Differential expression of polyamine biosynthetic pathways in skin lesions and in plasma reveals distinct profiles in diffuse cutaneous leishmaniasis

Abstract

Tegumentary leishmaniasis (TL) is a parasitic disease that can result in wide spectrum clinical manifestations. It is necessary to understand host and parasite determinants of clinical outcomes to identify novel therapeutic targets. Previous studies have indicated that the polyamine biosynthetic pathway is critical for Leishmania growth and survival. Despite its importance, expression of the such pathway has not been previously investigated in TL patients. We performed an exploratory analysis employing Systems Biology tools to compare circulating polyamines and amino acid concentration as well as polyamine pathway gene expression in cutaneous lesions patients presenting with distinct TL disease presentations. Diffuse cutaneous leishmaniasis (DCL) was associated with higher concentrations of amino acids, polyamines and its substrate transporters than mucosal cutaneous leishmaniasis or localized cutaneous leishmaniasis. In addition, the RNA expression of polyamine-related genes of patients lesions from two separate cohorts demonstrated that differential activation of this pathway is associated with parasite loads and able to discriminate the clinical spectrum of TL. Taken together, our findings highlight a new aspect of DCL immunopathogenesis indicating that the polyamine pathway may be explored as a novel therapeutic target to control disease burden.

Figure 1

Plasma concentrations of amino acids and polyamines in patients with tegumentary leishmaniasis. Plasma levels of ARG1, polyamines: Putrescin, Cadaverine, Spermidine and amino acids: Arginine, Ornithine, Citrulline, were compared among patients with localized (LCL; n = 29), mucosal (MCL; n = 13) and diffuse (DCL) leishmaniasis as well as healthy controls (n = 43). (A) A hierarchical clustering analysis (Ward’s method) was employed to show the amino acids and polyamines measured using a representative profile of geometric mean values (log2 -transformed) displayed for each clinical group. The color scale of the heatmap represents z-score by row. (B) Univariate analyzes with scatter plots of the comparisons are shown. Data were compared using the Kruskal–Wallis test with Dunn’s multiple comparisons ad hoc test (*P < 0.05, ***P < 0.001). Lines represent median values. Grey bars represent the percentiles 25 and 75 from healthy controls (C) Frequency of the indicated polyamines in healthy controls or those with MCL, LCL and DCL was compared the total free polyamines using the chi square test.

Figure 2

Differential expression of genes from polyamines pathways in lesions from patients with tegumentary leishmaniasis. Total RNA was extracted from lesion biopsy from patients with MCL (n = 4), LCL (n = 7) and those with DCL (n = 4). Indicated messenger RNA transcripts of host-specific cellular genes were quantified by nCounter (Nanostring) and were normalized by pan-leukocyte gene CD45 to account for detection of immune infiltration into tissues. (A) A hierarchical clustering analysis (Ward’s method) was employed to show the targeted genes of the polyamine pathway displaying a different profile for each clinical group. (B) A representative profile of geometric mean values (log2 transformed), for indicated genes, was compared among MCL, LCL and DCL patients. Data were compared using the Kruskal–Wallis test with Dunn’s multiple comparisons ad hoc test (*P < 0.05, **P < 0.01, ***P < 0.001). (C) Box- and-whisker plots of gene expression relative to CD45 are shown. Lines represent median values and interquartile ranges. Data were compared using the Mann–Whitney U test. *P < 0.05.

Figure 3

Tegumentary leishmaniasis patients display a unique profile of gene expression from the polyamine biosynthetic pathway. (A) Genes involved in the polyamine pathway were retrieved from the transcriptome dataset as described in “Methods” section and used in additional analyses to test whether its expression values were able to separate the distinct clinical groups. Fold-differences (DCL or LCL vs. healthy controls) were calculated and statistically significant differences are highlighted in colored bars. (B) A principal component analysis (PCA) model was employed to verify if the expression values of the genes from the polyamine biosynthetic pathway were able to classify the samples within the groups, regardless of the fact that some of these genes were not differentially expressed between the clinical groups. We used the normalized count table without the calculation of fold difference against the control group to input the PCA algorithm (see “Methods” section for details). (C) A Receiver Operator Characteristics (ROC) curve analysis was performed with these same gene expression values, to assess the sensibility and specificity of this classification. The ROC curve analysis used a multinomial model, in which the outcomes (HC, DCL and LCL) were binarized. Thus, this approach allowed us to compare the power of all the genes from polyamine biosynthetic pathway described above to discriminate between the following groups: (i) DCL vs. HC + LCL; (ii) HC vs. LCL + DCL; and (iii) LCL vs. HC + DCL. AUC: area under the curve.

Figure 4

Association between activation of genes from the polyamine pathway and parasite transcripts. (A) A hierarchical clustering analysis (Ward’s method) was employed to illustrate the overall expression profile of Leishmania genes in DCL and LCL lesion and its correlation with parasite loads (normalized leishmania transcripts as described in “Methods” section). Identifiers refer to putative functions (described in “Methods” section). (B) A similar statistical approach was used to evaluate the overall profile of genes of the polyamine biosynthetic pathway in TL patients vs. parasite loads. Correlations were tested using the Spearman rank. In (A) and (B), each column represents one patient. Spearman correlation rank coefficient (rho) values were displayed as bar plots, to demonstrate strength and directionality of the associations.

Figure 5

Polyamine pathway in patients with Diffuse Cutaneous Leishmaniasis. This illustration summarizes the data of Figs. 1 and 2. The figure shows the cascade of the polyamine biosynthesis and highlights the parameters which were statistically significant in plasma and/or skin lesions of Diffuse Cutaneous Leishmaniasis (DCL) patients compared to the other clinical forms of tegumentary leishmaniasis in our study settings.