Increased Abundance of Proteins Involved in Resistance to Oxidative and Nitrosative Stress at the Last Stages of Growth and Development of Leishmania amazonensis Promastigotes Revealed by Proteome Analysis

Abstract

Leishmania amazonensis is one of the major etiological agents of the neglected, stigmatizing disease termed american cutaneous leishmaniasis (ACL). ACL is a zoonosis and rodents are the main reservoirs. Most cases of ACL are reported in Brazil, Bolivia, Colombia and Peru. The biological cycle of the parasite is digenetic because sand fly vectors transmit the motile promastigote stage to the mammalian host dermis during blood meal intakes. The amastigote stage survives within phagocytes of the mammalian host. The purpose of this study is detection and identification of changes in protein abundance by 2DE/MALDI-TOF/TOF at the main growth phases of L. amazonensis promastigotes in axenic culture and the differentiation process that takes place simultaneously. The average number of proteins detected per gel is 202 and the non-redundant cumulative number is 339. Of those, 63 are differentially abundant throughout growth and simultaneous differentiation of L. amazonensis promastigotes. The main finding is that certain proteins involved in resistance to nitrosative and oxidative stress are more abundant at the last stages of growth and differentiation of cultured L. amazonensis promastigotes. These proteins are the arginase, a light variant of the tryparedoxin peroxidase, the iron superoxide dismutase, the regulatory subunit of the protein kinase A and a light HSP70 variant. These data taken together with the decrease of the stress-inducible protein 1 levels are additional evidence supporting the previously described pre-adaptative hypothesis, which consists of preparation in advance towards the amastigote stage.

Fig 1. Growth curve of L. amazonensis promastigotes.

Total protein samples were prepared and quantified at day 2 (early logarithmic phase), day 3 (mid logarithmic phase), day 5 (late logarithmic phase/early stationary phase) and day 7 (stationary phase).

Fig 2. Examples of 2DE of total protein extracts of L. amazonensis promastigotes.

(A) Early logarithmic (day 2) and (B) stationary phase (day 7). IEF was performed in a non-linear 3–10 pH interval. (C) 3D density graphs of spots Lam2801, Lam3501, Lam6102 and Lam7004 obtained with PD Quest software.

Fig 3. Differential abundance of proteins involved in L. amazonensis metabolism.

Constitutively expressed and differentially regulated proteins that participate in glycolysis, the Krebs cycle, amino acid metabolism and β-oxidation of fatty acids are depicted. Colour legend for up-regulation: green, day 2 (lag/early log); blue, day 3 (log); orange, day 5 (late log); red, day 7 (stat). The constitutively expressed proteins experimentally detected throughout the promastigote growth curve are highlighted in violet. Abbreviations not detailed in the text: ALD, fructose-1,6-bisphosphate aldolase; ECH, enoyl-CoA hydratase/isomerase; FMR, fumarase; MET6, methyltetrahydropteroyltriglutamate-homocysteine methyltransferase; MDH, malate dehydrogenase; PGK, phosphoglycerate kinase; PK, pyruvate kinase; SDH, succinate dehydrogenase.

Fig 4. Differential abundance of proteins involved in redox homeostasis.

The FE-SOD participates in ROS processing providing hydrogen peroxide, which is then reduced by the TryP with the assistance of the TXN1, trypanithione and TryR. TryP differential expression pattern is complex through the promastigote growth curve of L. amazonensis. A light TryP variant is up-regulated in stationary phase promastigotes. A heavier TryP variant is down-regulated at day 3 and another one up-regulated simultaneously. Finally, a fourth variant is constitutively expressed during promastigote growth. Colour legend for up-regulation: green, day 2 (lag/early log); blue, day 3 (log); orange, day 5 (late log); red, day 7 (stat). The constitutively expressed proteins experimentally detected throughout the promastigote growth curve are highlighted in violet.

Fig 5. Resistance to NO of logarithmic and stationary phase promastigotes of L. amazonensis.

The MTT assay was performed to compare the ability to resist increasing concentrations of NaNO₂ by logarithmic and stationary phase promastigotes of L. amazonensis. Three biological replicates of the experiment were performed and the differences, contrasted by the paired t-test, were significant at all NaNO₂ concentrations tested (p*<0.001).