Genome Plasticity in Cultured Leishmania donovani: Comparison of Early and Late Passages

Abstract

Leishmania donovani possesses a complex heteroxenic life cycle where infective metacyclic promastigotes are pre-adapted to infect their host and cope up with intracellular stress. Exploiting the similarities between cultured and sandfly derived promastigotes, we used early and late passage cultured promastigotes to show specific changes at genome level which compromise pathogen fitness reflected in gene expression and infection studies. The pathogen loses virulence mostly via transcriptional and translational regulations and long-time cultivation makes them struggle to convert to virulent metacyclics. At the genomic level very subtle plasticity was observed between the early and the late passages mostly in defense-related, nutrient acquisition and signal transduction genes. Chromosome Copy number variation is seen in the early and late passages involving several genes that may be playing a role in pathogenicity. Our study highlights the importance of ABC transporters and calpain like cysteine proteases in parasite virulence in cultured promastigotes. Interestingly, these proteins are emerging as important patho-adaptive factors in clinical isolates of Leishmania. We found that the currently available genome of Leishmania in the NCBI database are from late passages. Our early passage genome can act as a reference for future studies on virulent isolates of Leishmania. The annotated leads from this study can be used for virulence surveillance and therapeutic studies in the Indian subcontinent.

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

“…Thus, these results confirmed that early passaged parasites were potent enough to establish successful infection, but the late passaged ones were weak and unable to do so, both in vitro (in macrophages) as well as in vivo (in mice model). Previously, we have shown differences in the in vivo parasite burden in the spleen and liver of hamsters infected with the early and the late passage promastigotes (Sinha et al, 2018). We found that these differences were attributed to a differential expression of several virulence factors and cytoskeletal proteins in the parasites.…”

“…If we could identify those genes, they could serve as tools to develop potent antimicrobial interventions. In this study, 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 then performed genomic and transcriptomic studies on both the early passaged vAG83 and the late passaged nvAG83 parasites (Sinha et al, 2018).…”

“…In this study, 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 then 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.…”

Genomic Diversification, Structural Plasticity, and Hybridization in Leishmania (Viannia) braziliensis

“…Likewise, the local copy number variation may represent an evolutionarily important adaptation, to confer a degree of plasticity in the regulation and functional expression of genes or gene clusters involved in drug resistance, adaptation to different environmental, including survival in a variety of mammalian hosts and phenotypic diversity as has been observed in other species (L. donovani, L. tropica, L. infantum, and L. panamensis) (Laffitte et al, 2016;Iantorno et al, 2017;Sinha et al, 2018;Patino et al, 2020).…”

Genomic analysis of natural intra-specific hybrids among Ethiopian isolates of Leishmania donovani

“…both Ethiopian populations and the reference genome assembly rather than genuine variation within these populations. It is clear that structural variation appears rapidly during in vitro culture of Leishmania, so we cannot be sure that all of the variants we detect are present in natural populations rather than being an artefact of promastigote culture [39][40][41]. In the absence of gold standard known structural variation or any experimental validation it is possible our variant calls themselves are quite noisy.…”