Phylogenetic and evolutionary analysis of Chinese Leishmania isolates based on multilocus sequence typing

Abstract

Leishmaniasis is a debilitating infectious disease that has a variety of clinical forms. In China, visceral leishmaniasis (VL) is the most common symptom, and L. donovani and/or L. infantum are the likely pathogens. In this study, multilocus sequence typing (MLST) of five enzyme-coding genes (fh, g6pdh, icd, mpi, pgd) and two conserved genes (hsp70, lack) was used to investigate the phylogenetic relationships of Chinese Leishmania strains. Concatenated alignment of the nucleotide sequences of the seven genes was analyzed and phylogenetic trees were constructed using neighbor-joining and maximum parsimony models. A set of additional sequences from 25 strains (24 strains belong to the L. donovani complex and one strain belongs to L. gerbilli) were retrieved from GenBank to infer the molecular evolutionary history of Leishmania from China and other endemic areas worldwide. Phylogenetic analyses consolidated Chinese Leishmania into four groups: (i) one clade A population comprised 13 isolates from different foci in China, which were pathogenic to humans and canines. This population was subdivided into two subclades, clade A1 and clade A2, which comprised sister organisms to the remaining members of the worldwide L. donovani complex; (ii) a population in clade B consisted of one reference strain of L. turanica and five Chinese strains from Xinjiang; (iii) clade C (SELF-7 and EJNI-154) formed a population that was closely related to clade B, and both isolates were identified as L. gerbilli; and (iv) the final group, clade D, included Sauroleishmania (LIZRD and KXG-E) and was distinct from the other strains. We hypothesize that the phylogeny of Chinese Leishmania is associated with the geographical origins rather than with the clinical forms (VL or CL) of leishmaniasis. To conclude, this study provides further molecular information on Chinese Leishmania isolates and the Chinese isolates appear to have a more complex evolutionary history than previously thought.

Figure 1. Locations of endemic areas in Xinjiang, Gansu, Sichuan, Inner Mongolia and Shandong regions of China.

The Leishmania strains and their geographical origins are as follows: Xinjiang: Karamay (KXG-65, KXG-LIU, KXG-XU, KXG-Y, KXG-R, KXG-57, KXG-11, KXG-E); Kashgar (771, KS6, 801); Qitai (QITAI-15) and Turpan (SELF-7); Gansu: Wenxian (8801, WDD23) and (1101, 1102); Sichuan: Nanping (SC6, SC9); Inner Mongolia: Ejina (EJNI-154, LIZRD); Shandong: (9044).

Figure 2. Neighbor-joining unrooted tree constructed from hsp70 and lack genes for 28 isolates in this study.

The Kimura-2-parameter method was used. Numbers above branches correspond to bootstrap valued based on 1,000 replicates. The strains were designated by their names (See Table 1 for more details).

Figure 3. Phylogenetic trees constructed based on the sequences of the fh, g6pdh , icd, mpi and pgd genes for 25 isolates in this study and 24 isolates of the Leishmanaia donovani complex from other studies.

Neighbor-joining tree was constructed from five enzyme genes for the 49 isolates using MEGA 5.0 software. The Kimura-2-parameter method was used. Numbers above branches correspond to bootstrap values based on 1,000 replicates. The strains were designated by their names (See Table 1 for more details).