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Diversity of Protists I01:15

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Excavata is a diverse group of protists that includes both chemoorganotrophic and phototrophic species, with some thriving in anaerobic environments. Among the key groups within Excavata are diplomonads and parabasalids, which are flagellated protists that lack mitochondria and chloroplasts. These microorganisms typically inhabit anoxic environments, such as the intestines of animals, where they exist either symbiotically or as parasites, relying on fermentation for energy production. Some...

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High-throughput Gene Tagging in Trypanosoma brucei
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Published on: August 12, 2016

Cell density-dependent ectopic expression in bloodstream form Trypanosoma brucei.

Moazzam Ali1, Mark C Field

  • 1Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.

Experimental Parasitology
|March 30, 2013
PubMed
Summary

Ectopic protein expression in Trypanosoma brucei bloodstream forms decreases at high cell densities. This density-dependent reduction is linked to diffusible factors in conditioned media, impacting experimental quantification.

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Area of Science:

  • Molecular parasitology
  • Cell biology
  • Biochemistry

Background:

  • Ectopic protein expression is crucial for molecular parasitology and cell biology research.
  • Variability in protein expression levels was observed during studies of intracellular trafficking.

Purpose of the Study:

  • To systematically investigate the cause of highly variable ectopic protein expression in Trypanosoma brucei.
  • To determine the factors influencing protein expression levels in different cell densities and forms.

Main Methods:

  • Quantitative reverse transcription PCR (qRT-PCR)
  • Western blotting
  • Fluorescence microscopy
  • Culturing in conditioned media

Main Results:

  • Ectopic protein expression from a ribosomal promoter significantly diminished at high cell culture densities in bloodstream form Trypanosoma brucei.
  • This density-related reduction was observed across different cell lines, proteins, and vector backbones.
  • Procyclic form cells did not show detectable density-related expression changes.
  • Conditioned media experiments indicated a similar reduction at lower cell densities, suggesting a diffusible factor.

Conclusions:

  • A diffusible factor present in conditioned media influences ectopic protein expression in bloodstream form Trypanosoma brucei.
  • High cell density negatively impacts ectopic protein expression, with implications for accurate quantification in transgenic systems.