High-throughput chemical screening for antivirulence developmental phenotypes in Trypanosoma brucei.
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.
Centre for Immunity, Infection and Evolution, Institute for Immunology and Infection Research, School of Biological Sciences, University of Edinburgh, Edinburgh, United Kingdom.
Paula
P
MacGregor
Alasdair
A
Ivens
Steven
S
Shave
Iain
I
Collie
David
D
Gray
Manfred
M
Auer
Keith R
KR
Matthews
D057166:High-Throughput Screening Assays; D010641:Phenotype; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D054852:Small Molecule Libraries / Q000494:pharmacology*; D014346:Trypanosoma brucei brucei / Q000187:drug effects / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D014774:Virulence / Q000235:genetics
D015800:Protozoan Proteins; D012333:RNA, Messenger; D054852:Small Molecule Libraries