Demonstration of genetic exchange during cyclical development of Leishmania in the sand fly vector

Abstract

Genetic exchange has not been shown to be a mechanism underlying the extensive diversity of Leishmania parasites. We report here evidence that the invertebrate stages of Leishmania are capable of having a sexual cycle consistent with a meiotic process like that described for African trypanosomes. Hybrid progeny were generated that bore full genomic complements from both parents, but kinetoplast DNA maxicircles from one parent. Mating occurred only in the sand fly vector, and hybrids were transmitted to the mammalian host by sand fly bite. Genetic exchange likely contributes to phenotypic diversity in natural populations, and analysis of hybrid progeny will be useful for positional cloning of the genes controlling traits such as virulence, tissue tropism, and drug resistance.

Fig. 1. Genotyping of hybrid Leishmania

A, PCR for parental selectable drug markers SAT and HYG. Samples are F, FV1(SAT); L, LV39c5(HYG); M, 1 kb plus marker (Invitrogen, CA); -, no template control, 1, 1.10.B12; 2, 1.10.D9; 3, 4.3.G12; 4, 4.3.A12; 5, 1.14.E10; 6, 5.12.D9; 7, 5.12.F11; 8, 1.14.B11; 9, 5.22.10 B, SNP-CAPS analysis for loci on chromosomes 7, 25, and 35. F, L and M are as in panel A; mix a, b and c, parental templates mixed in different ratios (1:1, 3:1, or 1:3 F:L respectively) DNA content analysis showed that hybrids 5 and 8 are 3n (marked by *) and the remainder shown are 2n progeny. C, SNP-CAPS analysis of Maxicircle. Digestion with BfaI of the ND5 - divergent region PCR product is shown. Hybrids tested are 1, 4.7.A3; 2, 4.17.A3; 3, 4.9.E6; 4, 4.17.F4; 5, 1.10.B12; 6, 1.10.D9; 7, 4.3.G12; 8, 4.3.A12; 9, 1.14.E10; 10, 5.12.D9; 11, 5.12.F11; 12, 1.14.B11; and 13, 5.22.10.

Fig. 2. DNA contents of parental and 2n and 3n progeny clones

DNA contents were measured in log phase cells by flow cytometry after staining RNAse treated permeabilized cells with propidium iodide. A, parental lines (FV1(SAT), red; LV39c5(HYG), green. B, representative 2n (green; 4.3.G12, 1.10.D9) and 3n (blue; 1.14.E10; 4.9.C8) progeny. M1 refers to cells in the G1/G0 phase of the cell cycle and M2 refers to cells in G2/M phase. Small differences in the M1/M2 ratio reflect minor differences in cellular growth rate and growth phase and are not significant nor found in other studies of these lines.

Fig. 3. Lesion formation in BALB/c mice by parental and hybrid progeny clones. Mice were infected in the hind footpad by s.c. inoculation of 10⁴ metacyclic promastigtoes

Results are representative of three independent experiments.