Mutation Characteristics and Phylogenetic Analysis of Five Leishmania Clinical Isolates.

Zhiwan Zheng,Jiao Li,Dali Chen,Jianping Chen,Tao Luo,Shuangshuang Yin,Jie Luo,Qi Zhou,Jianhui Zhang,Jinlei He

doi:10.3390/ani12030321

Abstract

Simple SummaryLeishmaniasis, a neglected tropical disease, is caused by infection with the Leishmania species, threatening millions of people in approximately 100 endemic countries. The emergence of antimony-resistant Leishmania strains have brought difficulties to the treatment and elimination of leishmaniasis. This study performed genome-wide resequencing and phylogenetic analysis of five isolates from the Leishmania donovani complex, focusing on finding mutations related to antimony resistance and virulence of the newly isolated Leishmania strain L_HCZ in 2016. By combining whole-genome sequencing and whole-genome phylogenetic analysis, Leishmania isolates L_801, L_9044 and L_Liu were identified as Leishmania donovani, and L_HCZ as Leishmania infantum. By discovering genome-wide single-nucleotide polymorphisms and structural variations, we identified mutations of drug resistance-related genes in the antimony-resistant Leishmania isolate L_HCZ. The new Leishmania isolate L_HCZ has strong virulence and strong drug resistance, which should be taken seriously by the relevant health departments and scientific researchers.Leishmaniasis is a neglected tropical disease threatening millions of people worldwide. The emergence of antimony-resistant Leishmania strains have brought difficulties to the treatment and elimination of leishmaniasis. This study performed genome sequencing, phylogenetic analysis and mutation analysis of five Leishmania clinical isolates, especially the Leishmania strain L_HCZ isolated in 2016, which shows strong virulence and antimony resistance. By phylogenetic analysis, four isolates (L_DD8, L_801, L_Liu and L_9044) were identified as Leishmania donovani, the isolate L_HCZ was identified as Leishmania infantum and the isolate L_DD8 as a standard strain of L.donovani. Genome-wide mutation analysis was applied to identify mutations related to the drug resistance and virulence of the newly isolated L_HCZ. Compared with the other four Leishmania isolates, L_HCZ had the most mutations in genes associated with antimony resistance, including the ABC transporter, ascorbate-dependent peroxidase, gamma–glutamylcysteine synthetase, glucose-6-phosphate 1-dehydrogenase, ATP-binding cassette protein subfamily A and multi-drug resistance protein-like genes. Among the genes associated with virulence, L_HCZ had the most mutations in cysteine peptidase A, cysteine peptidase B, cysteine peptidase C, heat-shock protein 70, gp63, acid phosphatase, kinesin k39, kinesin, phosphoglycan beta 1, amastin-like surface protein and amastin-like proteins. The mutations in L_HCZ might possibly contribute to its antimony resistance and strong virulence in clinical patients. Whole-genome resequencing has exhibited broad application prospects and may be put into clinical use in the future for parasite identifying and epidemiological investigations.

Highlights

Visceral leishmaniasis (VL) is a neglected zoonotic parasitic disease and is caused by Leishmania parasites, which are transmitted by insects known as sandflies
The genomic coverages based on 1×, 4×, 10× depths for the samples were all greater than 90%, suggesting that the five isolates had high similarity with the reference genome (Table 3)
The number of single-nucleotide polymorphism (SNP), insertions, deletions, total Het/Hom ratio, SNP Het/Hom ratio and SNP Transitions/Transversions of L_9044, L_Liu and L_HCZ was approximately the same, and their homozygous variations were more than heterozygous variations, which was different from the results of L_801 and L_DD8

Summary

Introduction

Visceral leishmaniasis (VL) is a neglected zoonotic parasitic disease and is caused by Leishmania parasites, which are transmitted by insects known as sandflies. The main clinical symptoms of VL are long-term irregular fever, anemia, weight loss and hepatosplenomegaly [1]. Failure to treat the infection in time leads to the death of patients. The Leishmania spp. that causes VL is mainly derived from the Leishmania donovani complex (including Leishmania donovani and Leishmania infantum) [2]. VL is endemic in more than 60 countries worldwide and an estimated 50,000 to 90,000 new cases of VL occur worldwide annually [3]. A few anti-Leishmania drugs are available, and their effectiveness is severely limited by toxicity, cost and drug resistance [4]. Some resistant Leishmania strains have appeared in clinics, especially with resistance to antimony. Understanding the Leishmania genomes, their natural variation and genetic relationships is essential to support and strengthen public health monitoring and intervention strategies

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