Functional insights from a surface antigen mRNA-bound proteome

  1. Larissa Melo do Nascimento
  2. Franziska Egler
  3. Katharina Arnold
  4. Nina Papavasiliou
  5. Christine Clayton
  6. Esteban Erben  Is a corresponding author
  1. ZMBH, Germany
  2. Deutsche Krebsforschungszentrum (DKFZ), Germany
  3. DKFZ-ZMBH Alliance, Germany
  4. IIBIO - UNSAM, Argentina

Abstract

Trypanosoma brucei is the causative agent of human sleeping sickness. The parasites' Variant Surface Glycoprotein (VSG) enables them to evade adaptive immunity via antigenic variation. VSG comprises 10% of total cell protein and the high stability of VSG mRNA is essential for trypanosome survival. To determine how VSG mRNA stability is maintained, we used mRNA affinity purification to identify all its associated proteins. CFB2, an unconventional RNA-binding protein with an F-box domain, was specifically enriched with VSG mRNA. We demonstrate that CFB2 is essential for VSG mRNA stability, describe cis acting elements within the VSG 3'-untranslated region that regulate the interaction, identify trans-acting factors that are present in the VSG messenger ribonucleoprotein particle and mechanistically explain how CFB2 stabilizes the mRNA of this key pathogenicity factor. Beyond T. brucei, the mRNP purification approach has the potential to supply detailed biological insight into metabolism of relatively abundant mRNAs in any eukaryote.

Data availability

The RNASeq raw data is available at Array Express with the accession number E-MTAB-9700. The proteomics data are available via ProteomeXchange with identifier PXD021772.

The following data sets were generated

Article and author information

Author details

  1. Larissa Melo do Nascimento

    CC lab, ZMBH, Heidelberg, Germany
    Competing interests
    No competing interests declared.
  2. Franziska Egler

    CC lab, ZMBH, Heidelberg, Germany
    Competing interests
    No competing interests declared.
  3. Katharina Arnold

    CC lab, ZMBH, Heidelberg, Germany
    Competing interests
    No competing interests declared.
  4. Nina Papavasiliou

    Deutsche Krebsforschungszentrum (DKFZ), Heidelberg, Germany
    Competing interests
    No competing interests declared.
  5. Christine Clayton

    Zentrum fuer Molekulare Biologie der Universitaet Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany
    Competing interests
    Christine Clayton, Reviewing editor, eLife.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6384-0731
  6. Esteban Erben

    Molecular Parasitology, IIBIO - UNSAM, San Martín, Argentina
    For correspondence
    eerben@iib.unsam.edu.ar
    Competing interests
    No competing interests declared.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5179-7863

Funding

H2020 European Research Council (649019)

  • Nina Papavasiliou

Core Funding University of Heidelberg (ND)

  • Christine Clayton

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

© 2021, Melo do Nascimento et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 2,246
    views
  • 270
    downloads
  • 30
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Larissa Melo do Nascimento
  2. Franziska Egler
  3. Katharina Arnold
  4. Nina Papavasiliou
  5. Christine Clayton
  6. Esteban Erben
(2021)
Functional insights from a surface antigen mRNA-bound proteome
eLife 10:e68136.
https://doi.org/10.7554/eLife.68136

Share this article

https://doi.org/10.7554/eLife.68136

Further reading

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Ana Patrícia Graça, Vadim Nikitushkin ... Gerald Lackner
    Research Article

    Mycofactocin is a redox cofactor essential for the alcohol metabolism of mycobacteria. While the biosynthesis of mycofactocin is well established, the gene mftG, which encodes an oxidoreductase of the glucose-methanol-choline superfamily, remained functionally uncharacterized. Here, we show that MftG enzymes are almost exclusively found in genomes containing mycofactocin biosynthetic genes and are present in 75% of organisms harboring these genes. Gene deletion experiments in Mycolicibacterium smegmatis demonstrated a growth defect of the ∆mftG mutant on ethanol as a carbon source, accompanied by an arrest of cell division reminiscent of mild starvation. Investigation of carbon and cofactor metabolism implied a defect in mycofactocin reoxidation. Cell-free enzyme assays and respirometry using isolated cell membranes indicated that MftG acts as a mycofactocin dehydrogenase shuttling electrons toward the respiratory chain. Transcriptomics studies also indicated remodeling of redox metabolism to compensate for a shortage of redox equivalents. In conclusion, this work closes an important knowledge gap concerning the mycofactocin system and adds a new pathway to the intricate web of redox reactions governing the metabolism of mycobacteria.

    1. Microbiology and Infectious Disease
    Vandana Singh, Scot P Ouellette
    Research Article

    Chlamydia trachomatis is an obligate intracellular bacterial pathogen with a unique developmental cycle. It differentiates between two functional and morphological forms: the elementary body (EB) and the reticulate body (RB). The signals that trigger differentiation from one form to the other are unknown. EBs and RBs have distinctive characteristics that distinguish them, including their size, infectivity, proteome, and transcriptome. Intriguingly, they also differ in their overall redox status as EBs are oxidized and RBs are reduced. We hypothesize that alterations in redox may serve as a trigger for secondary differentiation. To test this, we examined the function of the primary antioxidant enzyme alkyl hydroperoxide reductase subunit C (AhpC), a well-known member of the peroxiredoxins family, in chlamydial growth and development. Based on our hypothesis, we predicted that altering the expression of ahpC would modulate chlamydial redox status and trigger earlier or delayed secondary differentiation. Therefore, we created ahpC overexpression and knockdown strains. During ahpC knockdown, ROS levels were elevated, and the bacteria were sensitive to a broad set of peroxide stresses. Interestingly, we observed increased expression of EB-associated genes and concurrent higher production of EBs at an earlier time in the developmental cycle, indicating earlier secondary differentiation occurs under elevated oxidation conditions. In contrast, overexpression of AhpC created a resistant phenotype against oxidizing agents and delayed secondary differentiation. Together, these results indicate that redox potential is a critical factor in developmental cycle progression. For the first time, our study provides a mechanism of chlamydial secondary differentiation dependent on redox status.