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ABSTRACT The African trypanosome has evolved mechanisms to adapt to changes in nutrient availability that occur during its life cycle. During transition from mammalian blood to insect vector gut, parasites experience a rapid reduction in environmental glucose. Here we describe how pleomorphic parasites respond to glucose depletion with a focus on parasite changes in energy metabolism and growth. Long slender bloodstream form parasites were rapidly killed as glucose concentrations fell, while short stumpy bloodstream form parasites persisted to differentiate into the insect-stage procyclic form parasite. The rate of differentiation was lower than that triggered by other cues but reached physiological rates when combined with cold shock. Both differentiation and growth of resulting procyclic form parasites were inhibited by glucose and nonmetabolizable glucose analogs, and these parasites were found to have upregulated amino acid metabolic pathway component gene expression. In summary, glucose transitions from the primary metabolite of the blood-stage infection to a negative regulator of cell development and growth in the insect vector, suggesting that the hexose is not only a key metabolic agent but also an important signaling molecule. IMPORTANCE As the African trypanosome Trypanosoma brucei completes its life cycle, it encounters many different environments. Adaptation to these environments includes modulation of metabolic pathways to parallel the availability of nutrients. Here, we describe how the blood-dwelling life cycle stages of the African trypanosome, which consume glucose to meet their nutritional needs, respond differently to culture in the near absence of glucose. The proliferative long slender parasites rapidly die, while the nondividing short stumpy parasite remains viable and undergoes differentiation to the next life cycle stage, the procyclic form parasite. Interestingly, a sugar analog that cannot be used as an energy source inhibited the process. Furthermore, the growth of procyclic form parasite that resulted from the event was inhibited by glucose, a behavior that is similar to that of parasites isolated from tsetse flies. Our findings suggest that glucose sensing serves as an important modulator of nutrient adaptation in the parasite.
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Genomic Occupancy of the Bromodomain Protein Bdf3 Is Dynamic during Differentiation of African Trypanosomes from Bloodstream to Procyclic Forms
ABSTRACT Trypanosoma brucei , the causative agent of human and animal African trypanosomiasis, cycles between a mammalian host and a tsetse fly vector. The parasite undergoes huge changes in morphology and metabolism during adaptation to each host environment. These changes are reflected in the different transcriptomes of parasites living in each host. However, it remains unclear whether chromatin-interacting proteins help mediate these changes. Bromodomain proteins localize to transcription start sites in bloodstream parasites, but whether the localization of bromodomain proteins changes as parasites differentiate from bloodstream to insect stages remains unknown. To address this question, we performed cleavage under target and release using nuclease (CUT&RUN) against bromodomain protein 3 (Bdf3) in parasites differentiating from bloodstream to insect forms. We found that Bdf3 occupancy at most loci increased at 3 h following onset of differentiation and decreased thereafter. A number of sites with increased bromodomain protein occupancy lie proximal to genes with altered transcript levels during differentiation, such as procyclins, procyclin-associated genes, and invariant surface glycoproteins. Most Bdf3-occupied sites are observed throughout differentiation. However, one site appears de novo during differentiation and lies proximal to the procyclin gene locus housing genes essential for remodeling surface proteins following transition to the insect stage. These studies indicate that occupancy of chromatin-interacting proteins is dynamic during life cycle stage transitions and provide the groundwork for future studies on the effects of changes in bromodomain protein occupancy. Additionally, the adaptation of CUT&RUN for Trypanosoma brucei provides other researchers with an alternative to chromatin immunoprecipitation (ChIP). IMPORTANCE The parasite Trypanosoma brucei is the causative agent of human and animal African trypanosomiasis (sleeping sickness). Trypanosomiasis, which affects humans and cattle, is fatal if untreated. Existing drugs have significant side effects. Thus, these parasites impose a significant human and economic burden in sub-Saharan Africa, where trypanosomiasis is endemic. T. brucei cycles between the mammalian host and a tsetse fly vector, and parasites undergo huge changes in morphology and metabolism to adapt to different hosts. Here, we show that DNA-interacting bromodomain protein 3 (Bdf3) shows changes in occupancy at its binding sites as parasites transition from the bloodstream to the insect stage. Additionally, a new binding site appears near the locus responsible for remodeling of parasite surface proteins during transition to the insect stage. Understanding the mechanisms behind host adaptation is important for understanding the life cycle of the parasite.
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- Award ID(s):
- 2041395
- PAR ID:
- 10408554
- Editor(s):
- Phillips, Margaret
- Date Published:
- Journal Name:
- mSphere
- Volume:
- 7
- Issue:
- 3
- ISSN:
- 2379-5042
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Kinetoplastid pathogens includingTrypanosoma brucei,T. cruzi, andLeishmaniaspecies, are early diverged, eukaryotic, unicellular parasites. Functional understanding of many proteins from these pathogens has been hampered by limited sequence homology to proteins from other model organisms. Here we describe the development of a high-throughput deep mutational scanning approach inT. bruceithat facilitates rapid and unbiased assessment of the impacts of many possible amino acid substitutions within a protein on cell fitness, as measured by relative cell growth. The approach leverages several molecular technologies: cells with conditional expression of a wild-type gene of interest and constitutive expression of a library of mutant variants, degron-controlled stabilization of I-SceI meganuclease to mediate highly efficient transfection of a mutant allele library, and a high-throughput sequencing readout for cell growth upon conditional knockdown of wild-type gene expression and exclusive expression of mutant variants. Using this method, we queried the effects of amino acid substitutions in the apparently non-catalytic RNase III-like domain of KREPB4 (B4), which is an essential component of the RNA Editing Catalytic Complexes (RECCs) that carry out mitochondrial RNA editing inT. brucei. We measured the impacts of thousands of B4 variants on bloodstream form cell growth and validated the most deleterious variants containing single amino acid substitutions. Crucially, there was no correlation between phenotypes and amino acid conservation, demonstrating the greater power of this method over traditional sequence homology searching to identify functional residues. The bloodstream form cell growth phenotypes were combined with structural modeling, RECC protein proximity data, and analysis of selected substitutions in procyclic formT. brucei. These analyses revealed that the B4 RNaseIII-like domain is essential for maintenance of RECC integrity and RECC protein abundances and is also involved in changes in RECCs that occur between bloodstream and procyclic form life cycle stages.more » « less
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/msphere.00023-22
