomarkers for detection and monitoring of African sleeping sickness, we have used a ‘deep-mining” proteomics approach to identify trypanosome proteins in human plasma. Abundant human plasma proteins were removed by immunodepletion. Depleted plasma samples were then digested to peptides with trypsin, fractionated by basic reversed phase and each fraction analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This sample processing and analysis method enabled identification of low levels of trypanosome proteins in pooled plasma from late stage sleeping sickness patients infected with Trypanosoma brucei rhodesiense. A total of 254 trypanosome proteins were confidently identified. Many of the parasite proteins identified were of unknown function, although metabolic enzymes, chaperones, proteases and ubiquitin-related/acting proteins were found. This approach to the identification of conserved, soluble trypanosome proteins in human plasma offers a possible route to improved disease diagnosis and monitoring, since these molecules are potential biomarkers for the development of a new generation of antigen-detection assays. The combined immuno-depletion/mass spectrometric approach can be applied to a variety of infectious diseases for unbiased biomarker identification.

Highlights

Several methods are currently used to diagnose human African trypanosomiasis (HAT), known as African sleeping sickness
Characterization of Plasma from HAT Patients To recapitulate previous literature reports, and to gain confidence that trypanosome antigens were present prior to undertaking our extensive MS analysis, plasma samples from HAT patients were examined for the presence of conserved, trypanosome antigens by indirect enzyme linked immunosorbent assays (ELISA) using polyclonal antiserum against T. b. rhodesiense Procyclic culture forms (PCF) as a probe
The anti-trypanosome antibodies were first characterized by immunoblotting and ELISA and reacted in both assays with antigens in lysates of the immunizing T. b. rhodesiense PCF and in lysates of T. congolense, T. b. brucei, and T. b. gambiense

Summary

Introduction

Several methods are currently used to diagnose human African trypanosomiasis (HAT), known as African sleeping sickness. This strategy, is made technically difficult by the high abundance of human plasma proteins [18,19], almost certainly explaining the failure over the past 30 years to identify trypanosome antigens in the blood of infected patients.

Results

Conclusion