RNA-Seq Revealed Expression of Many Novel Genes Associated With Leishmania donovani Persistence and Clearance in the Host Macrophage

Abstract

Host- as well as parasite-specific factors are equally crucial in allowing either the Leishmania parasites to dominate, or host macrophages to resist infection. To identify such factors, we infected murine peritoneal macrophages with either the virulent (vAG83) or the non-virulent (nvAG83) parasites of L. donovani. Then, through dual RNA-seq, we simultaneously elucidated the transcriptomic changes occurring both in the host and the parasites. Through Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of the differentially expressed (DE) genes, we showed that the vAG83-infected macrophages exhibit biased anti-inflammatory responses compared to the macrophages infected with the nvAG83. Moreover, the vAG83-infected macrophages displayed suppression of many important cellular processes, including protein synthesis. Further, through protein-protein interaction study, we showed significant downregulation in the expression of many hubs and hub-bottleneck genes in macrophages infected with vAG83 as compared to nvAG83. Cell signaling study showed that these two parasites activated the MAPK and PI3K-AKT signaling pathways differentially in the host cells. Through gene ontology analyses of the parasite-specific genes, we discovered that the genes for virulent factors and parasite survival were significantly upregulated in the intracellular amastigotes of vAG83. In contrast, genes involved in the immune stimulations, and those involved in negative regulation of the cell cycle and transcriptional regulation, were upregulated in the nvAG83. Collectively, these results depicted a differential regulation in the host and the parasite-specific molecules during in vitro persistence and clearance of the parasites.

Keywords: Leishmania donovani; RNA seq; macrophage; signaling/signaling pathways; transcriptome.

Figure 1

Dynamics of murine macrophage infection with virulent and non-virulent L. donovani promastigotes. Murine macrophages were infected with either virulent (2nd passage) or non-virulent (25th passage) L. donovani promastigotes for 3 h, washed, and further incubated until 24, 48, and 72 h p.i. The number of internalized parasites in 100 macrophages was determined microscopically by Giemsa staining. The results are expressed as mean S.E (n = 3). ^*P < 0.01, ^**P < 0.001.

Figure 2

Differentially expressed genes in (A) murine macrophage infected with virulent and non-virulent L. donovani for 12 h and in (B) L. donovani parasites. The numbers of DE genes in L. donovani-infected macrophages relative to uninfected controls and L. donovani depicted as horizontal bar plots. Box length depicts the number of DE genes either downregulated or upregulated at a P-value of < 0.05 with the total number of down- and up-regulated genes shown.

Figure 3

Analysis of differentially expressed (DE) genes in host macrophage at 12 h p.i. The DE gene lists for murine macrophage uninfected vs. infected with virulent and non-virulent parasites were compared and the overlap genes shown as a Venn diagram.

Figure 4

Validation of RNA-Seq-obtained DE genes by qPCR. (A) RNA-Seq data for 10 genes are represented in a bar graph. (B) The mRNA levels of the same set of genes were measured by qPCR and are represented as the fold change compared to non-infected control cells. (C) Fold difference values calculated by RNA-Seq (y axis) for the DE genes in macrophage infected with both parasites correlates to the mRNA expression of these genes using qPCR (x axis).

Figure 5

Heat Map of DE genes in infected macrophage compared to control macrophage. The DE genes involved in KEGG pathways downregulated by (A) virulent and (B) non-virulent parasites are represented as heat maps. Similarly, the DE genes involved in KEGG pathways upregulated by (C) virulent and (D) Non-virulent parasites are represented.

Figure 6

Pictorial representation of the constructed protein-protein interaction network of the deregulated macrophage genes in virulent and non-virulent stages of L. donovani infection and analyzing the important nodes of the network. (A) Shows the expression status of macrophage genes during virulent and non-virulent stages of L. donovani infection, where 54 (3 up-regulated and 51 down-regulated) and 116 (85 up-regulated and 31 down-regulated) genes were found to be expressed during the virulent and non-virulent stages of infection and 82 genes were expressed commonly in both stages. (B) Depicts the representation of the constructed virulent and non-virulent protein-protein interaction network. (C) Shows the schematic representation of the important nodes of the network. (D) Provides the number stage specific deregulated murine macrophage genes at virulent and non-virulent L. donovani infection stages. (E) Represents the constructed protein-protein interaction network of stage-specific (virulent and non-virulent) deregulated genes. (F) Shows the schematic representation of the important network nodes from the stage-specific networks.

Figure 7

Effect of L. donovani infection on MAPK and PI3K activation. Peritoneal macrophages were exposed to virulent and non-virulent L. donovani promastigotes for various times as indicated. LPS-treated macrophages were taken as a positive control and non-treated macrophages were taken as a negative control. Whole-cell lysates were prepared and subjected to western blotting using antibodies specific for (A,A1,B,B1) phospho-P38 MAPK, (C,C1,D,D1) phospho-ERK1/2 MAPK, and (E,E1,F,F1) phospho-AKT (ser473). Similarly, cell lysates of BMM exposed to virulent and non-virulent L. donovani promastigotes for various times as indicated were probed for (G,G1,G2) phospho-P38 MAPK and phospho-ERK1/2 MAPK, (H) phospho-AKT (ser473). β-actin was used as loading control for all the blots as described in materials and methods. The figures are representative of 2 independent experiments. Bands were analyzed densitometrically and bar graphs expressing arbitrary units are presented adjacent to corresponding western blots. Error bar represent mean ± S.D., n = 2. ^*P < 0.01, ^**P < 0.001, ^***P < 0.0001.