Proline Metabolism is Essential for Trypanosoma brucei brucei Survival in the Tsetse Vector

Abstract

Adaptation to different nutritional environments is essential for life cycle completion by all Trypanosoma brucei sub-species. In the tsetse fly vector, L-proline is among the most abundant amino acids and is mainly used by the fly for lactation and to fuel flight muscle. The procyclic (insect) stage of T. b. brucei uses L-proline as its main carbon source, relying on an efficient catabolic pathway to convert it to glutamate, and then to succinate, acetate and alanine as the main secreted end products. Here we investigated the essentiality of an undisrupted proline catabolic pathway in T. b. brucei by studying mitochondrial Δ1-pyrroline-5-carboxylate dehydrogenase (TbP5CDH), which catalyzes the irreversible conversion of gamma-glutamate semialdehyde (γGS) into L-glutamate and NADH. In addition, we provided evidence for the absence of a functional proline biosynthetic pathway. TbP5CDH expression is developmentally regulated in the insect stages of the parasite, but absent in bloodstream forms grown in vitro. RNAi down-regulation of TbP5CDH severely affected the growth of procyclic trypanosomes in vitro in the absence of glucose, and altered the metabolic flux when proline was the sole carbon source. Furthermore, TbP5CDH knocked-down cells exhibited alterations in the mitochondrial inner membrane potential (ΔΨm), respiratory control ratio and ATP production. Also, changes in the proline-glutamate oxidative capacity slightly affected the surface expression of the major surface glycoprotein EP-procyclin. In the tsetse, TbP5CDH knocked-down cells were impaired and thus unable to colonize the fly’s midgut, probably due to the lack of glucose between bloodmeals. Altogether, our data show that the regulated expression of the proline metabolism pathway in T. b. brucei allows this parasite to adapt to the nutritional environment of the tsetse midgut.

The zinc finger proteins ZC3H20 and ZC3H21 stabilise mRNAs encoding membrane proteins and mitochondrial proteins in insect‐form Trypanosoma brucei

“…When bloodstream forms reach high density, either in culture or in a mammal, they have a quorum sensing response (Rojas et al, ) and transform to nondividing stumpy form trypomastigotes. Upon uptake of the stumpy forms by Tsetse, or transfer to culture at 27°C in the presence of cis‐aconitate, stumpy forms develop into growing procyclic trypomastigotes, which rely mainly on mitochondrial pathways of ATP generation (Bringaud, Riviere, & Coustou, ; Mantilla et al, ). A protein called PAD1 (protein associated with differentiation), which is expressed by stumpy forms, is implicated in cis‐ aconitate sensing (Dean, Marchetti, Kirk, & Matthews, ).…”

The zinc finger proteins ZC3H20 and ZC3H21 stabilise mRNAs encoding membrane proteins and mitochondrial proteins in insect-formTrypanosoma brucei

“…When bloodstream forms reach high density, either in culture or in a mammal, they have a quorum sensing response (Rojas et al, 2018) and transform to non-dividing stumpy-form trypomastigotes. Upon uptake of the stumpy forms by Tsetse, or transfer to culture at 27°C in the presence of cis-aconitate, stumpy forms develop into growing procyclic trypomastigotes, which rely mainly on mitochondrial pathways of ATP generation (Mantilla et al, 2017, Bringaud et al, 2006. The trypanosomes later migrate via the proventriculus to the salivary glands, becoming first epimastigotes, then non-dividing metacyclic trypomastigotes which have variant surface glycoprotein and are infectious for mammals (Rotureau & Van Den Abbeele, 2013).…”

Characterisation of a putative glutamate 5‐kinase from Leishmania donovani

“…Metabolism of proline in leishmania has similarities and differences with its trypanosomatid cousins, the African trypanosome, Trypanosoma brucei and the South American trypanosome, Trypanosoma cruzi. While all three species are capable of using proline as an energy source, converting proline into glutamate and subsequently into tricarboxylic acid intermediates [6,18,34,35], metabolic labelling studies show that only leishmania is capable of synthesising proline de novo from either glutamate or glucose [14,18,34,36]. There is an absolute requirement for proline to support growth of T. brucei procyclic forms [18], whereas this is not the case for several Leishmania spp.…”