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.
Highlights
Macrophages are known to have microbicidal functions and are considered as the sentinels of the immune system (Franken et al, 2016)
To validate these in vitro results in an in vivo set-up, BALB/c mice were infected with promastigotes derived from different passages, and the parasite burden was determined in both the liver and the spleen, using methods like Leishman Donovan Units (LDU) and Limiting Dilution Assay (LDA)
We found that in the host macrophages, vAG83 and nvAG83 induced 456 and 473 DE genes, respectively, that were differentially expressed at a p-value cutoff of < 0.05 compared to the uninfected control
Summary
Macrophages are known to have microbicidal functions and are considered as the sentinels of the immune system (Franken et al, 2016) Their interaction with pathogens (of high and low virulence) varies significantly. We attempted to identify the host as well as the parasite-specific genes, which were modulated when the host macrophages interacted with the virulent and the non-virulent L. donovani parasites (vAG83 and nvAG83, respectively) (Sinha et al, 2018). To obtain nvAG83 parasites, we first cultured the vAG83 for several passages in medium, and performed genomic and transcriptomic studies on both the early passaged vAG83 and the late passaged nvAG83 parasites (Sinha et al, 2018) With these two parasites, we infected the non-elicited murine peritoneal macrophages (Ghosn et al, 2010), and measured the transcriptome of both the host as well and the infecting parasites with high-throughput deep sequencing (RNA-Seq) technology. RNA-Seq ensures a highly sensitive technique with high accuracy and provides a far more precise measurement of the level of transcripts than most other methods (Wang et al, 2009)
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