Comparative analyses of whole genome sequences of Leishmania infantum isolates from humans and dogs in northeastern Brazil

Abstract

The genomic sequences of 20 Leishmania infantum isolates collected in northeastern Brazil were compared with each other and with the available genomic sequences of 29 L. infantum/donovani isolates from Nepal and Turkey. The Brazilian isolates were obtained in the early 1990s or since 2009 from patients with visceral or non-ulcerating cutaneous leishmaniasis, asymptomatic humans, or dogs with visceral leishmaniasis. Two isolates were from the blood and bone marrow of the same visceral leishmaniasis patient. All 20 genomic sequences display 99.95% identity with each other and slightly less identity with a reference L. infantum genome from a Spanish isolate. Despite the high identity, analysis of individual differences among the 32 million base pair genomes showed sufficient variation to allow the isolates to be clustered based on the primary sequence. A major source of variation detected was in chromosome somy, with only four of the 36 chromosomes being predominantly disomic in all 49 isolates examined. In contrast, chromosome 31 was predominantly tetrasomic/pentasomic, consistent with its regions of synteny on two different disomic chromosomes of Trypanosoma brucei. In the Brazilian isolates, evidence for recombination was detected in 27 of the 36 chromosomes. Clustering analyses suggested two populations, in which two of the five older isolates from the 1990s clustered with a majority of recent isolates. Overall the analyses do not suggest individual sequence variants account for differences in clinical outcome or adaptation to different hosts. For the first known time, DNA of isolates from asymptomatic subjects were sequenced. Of interest, these displayed lower diversity than isolates from symptomatic subjects, an observation that deserves further investigation with additional isolates from asymptomatic subjects.

Keywords: Brazil; Copy number variation; Evolution; Intracellular parasite; Leishmania infantum; Parasite population structure.

Fig. 1

Aneuploidy in Leishmania Isolates. (A) Heatmap for aneuploidy status of Leishmania infantum chromosomes. Mosaic aneuploidy was found in the 20 Brazilian L. infantum isolates based on normalized chromosome read depth as shown in the key at the top left. The heatmap shows the estimated copy number of the 36 chromosomes (x-axis) in the isolates (y-axis) as disomic (value 2 in the key), pentasomic (value 5 in the key) or intermediate values (values between 2 and 5). The vertical bars on the left on the left of the heat map denote three groups of isolates formed by a hierarchical clustering analysis of the somy values. The 20 isolates indicated on the right are named as follows. Numbers 1–20 are the chronological order in which each isolate was collected during 1991–2013. “VL” isolates are from VL patients and from dogs; isolates with “A” in their name are from asymptomatic individuals; “CL” is an isolate from a CL patient; “h” isolates are from humans; d isolates are from dogs; “90” denotes isolates collected in the 1990s. Isolates 19VLh and 20VLh were collected from the blood and bone marrow, respectively, of the same individual. (B) Distributions of disomic chromosomes amongst three groups of L. infantum/Leishmania donovani. Chromosomes are shown that are predominately disomic in 20 L. infantum isolates from Brazil (this work), 17 L. donovani isolates from Nepal (Downing et al., 2011) and 12 L. infantum isolates from Turkey (Rogers et al., 2014). A total of 13 chromosomes in the Brazilian isolates, nine in the Nepalese isolates and six in the Turkish isolates were predominantly disomic. Four chromosomes (numbers 19, 28, 30 and 34) were predominantly disomic in all 49 isolates.

Fig. 2

Nucleotide diversity among Leishmania Infantum chromosomes. Nucleotide diversity and Tajimas’s D test for each chromosome according to isolate grouping characteristics. Chromosomes highlighted in grey bars are those that have a P value ≤0.05 for recombination under the PHI Test (Φ_w); those without bars (chromosomes 5,6, 8, 9, 14,17, 25, 26 and 31) showed no sign of recombination.

Fig. 3

Principal component analysis of the single nucleotide polymorphism dataset. Principal component analysis of genomic variation in the five Leishmania Infantum isolates from visceral leishmaniasis patients in the 1990s, five isolates from visceral leishmaniasis patients in 2012–13, four isolates from asymptomatic humans in 2011–12, five isolates from visceral leishmaniasis dogs in 2010–12 and one isolate from a cutaneous leishmaniasis patient in 2009. The two most significant PCs are related to 34.5% (21 + 13.5%) of the variation for all isolates. The 11 most closely related isolates, according to PCA, are indicated within the oval.