Dual transcriptome analysis reveals differential gene expression modulation influenced by Leishmania arginase and host genetic background

Abstract

The outcome of Leishmania infection is strongly influenced by the host's genetic background. BALB/c mice are susceptible to Leishmania infection, while C57BL/6 mice show discrete resistance. Central to the fate of the infection is the availability of l-arginine and the related metabolic processes in the host and parasite. Depending on l-arginine availability, nitric oxide synthase 2 (NOS2) of the host cell produces nitric oxide (NO) controlling the parasite growth. On the other hand, Leishmania can also use host l-arginine for the production of polyamines through its own arginase activity, thus favouring parasite replication. Considering RNA-seq data, we analysed the dual modulation of host and parasite gene expression of BALB/c or C57BL/6 mouse bone marrow-derived macrophages (BMDMs) after 4 h of infection with Leishmania amazonensis wild-type (La-WT) or L. amazonensis arginase knockout (La-arg^-). We identified 12 641 host transcripts and 8282 parasite transcripts by alignment analysis with the respective Mus musculus and L. mexicana genomes. The comparison of BALB/c_La-arg^-versus BALB/c_La-WT revealed 233 modulated transcripts, with most related to the immune response and some related to the amino acid transporters and l-arginine metabolism. In contrast, the comparison of C57BL/6_La-arg^-vs. C57BL/6_La-WT revealed only 30 modulated transcripts, including some related to the immune response but none related to amino acid transport or l-arginine metabolism. The transcriptome profiles of the intracellular amastigote revealed 94 modulated transcripts in the comparison of La-arg^-_BALB/c vs. La-WT_BALB/c and 45 modulated transcripts in the comparison of La-arg^-_C57BL/6 vs. La-WT_C57BL/6. Taken together, our data present new insights into the impact of parasite arginase activity on the orchestration of the host gene expression modulation, including in the immune response and amino acid transport and metabolism, mainly in susceptible BALB/c-infected macrophages. Moreover, we show how parasite arginase activity affects parasite gene expression modulation, including amino acid uptake and amastin expression.

Keywords: BALB/c; C57BL/6; Leishmania amazonensis; RNA-seq; arginine transport; immune response; macrophage infection.

Fig. 1.

Functional analysis of the differentially expressed gene profiles of BALB/c and C57BL/6 macrophages infected with La-WT or La-arg^- for 4 h. (a) Number of upregulated (light grey) and downregulated (dark grey) differentially expressed genes. (b) KEGG enrichment analysis shown as a heat map of the 14 most regulated pathways based on the indicated comparisons. (c) Venn diagram of the 263 DEGs based on the comparisons of BALB/c_La-arg^- vs. BALB/c_La-WT and C57BL/6_La-arg^- vs. C57BL/6_La-WT. (d) List of the exclusively and commonly transcripts for each comparison according to the Venn diagram analysis. The red transcripts are related to the immune response, and arginine transport and metabolism. (e) DEG profiles as indicated by log₂-fold changes of the exclusively modulated genes involved in the immune response and arginine transport and metabolism in the comparisons of BALB/c_La-arg^- vs. BALB/c_La-WT and C57BL/6_La-arg^- vs. C57BL/6_La-WT. L. amazonensis wild-type (La-WT) and L. amazonensis arginase knockout (La-arg^-). The data are from five independent biological replicates, considering fold change ≥2 and a p-value <0.05.

Fig. 2.

Fold-change expression profiles of the differentially expressed genes in BALB/c and C57BL/6 macrophages infected with La-WT or La-arg^- and in La-WT and La-arg^- intracellular amastigotes in BALB/c and C57BL/6 macrophages after 4 h of infection. (a) Volcano plots of the comparisons of BALB/c and C57BL/6 macrophages infected with La-WT or La-arg^-, considering genes with a fold change ≥2 and a p-value <0.05. Genes significantly upregulated (yellow dots) are in the upper right square of each graph (positive log-fold-change value). Genes significantly downregulated (blue dots) are in the upper left square of each graph (negative log-fold-change value). (b) Volume plots of the comparisons of BALB/c and C57BL/6 macrophages infected with La-WT or La-arg^-, based on genes with log₂-fold change ≥2 and a p-value <0.05 (blue dots). The top five significantly upregulated and downregulated genes are represented by red dots. (c) Volcano plots of the comparisons of La-WT and La-arg^- intracellular amastigotes in BALB/c and C57BL/6 macrophages, considering genes with fold change ≥2 and a p-value <0.05. Genes significantly upregulated (yellow dots) are in the upper right square of each graph (positive log-fold-change value). Genes significantly downregulated (blue dots) are in the upper left square of each graph (negative log-fold-change value). (d) Volume plots of the comparisons of La-WT and La-arg^- intracellular amastigotes in BALB/c and C57BL/6 macrophages, considering genes with log₂-fold change ≥2 and a p-value <0.05 (blue dots). The top five significantly upregulated and downregulated genes are represented by red dots. L. amazonensis wild-type (La-WT) and L. amazonensis arginase knockout (La-arg^-).

Fig. 3.

Gene network analysis of BALB/c and C57BL/6 macrophages infected with La-WT or La-arg^- for 4 h. (a) Network mapping based on co-expression analysis of the exclusively modulated genes based on the comparison of BALB/c_La-arg^- vs. BALB/c_La-WT. (b) Network mapping based on the co-expression analysis of the exclusively modulated genes for the comparison of C57BL/6_La-arg^- vs. C57BL/6_La-WT. L. amazonensis wild-type (La-WT) and L. amazonensis arginase knockout (La-arg^-).

Fig. 4.

Functional analysis of the differentially expressed gene profile of La-WT and La-arg^- infecting BALB/c or C57BL/6 macrophages after 4 h. (a) Count of the differentially expressed genes (DEGs) upregulated (light grey) or downregulated (dark grey). (b) Venn diagram of the 108 DEGs in La-arg^-_BALB/c vs. La-WT_BALB/c and La-arg^-_C57BL/6 vs. La-WT_C57BL/6. (c) List of the exclusive and common transcripts for each comparison according to the Venn diagram analysis. (d) DEG profiles represented by log₂-fold changes of the exclusively modulated genes in the comparisons of BALB/c_La-arg^- vs. BALB/c_La-WT and C57BL/6_La-arg^- vs. C57BL/6_La-WT. L. amazonensis wild-type (La-WT) and L. amazonensis arginase knockout (La-arg^-). The data are from five independent biological replicates, considering fold change ≥2 and a p-value <0.05.