High-Throughput Chemical Screening for Antivirulence Developmental Phenotypes in Trypanosoma brucei

Paula Macgregor,Manfred Auer,David Gray,Steven Shave,Alasdair Ivens,Keith R Matthews,Iain Collie

doi:10.1128/ec.00335-13

Abstract

In the bloodstream of mammalian hosts, the sleeping sickness parasite, Trypanosoma brucei, exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form. The transition between these developmental forms is controlled by a density-dependent mechanism that is important for the parasite's infection dynamics, immune evasion via ordered antigenic variation, and disease transmissibility. However, stumpy formation has been lost in most laboratory-adapted trypanosome lines, generating monomorphic parasites that proliferate uncontrolled as slender forms in vitro and in vivo. Nonetheless, these forms are readily amenable to cell culture and high-throughput screening for trypanocidal lead compounds. Here, we have developed and exploited a high-throughput screen for developmental phenotypes using a transgenic monomorphic cell line expressing a reporter under the regulation of gene control signals from the stumpy-specific molecule PAD1. Using a whole-cell fluorescence-based assay to screen over 6,000 small molecules from a kinase-focused compound library, small molecules able to activate stumpy-specific gene expression and proliferation arrest were assayed in a rapid assay format. Independent follow-up validation identified one hit able to induce modest, yet specific, changes in mRNA expression indicative of a partial differentiation to stumpy forms in monomorphs. Further, in pleomorphs this compound induced a stumpy-like phenotype, entailing growth arrest, morphological changes, PAD1 expression, and enhanced differentiation to procyclic forms. This not only provides a potential tool compound for the further understanding of stumpy formation but also demonstrates the use of high-throughput screening in the identification of compounds able to induce specific phenotypes, such as differentiation, in African trypanosomes.

Highlights

In the bloodstream of mammalian hosts, the sleeping sickness parasite, Trypanosoma brucei, exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form
In the bloodstream of their mammalian host, African trypanosomes differentiate from the proliferative slender form to the transmissible stumpy form [15], a transition that is key to the within-host infection dynamics and transmissibility of the parasite [16]
We have previously demonstrated that transgenic cell lines that utilize reporter genes [12] coupled to the 3= untranslated region (UTR) of the PAD1 gene (Tb927.7.5930), a functional molecular marker for stumpy forms [23], can report on the response of monomorphic slender cells to conditions that promote the production of stumpy-like forms [16]

Summary

Introduction

In the bloodstream of mammalian hosts, the sleeping sickness parasite, Trypanosoma brucei, exists as a proliferative slender form or a nonproliferative, transmissible, stumpy form. This assay has been used to screen over 6,000 kinase-focused inhibitors for their ability to induce PAD1 3= UTR-regulated reporter expression as a proxy for the induction of stumpy formation This led to the identification of two structurally similar compounds that caused an unspecific increase in reporter expression as well as one chemically distinct compound that caused an upregulation of PAD1 mRNA expression and generated consistent changes in mRNA expression for a small set of genes that are representative of partial stumpy formation in monomorphs and that generate a stumpy-like phenotype in pleomorphic cell lines. This demonstrates the validity of a reporter screen for stumpy formation and of the use of high-throughput screening for the identification of small compounds that induce the cytocidal or cytostatic outcomes routinely analyzed and specific phenotypic responses useful for the molecular analysis of trypanosome biology

Methods

Results

Conclusion