Minor temperature shifts do not affect chromosomal ploidy but cause transcriptomic changes in Leishmania braziliensis promastigotes in vitro

Abstract

Background: The leishmaniases are complex neglected diseases caused by protozoan parasites of the genus Leishmania. Leishmania braziliensis is the main etiological agent of cutaneous leishmaniasis in the New World. In recent studies, genomic changes such as chromosome and gene copy number variations (CNVs), as well as transcriptomic changes have been highlighted as mechanisms used by Leishmania species to adapt to stress situations.

Objectives: The aim of this study was to determine the effect of short-term minor temperature shifts in the genomic and transcriptomic responses of L. braziliensis promastigotes in vitro.

Methods: Growth curves, genome and transcriptome sequencing of L. braziliensis promastigotes were conducted from cultures exposed to three different temperatures (24ºC, 28ºC and 30ºC) compared with the control temperature (26ºC).

Findings: Our results showed a decrease in L. braziliensis proliferation at 30ºC, with around 3% of the genes showing CNVs at each temperature, and transcriptomic changes in genes encoding amastin surface-like proteins, heat shock proteins and transport proteins, which may indicate a direct response to temperature stress.

Main conclusions: This study provides evidence that L. braziliensis promastigotes exhibit a decrease in cell density, and noticeable changes in the transcriptomic profiles. However, there were not perceptible changes at chromosome CNVs and only ~3% of the genes changed their copies in each treatment.

Fig. 1:. effect of temperature on the growth curve of Leishmania braziliensis promastigotes. Parasites were cultured at four different temperatures: 26ºC (orange), as the control temperature; 24ºC (light blue); 28ºC (light green); and 30ºC (yellow). The beginning of the logarithmic phase (BLP) for each treatment is indicated. Parasite growth was monitored daily for seven days through a Neubauer chamber. Bars represent standard errors obtained from three independent experiments. The cultures had an initial concentration of 1 × 10⁶ parasites/mL. The days of quantification are represented on the x-axis and the concentration of parasites per mL is expressed in a logarithmic scale on the y-axis.

Fig. 2:. DNA-based and RNA-based ploidy values. (A) The DNA-based heatmap shows the ploidy of each of the 35 chromosomes calculated through the results obtained by high-throughput DNA sequencing. Each sample is indicated by the letters Lb (abbreviation for Leishmania braziliensis) and the temperature assessed. (B) The RNA-based heatmap shows the ploidy of each of the 35 chromosomes calculated through the results obtained by high-throughput RNA sequencing. Four replicates (two biological and two technical) are included and this is indicated between the letters Lb (abbreviation for L. braziliensis) and the last number (the temperature assessed); for example, Lb_1_30 is the first replicate at a temperature of 30ºC. The colour key indicates the chromosome ploidy value (p), which ranges from 1 to 5, and for which haploid is when p < 1.5, diploid 1.5 ≤ p < 2.5, triploid 2.5 ≤ p < 3.5, tetraploid 3.5 ≤ p < 4.5 and pentaploid 4.5 ≤ p < 5, as previously described. Both heatmaps include the three assessed temperatures along with the control temperature.

Fig. 3:. (A) Heatmap representing the number of genes per chromosome with copy number variations (CNVs) compared with the control. The coloured box indicates the ranges of genes with CNVs. The number below the heatmap indicates the temperature tested and the numbers on the right indicate each chromosome. (B) Venn diagram shows the number of genes with CNVs compared with the control and the number of overlapping genes of Leishmania braziliensis under different temperatures: 24ºC (light blue), 28ºC (light green) and 30ºC (yellow), with a fold change of Z score > 2 compared with the copy number at the control temperature (26ºC). The numbers in brackets are the total number of genes with CNVs at each temperature.

Fig. 4:. percentage of genes per ontology term for each temperature based on the DNA sequencing results. Unique genes with a fold-change in the copy number of 2 compared with the control (at 26ºC) (control temperature) were evaluated for each temperature: 24ºC (102 genes), 28ºC (92 genes) and 30ºC (114 genes), to obtain the ontology terms. Only the first 10 terms were used for each temperature. We calculated the percentage considering the total number of genes with CNVs for each case and determined the decreased and increased copy numbers. (A) The ontology of the increased read depth compared with the control. (B) The ontology of the decreased read depth compared with the control. The x-axis corresponds to the percentage of genes classified for an ontology term (the percentage was calculated from the total number of genes in the category of temperature and the increased or decreased read depth).

Fig. 5:. read depth of genes compared with those at the control temperature based on the DNA sequencing results. The ontology classifications of these genes are illustrated in Fig. 4. The genes with increased read depth compared with the control at each temperature are presented in the upper three graphs in the figure (A, C, E), while the genes with a decreased read count are shown in the lower three graphs in the figure (B, D, F). Joined genes are shown in the Supplementary data (Figs 2, 3).

Fig. 6:. MA plots representing the differentially expressed genes (DEGs) of Leishmania braziliensis promastigotes under each treatment, (A) at 24ºC, (B), 28ºC and (C) 30ºC. The up- and downregulated DEGs are highlighted in red and the genes with steady-state levels of RNA are represented by grey dots. The dotted black lines represent the cut-off of the fold change (log fold change > 1 and < ˗1).

Fig. 7:. venn diagrams and gene ontology terms of the differentially expressed genes (DEGs). Illustrations of the number of DEGs shared between two or three temperature treatments and the number of unique DEGs at each temperature assessed for both DEG classifications: (A) upregulated and (C) downregulated. The numbers in brackets are the total number of DEGs at each temperature. Representation of the gene ontology (GO) terms with the highest proportion of genes associated with them; GO terms for (B) upregulated and (D) downregulated genes. The x axis shows the percentage of genes associated with each GO term calculated considering the total number of DEGs for each classification (up- and downregulated genes). Each colour represents a temperature, 24°C (light blue), 28°C (light green) and 30°C (yellow).