Temperature increase prevails over acidification in gene expression modulation of amastigote differentiation in Leishmania infantum

Abstract

Background: The extracellular promastigote and the intracellular amastigote stages alternate in the digenetic life cycle of the trypanosomatid parasite Leishmania. Amastigotes develop inside parasitophorous vacuoles of mammalian phagocytes, where they tolerate extreme environmental conditions. Temperature increase and pH decrease are crucial factors in the multifactorial differentiation process of promastigotes to amastigotes. Although expression profiling approaches for axenic, cell culture- and lesion-derived amastigotes have already been reported, the specific influence of temperature increase and acidification of the environment on developmental regulation of genes has not been previously studied. For the first time, we have used custom L. infantum genomic DNA microarrays to compare the isolated and the combined effects of both factors on the transcriptome.

Results: Immunofluorescence analysis of promastigote-specific glycoprotein gp46 and expression modulation analysis of the amastigote-specific A2 gene have revealed that concomitant exposure to temperature increase and acidification leads to amastigote-like forms. The temperature-induced gene expression profile in the absence of pH variation resembles the profile obtained under combined exposure to both factors unlike that obtained for exposure to acidification alone. In fact, the subsequent fold change-based global iterative hierarchical clustering analysis supports these findings.

Conclusions: The specific influence of temperature and pH on the differential regulation of genes described in this study and the evidence provided by clustering analysis is consistent with the predominant role of temperature increase over extracellular pH decrease in the amastigote differentiation process, which provides new insights into Leishmania physiology.

Figure 1

Average growth curves of control and temperature/pH-treated L. infantum promastigotes. Three replicates of the cultures were performed for each of the conditions assayed. RNA samples were extracted and processed for transcriptome analysis on day 4. Growth arrest is induced by pH decrease.

Figure 2

gp46 IFA. Samples of all the experimental conditions described in this article were collected on day 4 for IFA analysis. (A-D) CC; (E-H) TPS; (I-L) TS; and (M-P) PS. Incubations were performed with: PBS as negative control for the FITC-conjugated anti-mouse IgG secondary antibody (A, E, I, M); monoclonal anti-rabbit complement factor H primary antibody negative control (B, F, J, N); SIM110 monoclonal anti-SLA as positive control (C, G, K, O); and monoclonal anti-gp46 (D, H, L, P). As a summary, gp46 is expressed under CC, TS and PS but not in TPS-treated AL.

Figure 3

Multilevel sector charts of α-scores for GO molecular functions annotated on differentially regulated genes under TPS. (A) Molecular function GO terms annotated on down-regulated genes under TPS. (B) Molecular function GO terms annotated on up-regulated genes under TPS. (C) Biological process GO terms annotated on down-regulated genes under TPS. (D). Biological process GO terms annotated on up-regulated genes under TPS.

Figure 4

HCL-ST of genes differentially modulated under TPS, TS and/or PS. After performing SAM for all experimental groups, HCL-ST analysis was performed independently for (A) genes with (B) and without significant differences between groups according to SAM. Support Tree algorithm with a jackknifing resampling option and 100 iterations for the construction and clustering of gene expression matrix were applied in HCL-ST. Clones in (A) were grouped into 26 clusters and clones in (B) in two clusters depending on differential regulation. This analysis confirms that expression profile similarity is higher between TPS and TS than between TPS or TS and PS. Control spots LiA2, LdoA2, Lip36, Lipolb, Lihsp70, Ldohsp70 and Lmahsp70 show significant differences in gene expression between the experimental groups (A2 gene is up-regulated under TPS and hsp70 under PS) and LiTopoII, LiDNAg, Lamhsp70, LiGAPDH, LdoGAPDH and herring DNA do not. Clones with significant differences between the experimental conditions are identified in Additional file 6.

Figure 5

Scheme representing differentially regulated genes under TPS and their subcellular localisation and/or functional relations. Up-regulated genes are represented in red colour (Cy5) and down-regulated in green (Cy3). Further explanations in the TPS expression profile subsection, which is included in the Results and Discussion section.

Figure 6

Amino acid sequence and domain analysis of amastin genes found to be differentially regulated under TPS and TS. (A) MEV comparison of differential regulation under TPS, TS and PS. (B) Sequence similarity tree representing distances between amastin genes found to be up-regulated under TPS and TS. (C) Amino acid sequences were aligned with CLUSTALW2 software. The darker the position is highlighted the more conserved the residue is. The boundaries of inner, transmembrane and outer domain sequences predicted with TMHMM 2.0 software are represented below sequence alignments.