Analysis of the Leishmania donovani transcriptome reveals an ordered progression of transient and permanent changes in gene expression during differentiation

Abstract

Leishmania donovani is an intracellular protozoan parasite that causes kala-azar in humans. During infection the extracellular insect forms (promastigotes) undergo rapid differentiation to intracellular amastigotes that proliferates in phagolysosomes of mammalian macrophages. We used microarray-based expression profiling to investigate the time-course of changes in RNA abundance during promastigote-to-amastigote differentiation in a host-free system that mimics this process. These studies revealed that several hundred genes underwent an ordered progression of transient or permanent up- and down-regulation during differentiation. Genes that were permanently up-regulated in amastigotes were enriched for transporters and surface proteins, but under-represented in genes involved in protein and other metabolism. Most of these changes occurred late in the differentiation process, when morphological differentiation was essentially complete. Down-regulated genes were over-represented in those involved in cell motility, growth and/or maintenance, and these changes generally occurred earlier in the process. Genes that were transiently up- or down-regulated during differentiation included those encoding heat shock proteins, ubiquitin hydrolases, RNA binding proteins, protein kinases, a protein phosphatase, and a histone deacetylase. These results suggest that changes in mRNA abundance may be important in signal transduction, as well as protein and mRNA turnover, during differentiation. In addition to these mRNA changes, other transcripts including one or more rRNAs and snoRNAs, and non-coding RNAs from several telomeres, also showed substantial changes in abundance during the differentiation process. This paper provides the first genome-scale quantitative analysis of gene expression during the transition from promastigotes to amastigotes and demonstrates the utility of the host-free differentiation system.

Figure 1

Pair-wise comparison of signal intensity on DNA microarrays hybridized with RNA from Leishmania donovani at different times after exposure to the differentiation signal. Each panel shows a log₁₀ plot of Cy3 (532 nm) vs. Cy5 (635 nm) calibrated fluorescent response from representative hybridizations for (A) Cy3- vs. Cy5-labeled promastigotes (P) immediately before exposure to the differentiation signal, (B) promastigotes vs. parasites 5 hr after exposure to the differentiation signal, (C) promastigotes vs. 10 hr, (D) promastigotes vs. 24 hr and (E) promastigotes vs. axenic amastigotes (A).

Figure 2

Patterns of gene expression changes during differentiation. (A) The mean fold-change in signal at each time-point during differentiation (relative to promastigotes) was averaged for the 9078 GSS elements with data from at least three time points and the standard deviation (y-axis) plotted against the cumulative number of GSSs (x-axis). (B) The 907 GSS showing the greatest standard deviation were clustered using the TMeV software package. The columns (from left to right) represent promastigotes (P), the 5, 10, 24 hr time-points after exposure to the differentiation signal and amastigotes (A), while the rows represent the fold-change values for each gene. Up-regulation is represented by red, down-regulation is represented by green and no change is represented by black. Genes showing statistically significant (by panel data or 2-group analysis) differences from promastigotes at one or more time-points are indicated by a blue line in the rightmost column. Clustered genes are indicated by the blue triangles to the left. (C) Expression graphs for each of the 12 clusters from panel B, plotting the log₂ fold-change values (y-axis) at each of the five time-points (x-axis). The average for all genes in each cluster is shown by the magenta line. Orange and yellow lines indicate GSS elements containing TAS and THR sequences, green lines indicate GSSs containing snoRNA genes, and blue lines indicate GSSs containing kDNA sequence.

Figure 3

Northern analysis of differentially expressed genes. (A) RNA (10 μg in each lane) from metacyclic promastigotes maintained in stationary phase for 6 (6s) or 4 (4s) days; procyclic promastigotes in late-log phase (P), and 1, 5 and 24 hr after exposure to differentiation signal were hybridized with [³²P]-labeled probe for SHERP1 (top panel). rRNA was visualized by ethidium bromide staining (bottom panel). (B) RNA from parasites at immediately before exposure to the differentiation signal (P), as well as 5, 10 and 24 hr after exposure, and axenic amastigotes (A) were hybridized with probes for LmjF34.0070 (top panel I), LmjF35.1200 (panel II) LmjF17.1090 (panel III). rRNAs are shown in the bottom panel. The fold-change values from the microarray results (μA) are indicated below each panel.