Integrative genomic, proteomic and phenotypic studies of Leishmania donovani strains revealed genetic features associated with virulence and antimony-resistance

Abstract

Background Leishmaniasis is a neglected tropical disease affecting millions of people worldwide. Emerging drug resistance of Leishmania species poses threaten to the effective control and elimination programme of this neglected tropical disease. Methods In this work, we conducted drug-resistance testing, whole genome resequencing and proteome profiling for a recently reported clinical isolate with supposed drug resistance (HCZ), and two reference sensitive strains (DD8 and 9044) of Leishmania donovani, to explore molecular mechanisms underlying drug resistance in this parasite. Results With reference to DD8 and 9044 strains, HCZ isolate showed higher-level virulence and clear resistance to antimonials in promastigote culture, infected macrophages and animal experiment. Pairwise genomic comparisons revealed genetic variations (86 copy number variations, 271 frameshift mutations in protein-coding genes and two site mutations in non-coding genes) in HCZ isolate that were absent from the reference sensitive strains. Proteomic analysis indicated different protein expression between HCZ isolate and reference strains, including 69 exclusively detected proteins and 82 consistently down-/upregulated molecules in the HCZ isolate. Integrative analysis showed linkage of 12 genomic variations (gene duplication, insertion and deletion) and their protein expression changes in HCZ isolate, which might be associated with pathogenic and antimony-resistant phenotype. Functional annotation analyses further indicated that molecules involved in nucleotide-binding, fatty acid metabolism, oxidation-reduction and transport might play a role in host-parasite interaction and drug-resistance. Conclusions This comprehensive integrative work provided novel insights into the genetic basis underlying virulence and resistance, suggesting new aspects to be investigated for a better intervention against L. donovani and associated diseases.

Impact of Genetic Diversity and Genome Plasticity of Leishmania spp. in Treatment and the Search for Novel Chemotherapeutic Targets

“…Variations in the parasite genome can be associated with its geographical distribution and clinical manifestations, which can influence leishmaniasis management. Interestingly, large-scale research involving a large number of samples and wide geographic range has revealed that genetic diversity is significantly higher than that previously reported ( Bussotti et al., 2018 ; Zackay et al., 2018 ; Franssen et al., 2020 ; Imamura et al., 2020 ; Patino et al., 2020 ; Salloum et al., 2020 ; Zheng et al., 2020 ; Patino et al., 2021 ; Schwabl et al., 2021 ). Recently, single-cell sequencing has demonstrated the presence of several distinct karyotypes within the same Leishmania clone ( Imamura et al., 2020 ; Negreira et al., 2020 ), and multiple-genotype infections have been demonstrated to occur even within the same host and tissue ( Cupolillo et al., 2020 ).…”

Drug-Induced Lipid Remodeling in Leishmania Parasites

“…Otherwise, there are evidences to suggest that a non-inhibition of the fatty acid βoxidation is associated with an antimony-resistant profile. Not only genetic variations but also a predominant upregulation in proteins involved in fatty acid β-oxidation have been described in antimony resistant parasites [22,58,59]. Particularly, the long-chain fatty acyl-CoA ligase (LCFA) a key enzyme involved in β-oxidation of long fatty acids has been found to be differentially increased in antimony-resistant Leishmania donovani amastigotes by microarray analysis [60,61].…”

Mutation Characteristics and Phylogenetic Analysis of Five Leishmania Clinical Isolates

“…According to these two papers, some interesting results attracted our attention. In this study, we found the genes of protein kinase, kinesin, amastin-like surface protein, N-acyl-L-amino acid amidohydrolase protein and sodium stibogluconate resistance protein in L_HCZ had more mutations, and in our previous study we found the expression of these genes at the protein level was up-regulated [65]. Similarly, the mutation of mitogen-activated protein kinase in L_HCZ was less than that in L_9044 and L_Liu, and the protein expressed by this gene was down-regulated.…”

“…The datasets of the five Leishmania clinical isolates will provide a molecular basis for future research on the pathogenic mechanism, drug resistance mechanism and vaccine development of Leishmania. In our previous study, we focused on verifying the virulence and antimony resistance of the Leishmania clinical isolate (L_HCZ) at molecular levels and proved that L_HCZ has high-level pathogenicity and evident antimony resistance in vitro, using an integrative approach of genome sequencing, proteomic profiling and phenotypic analysis to find the target spot of virulence and antimony resistance of L_HCZ [65]. According to these two papers, some interesting results attracted our attention.…”