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Functional insights from a surface antigen mRNA-bound proteome.

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 (cyclin F-box protein 2), 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.

SUBMITTER: Melo do Nascimento L

PROVIDER: S-EPMC8051951 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Surface proteins of the obligate intracellular bacterium Rickettsia typhi, the agent of murine or endemic typhus fever, comprise an important interface for host-pathogen interactions including adherence, invasion and survival in the host cytoplasm. In this report, we present analyses of the surface exposed proteins of R. typhi based on a suite of predictive algorithms complemented by experimental surface-labeling with thiol-cleavable sulfo-NHS-SS-biotin and identification of labeled peptides by LC MS/MS. Further, we focus on proteins belonging to the surface cell antigen (Sca) autotransporter (AT) family which are known to be involved in rickettsial infection of mammalian cells. Each species of Rickettsia has a different complement of sca genes in various states; R. typhi, has genes sca1 thru sca5. In silico analyses indicate divergence of the Sca paralogs across the four Rickettsia groups and concur with previous evidence of positive selection. Transcripts for each sca were detected during infection of L929 cells and four of the five Sca proteins were detected in the surface proteome analysis. We observed that each R. typhi Sca protein is expressed during in vitro infections and selected Sca proteins were expressed during in vivo infections. Using biotin-affinity pull down assays, negative staining electron microscopy, and flow cytometry, we demonstrate that the Sca proteins in R. typhi are localized to the surface of the bacteria. All Scas were detected during infection of L929 cells by immunogold electron microscopy. Immunofluorescence assays demonstrate that Scas 1-3 and 5 are expressed in the spleens of infected Sprague-Dawley rats and Scas 3, 4 and 5 are expressed in cat fleas (Ctenocephalides felis). Sca proteins may be crucial in the recognition and invasion of different host cell types. In short, continuous expression of all Scas may ensure that rickettsiae are primed i) to infect mammalian cells should the flea bite a host, ii) to remain infectious when extracellular and iii) to infect the flea midgut when ingested with a blood meal. Each Sca protein may be important for survival of R. typhi and the lack of host restricted expression may indicate a strategy of preparedness for infection of a new host.

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Functional insights from a surface antigen mRNA-bound proteome.

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