Elsevier

Journal of Proteomics

Volume 75, Issue 5, 16 February 2012, Pages 1463-1471
Journal of Proteomics

Comparative proteomics profiling of a gentamicin-attenuated Leishmania infantum cell line identifies key changes in parasite thiol-redox metabolism

https://doi.org/10.1016/j.jprot.2011.11.018Get rights and content

Abstract

We have previously described an attenuated line of Leishmania infantum (H-line), selected by culturing promastigotes in vitro in the presence of gentamicin. To elucidate the molecular basis for this attenuation, we undertook a comparative proteomic analysis using multiplex 2-dimensional (2D) difference gel electrophoresis. Eighteen proteins that showed significant and reproducible changes in expression were identified. Many of these were components of the thiol-redox control system in Leishmania and this observation, validated by Western blot, prompted us to investigate the sensitivity of the attenuated line to oxidative stress. The attenuated line was found to be significantly more susceptible to hydrogen peroxide, a change which may explain the loss of virulence. In a direct assay of trypanothione-dependent peroxidase activity, hydrogen peroxide metabolism in the H-line was significantly lower than in wild type. Furthermore, trypanothione reductase activity was significantly lower in the H-line, suggesting that gentamicin selection may result in pleiotropic affects on thiol metabolism in Leishmania. A putative RNA-binding protein was very strongly up-regulated in the attenuated line, suggesting a possible target for gentamicin in Leishmania.

Graphical abstract

Highlights

► Comparative proteomic analysis was applied to investigate changes in protein expression in an attenuated line of Leishmania infantum. ► 18 protein species were found to be significantly and reproducibly modulated. ► Several changes were in components of the thiol-redox control system. ► The attenuated line was shown to be more susceptible to oxidative stress.

Introduction

Leishmania infantum, the causative agent of visceral leishmaniasis in humans and dogs, is an obligate intracellular protozoan of mammalian macrophages. Control of visceral leishmaniasis in the dog can lead to a reduced prevalence of disease in associated human populations [1]. No effective vaccine is currently available against visceral leishmaniasis and control by chemotherapy is compromised because existing drugs are toxic and drug resistance is prevalent. We previously reported that a cultured attenuated line of L. infantum, identified as L. infantum H-line, was selected by culturing promastigotes in vitro under pressure of the aminoglycoside antibiotic gentamicin [2]. The attenuated line of L. infantum was phagocytosed by, but was unable to survive within, bone marrow-derived macrophages of BALB/c mice while the unselected L. infantum wild-type (WT) survived and multiplied within these macrophages [2]. The attenuated line failed to disseminate into visceral organs, whereas L. infantum WT spread into the visceral organs of infected BALB/c mice [3]. We have recently reported that L. infantum H-line induced no clinical signs and pathological abnormalities in dogs [3]. In addition, infection with H-line parasites elicited a Th1 response in these animals and protected against subsequent infection with wild-type parasites [4]. The attenuated line thus represents a potentially attractive route for the development of a vaccine.

One of the hurdles that must be overcome in order to realize this potential is to gain insight into the molecular changes that underpin selection of attenuated Leishmania under gentamicin pressure. Gentamicin, which is frequently added to in vitro cultures of Leishmania to control bacterial contamination [5], [6], is an aminoglycoside that interacts with RNA in prokaryotic cells [7]. The precise mechanism of bactericidal activity of aminoglycosides is not fully understood, but some hypotheses include disruption of ribosomal activity by breaking up polysomes, misreading of mRNA during protein synthesis and production of abnormal or nonfunctional proteins. Aminoglycosides are less effective against eukaryotic cells, and this selectivity may reflect divergence of the translation machinery between prokaryotes and eukaryotes. Nevertheless, aminoglycosides do exhibit antileishmanial activity and one such compound, paromomycin, has recently been approved for the treatment of human leishmaniasis [8], although the mechanism of action is not understood.

Gentamicin inhibits in vitro growth of Leishmania, albeit at a higher concentration than that required to limit bacterial growth (Burchmore, unpublished observation), and we have previously shown that passage of Leishmania in concentrations of gentamicin that marginally inhibit growth results in the selection of cells that are avirulent [2]. Our previous data indicate that this attenuation of virulence is stable, but that attenuated parasites are able to elicit a protective immune response [9].

In the present study, a comparative proteomic analysis of L. infantum H-line and L. infantum WT, showed that expression of a number of proteins was strongly and reproducibly modulated upon attenuation. Altered expression of the redox active enzyme tryparedoxin peroxidase was prominent. We have validated this proteomic difference by Western blotting and also demonstrated that the L. infantum H-line express reduced tryparedoxin dependent peroxidase activity. In addition, a very strong and reproducible down regulation of a putative component of RNase P [10] was observed, a known target of aminoglycosides in eukaryotes [11]. This protein may be a target for aminoglycosides such as gentamicin and paromomycin in Leishmania.

Section snippets

Parasites

Promastigotes of L. infantum JPCM5 (MCAN/ES/98/LIM-877) were cultivated in complete haemoflagellate minimal essential medium (HOMEM) (GIBCO) supplemented with 10% (vol/vol) heat-inactivated fetal calf serum (HI-FCS) (Labtech International) [12]. The attenuated line of L. infantum H-line was generated in the same medium supplemented with 10% (vol/vol) HI-FCS and 20 μg/mL gentamicin (Sigma) [2]. Briefly, amastigotes of L. infantum derived from the spleen of infected hamster were transferred into

Comparative analysis of 2D gels

Difference gel electrophoresis was used to highlight global changes in protein expression in an attenuated Leishmania line, derived from the wild type L. infantum strain for which genome sequence is available. Comparative analysis of 2D gels of L. infantum H-line and WT-line revealed a number of differentially expressed spots (Fig. 1). The application of multiplex DiGE technology with Decyder version 6 software (GE Healthcare) facilitated relative quantitation of matched spots in replicate gels

Discussion

We have previously reported the generation and immunological characterization of L. infantum, attenuated by selection with gentamicin. We aim to exploit this strain as a vaccine to control canine leishmaniasis and we therefore wished to gain insight into the molecular changes that underpin attenuation of Leishmania by gentamicin selection. Aminoglycoside antibiotics, including gentamicin, are known to target the 30S ribosomal subunit of bacteria, compromising the fidelity of protein translation

Acknowledgement

SW is funded by the Wellcome Trust (WT 079838).

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