Flagellar cAMP signaling controls trypanosome progression through host tissues

Abstract

The unicellular parasite Trypanosoma brucei is transmitted between mammals by tsetse flies. Following the discovery that flagellar phosphodiesterase PDEB1 is required for trypanosomes to move in response to signals in vitro (social motility), we investigated its role in tsetse flies. Here we show that PDEB1 knockout parasites exhibit subtle changes in movement, reminiscent of bacterial chemotaxis mutants. Infecting flies with the knockout, followed by live confocal microscopy of fluorescent parasites within dual-labelled insect tissues, shows that PDEB1 is important for traversal of the peritrophic matrix, which separates the midgut lumen from the ectoperitrophic space. Without PDEB1, parasites are trapped in the lumen and cannot progress through the cycle. This demonstrates that the peritrophic matrix is a barrier that must be actively overcome and that the parasite’s flagellar cAMP signaling pathway facilitates this. Migration may depend on perception of chemotactic cues, which could stem from co-infecting parasites and/or the insect host.

A transient CRISPR/Cas9 expression system for genome editing in Trypanosoma brucei

“…The repair template, in the form of a purified PCR product, including homology arms of 30 bp on each side, was amplified from pPOTv7 [7]. As a positive control we transfected a derivative of T. brucei Lister 427 procyclic forms that already expresses Cas9 and T7RNAP constitutively (Lister 427/Cas9) [5]. This gave rise to cells with a fluorescent flagellum as expected (tryptag.org) ( Figure 2A).…”

“…We modified this plasmid by replacing the puromycin acetyltransferase gene with that of T7RNAP, giving rise to pTB011_Cas9_T7RNAP_blast ( Figure 1A). This plasmid allows generation of CRISPR/Cas9competent cell lines, constitutively expressing T7RNAP and Cas9 from the tubulin locus, in a single round of transfection [5]. Further modification of this plasmid, and optimisation of the transfection protocol for transient expression of the proteins and guide RNAs, are described below.…”

Evaluation of modulators of cAMP-response in terms of their impact on cell cycle and mitochondrial activity ofLeishmania donovani

“…Since etazolate apparently have noimpact of total cellular cAMP pool [5]), it is anticipated that anti-parasitic activity of etazolate is due to microdomain-specific modulation of cAMP response or by binding to some unidentified effector(s). Etazolate, primarily a PDE4 inhibitor [24,28], is a well-established drug of choice with no major side effects reported [28] in preclinical studies as well as pharmacokinetic and safety profiles in Phase I and Phase IIaclinical studies. Etazolate produced antidepressant like effects in animal models of depression and at the same time it could be used in the treatment of Alzheimer’s disease.…”

APEX2 proximity proteomics resolves flagellum subdomains and identifies flagellum tip-specific proteins inTrypanosoma brucei

“…One, previously identified as “cAMP response protein 3” (CARP3), is a candidate cAMP effector [59] and over half of the proteins are adenylate cyclases. A role for the flagellum tip in cAMP signaling has been previously demonstrated [10, 11] and flagellar cAMP signaling is required for the T. brucei transmission cycle in the tsetse fly [12]. Another protein group well-represented in the AC1s proximity proteome is calpain-like proteins.…”