Macromolecular biosynthetic parameters and metabolic profile in different life stages of Leishmania braziliensis: Amastigotes as a functionally less active stage.

Marlene Jara,Jorge Arevalo,Guy Caljon,Ilse Maes,Jean-Claude Dujardin,Maya Berg,Bart Cuypers,Manu Vanaerschot,Geraldine De Muylder,María Del Carmen Orozco,Denis Castillo,Vyacheslav Yurchenko

doi:10.1371/journal.pone.0180532

Abstract

It was recently hypothesized that Leishmania amastigotes could constitute a semi-quiescent stage characterized by low replication and reduced metabolic activity. This concept developed with Leishmania (Leishmania) mexicana and Leishmania (Leishmania) major models might explain numerous clinical and sub-clinical features of Leishmania (Viannia) braziliensis infections, like reactivation of the disease, non-response to chemotherapy or asymptomatic infections. We compared here in vitro the proliferative capability of L. (V.) braziliensis amastigotes and promastigotes, assessed the expression of key molecular parameters and performed metabolomic analysis. We found that contrary to the highly proliferative promastigotes, amastigotes (axenic and intracellular) do not show evidence of extensive proliferation. In parallel, amastigotes showed a significant decrease of (i) the kDNA mini-circle abundance, (ii) the intracellular ATP level, (iii) the ribosomal components: rRNA subunits 18S and 28S α and ribosomal proteins RPS15 and RPL19, (iv) total RNA and protein levels. An untargeted metabolomic study identified clear differences between the different life stages: in comparison to logarithmic promastigotes, axenic amastigotes showed (a) a strong decrease of 14 essential and non-essential amino acids and eight metabolites involved in polyamine synthesis, (b) extensive changes in the phospholipids composition and (c) increased levels of several endogenous and exogenous sterols. Altogether, our results show that L. (V.) braziliensis amastigotes can show a phenotype with negligible rate of proliferation, a lower capacity of biosynthesis, a reduced bio-energetic level and a strongly altered metabolism. Our results pave the way for further exploration of quiescence among amastigotes of this species.

Highlights

Leishmania is a Protozoan parasite with a life cycle involving invertebrate and vertebrate hosts: extracellular promastigotes proliferate in the sand fly mid-gut, while intracellular amastigotes are adapted to live within the phagolysosomes of mammalian macrophages [1]
Our results show that in vitro, L. (V.) braziliensis amastigotes can show a phenotype with negligible rate of proliferation, a lower capacity of biosynthesis, a reduced bio-energetic level and a strongly altered metabolism
Promastigotes of the reference strain M2904 reached their stationary phase on the fourth day with a density of 52.3 x 106 ± 5.0 parasites/mL (Mean ± standard errors of the means (SEM)) of culture (Fig 1A)

Summary

Introduction

Leishmania is a Protozoan parasite with a life cycle involving invertebrate and vertebrate hosts: extracellular promastigotes proliferate in the sand fly mid-gut, while intracellular amastigotes are adapted to live within the phagolysosomes of mammalian macrophages [1]. Chronic persistence of parasites is a striking feature of Leishmania infections This is among others illustrated by the presence of viable amastigotes in scars of CL lesions after therapy or even years after clinical cure [3,4,5,6] that eventually could produce reactivation of the lesion [7]. A recent report on Leishmania (Leishmania) major showed that the picture could be even more complex, with the co-existence of ‘fast’ (60 hours doubling time) and low/non-replicating amastigotes in infected mice [10] but the authors could not assess the metabolic status of these different types of parasites

Methods

Results

Conclusion