Stress conditions promote Leishmania hybridization in vitro marked by expression of the ancestral gamete fusogen HAP2 as revealed by single-cell RNA-seq

Abstract

Leishmania are protozoan parasites transmitted by the bite of sand fly vectors producing a wide spectrum of diseases in their mammalian hosts. These diverse clinical outcomes are directly associated with parasite strain and species diversity. Although Leishmania reproduction is mainly clonal, a cryptic sexual cycle capable of producing hybrid genotypes has been inferred from population genetic studies, and directly demonstrated by laboratory crosses. Experimentally, mating competence has been largely confined to promastigotes developing in the sand fly midgut. The ability to hybridize culture promastigotes in vitro has been limited so far to low efficiency crosses between two L. tropica strains, L747 and MA37, that mate with high efficiency in flies. Here, we show that exposure of promastigote cultures to DNA damage stress produces a remarkably enhanced efficiency of in vitro hybridization of the L. tropica strains, and extends to other species, including L. donovani, L. infantum, and L. braziliensis, a capacity to generate intra- and interspecific hybrids. Whole genome sequencing and total DNA content analyses indicate that the hybrids are in each case full genome, mostly tetraploid hybrids. Single-cell RNA sequencing of the L747 and MA37 parental lines highlights the transcriptome heterogeneity of culture promastigotes and reveals discrete clusters that emerge post-irradiation in which genes potentially involved in genetic exchange are expressed, including the ancestral gamete fusogen HAP2. By generating reporter constructs for HAP2, we could select for promastigotes that could either hybridize or not in vitro. Overall, this work reveals that there are specific populations involved in Leishmania hybridization associated with a discernible transcriptomic signature, and that stress facilitated in vitro hybridization can be a transformative approach to generate large numbers of hybrid genotypes between diverse species and strains.

Experimental Hybridization in Leishmania: Tools for the Study of Genetic Exchange

“…In fact, recent evidence in T. brucei and Leishmania suggests that while maxicircle contribution is uniparental, minicircle inheritance is biparental [ 8 , 30 ]. The same has been observed for in vitro Leishmania hybrids [ 15 , 16 ]. It is proposed that maxicircle composition in hybrids is derived from both parents upon completion of genetic exchange, while rapid DNA segregation in the following mitotic divisions may lead to the observed loss of contribution by one of the parents.…”

“…All three in vitro hybrids from a diploid L. donovani × L. major cross were triploid, and two in vitro hybrids obtained from a diploid L. donovani × triploid L. tropica cross were tetraploid [ 15 ]. In the case of a recently published method of irradiation-facilitated in vitro hybridization (discussed below), almost all of the progeny were tetraploid [ 16 ]. Although classical meiosis entails the production of haploid gametic cells after reductional division and subsequent fusion of two different gametes, alternative models can be proposed to explain the generation of polyploid hybrids.…”

“…In 2022, a modified protocol provided a reproducible enhanced recovery rate of in vitro hybrids by several times over through the treatment of parental Leishmania cells with low levels of DNA-damaging agents prior to co-culture [ 16 ]. The rationale for these studies was that in organisms that are known to be facultatively sexual, they can be triggered to initiate their sexual cycle by conditions that produce DNA damage [ 40 , 41 , 42 ].…”

“…L. tropica exposed to 6.5 Gy of gamma-radiation revealed an increase in hybrid recovery of >100-fold in vitro [ 16 ]. The iHybrid approach allowed for the in vitro generation of intraspecific hybrids of L. braziliensis and L. donovani as well as interspecific hybrids between L. tropica and L. infantum ( Figure 1 ).…”

“…Following this initial report, experimental intraspecific, interspecific, and intraclonal hybrids have been recovered from or observed in sand flies co-infected with recombinant lines of Leishmania containing different antibiotic resistance and/or fluorescence genes [ 10 , 11 , 12 , 13 , 14 ]. Most recently, both ourselves and others have reported that axenic culture forms of promastigotes can form stable hybrids entirely in vitro [ 15 , 16 , 17 ]. Possible hybrids formed between intracellular amastigotes residing in a common macrophage vacuole have been described as well [ 18 ].…”