A search for Trypanosoma brucei rhodesiense diagnostic antigens by proteomic screening and targeted cloning

Abstract

Background: The only available diagnostic method for East African trypanosomiasis is light microscopy of blood samples. A simple immunodiagnostic would greatly aid trypanosomiasis control.

Methodology and principal findings: To find trypanosome proteins that are specifically recognised by sera from human sleeping sickness patients, we have screened the Trypanosoma brucei brucei proteome by Western blotting. Using cytosolic, cytoskeletal and glycosomal fractions, we found that the vast majority of abundant trypanosome proteins is not specifically recognised by patient sera. We identified phosphoglycerate kinase (PGKC), heat shock protein (HSP70), and histones H2B and H3 as possible candidate diagnostic antigens. These proteins, plus paraflagellar rod protein 1, rhodesain (a cysteine protease), and an extracellular fragment of the Trypanosoma brucei nucleoside transporter TbNT10, were expressed in E. coli and tested for reactivity with patient and control sera. Only TbHSP70 was preferentially recognized by patient sera, but the sensitivity and specificity were insufficient for use of TbHSP70 alone as a diagnostic. Immunoprecipitation using a native protein extract revealed no specifically reacting proteins.

Conclusions: No abundant T. brucei soluble, glycosomal or cytoskeletal protein is likely to be useful in diagnosis. To find useful diagnostic antigens it will therefore be necessary to use more sophisticated proteomic methods, or to test a very large panel of candidate proteins.

Figure 1. Interaction of human sera with trypanosome fractions.

Trypanosome extracts were analysed by Western blotting using sera from sleeping sickness patients (left) or controls (right) diluted at 1∶1000. Bands at the positions indicated were analysed by mass spectrometry; the identified protein is indicated. Serum sample numbers are beneath the lanes and marker positions (kD) are shown to the left. (A) Bloodstream-form cytosol. (B) Procyclic-form cytosol. (C) Procyclic-form detergent-soluble fraction. (D) Procyclic-form cytoskeleton fraction.

Figure 2. Interaction of human sera with recombinant T. brucei HSP70.

(A) T. brucei HSP70 was produced in E. coli as a fusion with glutathione-S-transferase (GST). Panel (A) shows the results using the serum batch 1, and panels (B) and (C) different pairs of Western blots for serum batch 2. (D) Reactivity of all sera against HSP70. The signal of the HSP70 band was measured, and background from immediately above and below the band was subtracted. In each experiment, 20 control sera and 20 infection sera were examined, and the average signal was calculated. The result for each serum was then calculated relative to the average. Each spot represents the average for 3 or 4 Western blots.