Dataset Information

Proteomic analysis of the bloodstream forms (slender, stumpy-QS, stumpy-Glyc, intermediate-Glyc) and procyclic forms of Trypanosoma brucei.

ABSTRACT: In the mammalian bloodstream, proliferative slender forms of T. brucei differentiate into cell cycle-arrested stumpy forms (stumpy-QS) via a quorum sensing mechanism triggered by high parasite density. This transition is thought to protect the host from excessive parasitaemia while preparing the parasites for transmission to the tsetse fly. In this study, we demonstrated that glycerol can also induce the differentiation of slender forms into stumpy forms (stumpy-Glyc), mimicking quorum sensing-induced stumpy forms (stumpy-QS), but with an extended lifespan. Moreover, under tissue-like conditions mimicking the interstitial fluids of adipocytes in the skin (4 mM glucose, 0.25 mM glycerol), glycerol generated proliferative intermediate forms (intermediate-Glyc) capable of differentiating into procyclic forms and establishing infections in tsetse flies. Notably, glycerol also prolonged the survival of quorum-sensing-induced stumpy forms by delaying cell death. Our findings suggest a revised model for T. brucei transmission where quorum sensing controls parasitaemia by producing short-lived stumpy-QS forms, while glycerol secreted by adipocytes in the skin supports the formation of transmissible intermediate-Glyc forms and extends the lifespan of stumpy forms, thus enhancing parasite transmission to tsetse flies.

INSTRUMENT(S):

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Dupuy Jean-William

LAB HEAD: Frédéric Bringaud

PROVIDER: PXD056405 | Pride | 2026-06-23

REPOSITORIES: Pride

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Dataset's files

Source:

			Action	DRS
	Link_between_files.xlsx	Xlsx
	TriTrypDB-65_TbruceiTREU927_AnnotatedProteins.fasta	Fasta
	checksum.txt	Txt
	fb230322_01.msf	Msf
	fb230322_01.pdResult	Other

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Project description:The protozoan parasites Trypanosoma brucei spp. are responsible for important human and livestock diseases in sub Saharan Africa. In the mammalian blood, two developmental forms of the parasite exist: proliferative ?slender? forms and transmissible ?stumpy? forms that are quiescent, awaiting uptake in a tsetse fly bloodmeal. The slender to stumpy differentiation is a density-dependent response that resembles quorum sensing in microbial systems and is crucial for the parasite life cycle, ensuring both infection chronicity and disease transmission. The response is triggered by an elusive ?stumpy induction factor? (SIF) whose intracellular signaling pathway is also completely uncharacterized. Laboratory-adapted (monomorphic) trypanosome strains cannot respond to SIF, but can generate forms with stumpy characteristics when exposed to cell permeable cAMP and AMP analogues. Exploiting this, we have used a genome-wide RNAi library screen to identify the signaling components driving stumpy formation. In separate screens, monomorphic parasites were exposed to cell permeable cAMP or AMP analogues to select cells that remained proliferative and so were unresponsive to these signals. Genome-wide ion torrent-based RNA interference Target sequencing (RIT-seq) identified a cohort of genes implicated in all steps of the signaling pathway, from purine metabolism, through signal transducers (kinases, phosphatases) to gene expression regulators. The identified genes at each step have been validated in cells naturally capable of stumpy formation, confirming their role in SIF-induced density sensing and cellular quiescence. RNA was isolated from AnTat1.1 cells grown in mice with or without doxycycline. RBP7 (Tb927.10.12100) RNAi lines were grown in mice with or without doxycycline. Since the RBP7 RNAi is leaky (six independent cell lines were analysed and all were significantly leaky), transcripts that showed elevated or reduced abundance in both RNAi induced and uninduced populations were identified in comparison to the AnTat1.1 control. A similar approach was adopted for the RBP7 over-expressing lines.

Project description:Introduction: Pantoea stewartii subsp. stewartii is a proteobacterium that causes Stewart’s wilt disease in corn plants. The bacteria form a biofilm in the xylem of infected plants and produce capsule that blocks water transport, eventually causing wilt. At low cell densities, the quorum-sensing (QS) regulatory protein EsaR is known to directly repress expression of esaR itself as well as the capsular synthesis operon transcription regulator, rcsA, and a 2,5-diketogluconate reductase, dkgA. It simultaneously directly activates expression of genes for a putative small RNA, esaS, the glycerol utilization operon, glpFKX, and another transcriptional regulator, lrhA. At high bacterial cell densities, all of this regulation is relieved when EsaR binds an acylated homoserine lactone signal, which is synthesized constitutively over growth. QS-dependent gene expression is critical for the establishment of disease in the plant. Objective: However, the identity of the full set of genes controlled by EsaR/QS is unknown. A proteomic approach previously identified around 30 proteins in the QS regulon. In this study, a whole-transcriptome, next-generation sequencing analysis of rRNA-depleted RNA from QS-proficient and -deficient P. stewartii strains was performed to identify additional targets of EsaR. Result: EsaR-dependent transcriptional regulation of a subset of differentially expressed genes was confirmed by quantitative reverse transcription-PCR (qRT-PCR). Electrophoretic mobility shift assays demonstrated that EsaR directly bound 10 newly identified target promoters. Overall, the QS regulon of P. stewartii orchestrates three major physiological responses: capsule and cell envelope biosynthesis, surface motility and adhesion, and stress response. Comparison of gene expression in two strains of Pantoea stewartii, QS-proficient DC283 ES∆IR (pSVB60) and QS-deficient DC283 ES∆IR (pBBR1MCS-3) to identify those genes that are directly regulated by the master Quorum-sensing regulator, EsaR.

Dataset Information

Proteomic analysis of the bloodstream forms (slender, stumpy-QS, stumpy-Glyc, intermediate-Glyc) and procyclic forms of Trypanosoma brucei.

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