Leptomonas seymouri: Adaptations to the Dixenous Life Cycle Analyzed by Genome Sequencing, Transcriptome Profiling and Co-infection with Leishmania donovani

Abstract

The co-infection cases involving dixenous Leishmania spp. (mostly of the L. donovani complex) and presumably monoxenous trypanosomatids in immunocompromised mammalian hosts including humans are well documented. The main opportunistic parasite has been identified as Leptomonas seymouri of the sub-family Leishmaniinae. The molecular mechanisms allowing a parasite of insects to withstand elevated temperature and substantially different conditions of vertebrate tissues are not understood. Here we demonstrate that L. seymouri is well adapted for the environment of the warm-blooded host. We sequenced the genome and compared the whole transcriptome profiles of this species cultivated at low and high temperatures (mimicking the vector and the vertebrate host, respectively) and identified genes and pathways differentially expressed under these experimental conditions. Moreover, Leptomonas seymouri was found to persist for several days in two species of Phlebotomus spp. implicated in Leishmania donovani transmission. Despite of all these adaptations, L. seymouri remains a predominantly monoxenous species not capable of infecting vertebrate cells under normal conditions.

Fig 1. Growth kinetics of Leptomonas pyrrhocoris and L. seymouri at 23°C, 29°C, and 35°C in SDM and blood-agar media.

Data from 3 independent biological replicates are presented.

Fig 2. Morphology of Leptomonas seymouri cells cultivated in vitro in SDM and blood-agar media at low (23°C) and high (35°C) temperature.

A, Giemsa-stained slides, scale bar is 10 μm. B, ANOVA statistical analysis of 100 cells, average and standard error are presented in micrometers (μm). L–length, W–width, N–length of the nucleus, AK–distance between the kinetoplast and anterior end of the cell, AN–distance between the nucleus and anterior end of the cell, FF–free flagellum length.

Fig 3. Orthologous group presence (denoted by "+")/absence (denoted by "-") patterns for Leptomonas seymouri, Leptomonas pyrrhocoris, Crithidia fasciculata, and several Leishmania species.

The number of present OGs is indicated on the right.

Fig 4. Leptomonas seymouri harbors dsRNA viruses.

A, Cells were stained with DAPI, mitotracker Red and anti-dsRNA antibody for viral detection. Scale bar is 10 μm. B, Total RNA samples isolated from Leptomonas seymouri (Ls) and Blechomonas pulexsimulantis (Bp) were treated with DNase I and S1 nuclease and separated by gel electrophoresis. M– 1 kb ladder (Thermo Fisher Scientific).

Fig 5. Experimental infection and co-infection of Phlebotomus spp.

Phlebotomus argentipes and P. orientalis were infected with Leptomonas. seymouri using sugar- (A) or blood- (B) meal method. Intensity of infection was assayed on days 2, 6, and 9 post infection and defined as light (less than 100 promastigotes, white bar), moderate (100–1,000 promastigotes, grey bar), or heavy (over 1,000 promastigotes, black bar) depending on the number of parasites per gut. C, Experimental co-infection of P. argentipes with mCherry Leptomonas seymouri and GFP Leishmania donovani GR-374. Intensity of infection was assayed on days 2 and 5 post infection with blood meal and defined as light, moderate, or heavy as above. Numbers above each bar indicate the number of dissected females.