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.

Keywords: Leishmania donovani; genome plasticity; genomics; in vitro passage; promastigotes.

FIGURE 1

In vitro hamster derived macrophage infection with different passages. Hamster macrophages were infected with stationary promastigotes of Leishmania donovani AG83, as described in Section “Materials and Methods” and the cells were incubated for 24 h and 72 h at 37°C, 5% CO₂. (A) The number of amastigotes in each passage and (B) the percentage of infected cells were analyzed by counting 100 cells. Data shown are mean ± SE of experiment performed in quadruplet.

FIGURE 2

Infection in hamsters. Hamsters were infected by intracardiac injection of 2 × 10⁷ L. donovani AG83 promastigotes. Animals were sacrificed at 8 week post-infection and (A,C) liver and (B,D) spleen parasite burden was determined by (A,B) LDU and (C,D) LDA. Data represent mean ± SE of two independent experiments (n = 4–6). (E) The mean weights of liver and spleen were compared with respective organ weights of uninfected animals.

FIGURE 3

Mummerplot comparisons and Gene Ontology analysis of early and late passage genomes to the NCBI reference genomes. (Upper) The y-axis represents HTI4 (left) or HTI5 (middle) whole genome assemblies compared to x-axis representingLdBPK282A1 assembly. The HTI4 (x-axis) versus HTI5 (y-axis) chromosomal assembly comparison is given in the right panel. The dots represent the positions of conserved DNA sequences on the genomes. (Lower) REVIGO was used to visualize the summary of significantly enriched GO terms. The scatterplots show the cluster representatives in a two dimensional space derived by applying multidimensional scaling to a matrix of the GO terms’ semantic similarities for early (left panel) and late (right panel) passages. Clustal differences are represented in boxes. Bubble color indicates the user-provided p-value; size indicates the frequency of the GO term in the underlying GOA database (bubbles of more general terms are larger).

FIGURE 4

Nucleotide and protein sequence comparison in L. donovani ABC transporter gene in Chr 23 of early and late passages. (Upper) Shows multiple nucleotide polymorphisms in HTI4 and HTI5 including indels and nucleotide substitutions. (Lower) Describes the protein sequence polymorphism as a result of sequence changes. The differences leading to functional loss are marked as boxes.

FIGURE 5

Synonymous and non-synonymous substitution in early and late passages leading to altered ABC transporter genes in chromosome 31. (A) A pair wise comparison between the protein coding genes in HTI4 and HTI5 showing single amino acid mismatches. (B) Nucleotide comparison in two genes indicates few substitutions, out of which the first one is a synonymous substitution and the second one is a non-synonymous substitution. A comparison between HTI4, HTI5 and Genbank strain (C) clearly indicates change of G–A in late passage same as the reference genome.

FIGURE 6

Schematic showing domain copy of MFS gene in Chromosome 29. A pseudo gene of MFS class of proteins present in HTI4 is missing in HTI5. However, a small domain from region 297–360 is being copied at two different locations in chromosome 29 with a non-synonymous substitution (AAC->AGC).

FIGURE 7

Comparison of Acetyl Co-A synthetase genes in early and late passage. Gene coding for Acetyl CoA Synthetase undergoes several substitutions in the late passage which makes it non-functional.

FIGURE 8

Frameshift mutation in Calpain like cysteine protease gene due to a single insertion in late passage. A single insertion in HTI5 (as well as Genbank LDBPK 282 A1) completely changes the coding frame of the gene (Upper panel) making it inactive. The changes in late passage and Genbank strain is consistent.

FIGURE 9

Hierarchical Clustering Heatmap of differentially expressed genes in early, intermediate and late passages. Heat maps were generated to show the comparative log 2 abundance ratios between early, intermediate, and late passages using RNAseq data. Upregulated proteins are depicted by blue bars, downregulated proteins by red bars.