Protozoan parasites of the Leishmania genus produce a spectrum of diseases in their mammalian hosts, including humans, ranging from self-limiting cutaneous lesions and tissue-destructive mucosal involvement to infection of the deep viscera that is fatal in the absence of treatment. These clinical outcomes are associated with an extraordinary diversity of Leishmania species and strains, with more than 20 species that are pathogenic to humans. All members of the genus have a dimorphic life cycle consisting of extracellular promastigotes that multiply asexually in the digestive tract of their sand fly vectors, and amastigotes that multiply asexually in the phagosomes of their mammalian host cells, primarily macrophages. Leishmania were long considered to be essentially clonal, with genetic diversity arising from gradual accumulation of somatic mutations (Tibayrenc et al., 1993).

The reproductive strategies of Leishmania are now known to include a cryptic sexual cycle that has been inferred from the analysis of hybrid genotypes observed in natural isolates (Bañuls et al., 1997; Dujardin et al., 1995; Nolder et al., 2007; Odiwuor et al., 2011; Ravel et al., 2006; Rogers et al., 2014) and directly demonstrated by the generation of hybrids between different Leishmania strains and species in the laboratory (Akopyants et al., 2009; Inbar et al., 2013; Romano et al., 2014; Sadlova et al., 2011). The latter involved mating between extracellular promastigotes stages developing in the sand fly and was achieved by coinfection of flies with two parental lines bearing different drug resistance markers, with subsequent selection of double-drug-resistant cells. Experimentally at least, the sexual cycle is nonobligatory, relatively rare, and confined to life-cycle stages present in the sand fly midgut. Based on whole-genome sequencing analyses, the allele inheritance patterns of experimental hybrids provide strong evidence that the system of genetic exchange in Leishmania is Mendelian and involves meiosis-like sexual recombination (Inbar et al., 2019). Homologues of meiosis-specific genes are found within the genomes of Leishmania and are expressed by stages in the sand fly, including the core meiotic genes SPO11, HOP1, and DMC1 involved in creating DNA double-strand breaks, homologous chromosome alignment, and recombination (Inbar et al., 2017; Ramesh et al., 2005). Homologues of the cell and nuclear fusion proteins HAP2 and GEX1, respectively, have also been found in ancestral trypanosomatid lineages and extant Leishmania species, providing indirect evidence that sex was already present in the last eukaryotic common ancestor (Speijer et al., 2015). Importantly, there has been no direct evidence that any of these genes or their products are involved in Leishmania genetic exchange, and due to the difficulty of making direct observations of the mating events in the sand fly, the precise nature of this reproductive process, including the identification of putative gametic cells, has been difficult to study.

We recently reported that Leishmania promastigotes, which can be grown readily in axenic culture, can form stable hybrids entirely in vitro (Louradour et al., 2020). While this result proved that mating competent cells can arise in culture, its broader applicability to study the biology of mating and to generate recombinant parasites for genetic linkage analysis was limited by the fact that the in vitro hybridization was confined to only two strains of Leishmania tropica and occurred at frequencies far lower than in the sand fly. In the current studies, we have investigated the conditions that might potentiate the mating success of Leishmania in vitro. It is common for organisms that are facultatively sexual to undergo sex in response to environmental stress, including conditions that produce DNA damage (Ram and Hadany, 2016; Schoustra et al., 2010). Thus, a primary function of sex is proposed to be an adaptation for DNA repair, for which a homologous, undamaged chromosome serves as a template (Bernstein et al., 2018). Amongst eukaryotic microbes, oxidative DNA-damaging conditions have been shown to promote sexual spore formation in the yeast Schizosaccharomyces pombe (Bernstein and Johns, 1989) and in the multicellular green alga Volvox carteri (Nedelcu et al., 2004). Oxidative stress also efficiently induced the expression of sexual pheromone precursors and same-sex mating in Candida albicans (Guan et al., 2019). DNA damage caused by X-irradiation induced meiotic recombination in the budding yeast Sacchromyces cerevisiae (Thorne and Byers, 1993) and in the nematode Caenorhabditis elegans (Dernburg et al., 1998). In the current studies, we have used DNA-damaging conditions to promote in vitro hybridization of Leishmania. Exposure to H₂O_2, methyl methanesulfonate (MMS), or to γ-radiation produced a remarkably enhanced efficiency in the in vitro hybridization of the L. tropica strains, and extended to other species and strains a capacity for in vitro hybridization. Single-cell RNA-seq comparisons of untreated and irradiated promastigotes revealed greatly expanded subpopulations amongst the irradiated cells that expressed homologues of genes involved in genetic exchange. We identified expression of the ancestral gamete fusogen HAP2 as a selectable marker for promastigotes able to hybridize in vitro.

Reproduction in Leishmania includes a cryptic sexual cycle that operates among the extracellular promastigote stages developing in the sand fly vector. The mating-competent cells, and in particular the existence of a gametic stage, have yet to be identified. Studying the biology of mating in Leishmania would be greatly facilitated if these events could be reproduced using cells from axenic cultures, and we have recently achieved the recovery of stable hybrids generated between cultured promastigotes entirely in vitro (Louradour et al., 2020). While these initial findings established that mating-competent forms can arise in axenic culture, the mating frequencies were extremely low, and only a single pairwise combination of L. tropica parental lines would successfully hybridize. In the current studies, we demonstrate that under various conditions of environmental stress, including exposure to H₂O₂, MMS, or γ-radiation, the in vitro hybridization capacity of the L. tropica strains could be markedly enhanced and extended to other L. tropica strains and multiple Leishmania species, including L. infantum, L. donovani, and L. braziliensis. The products of these cell fusion events were in each case full-genome, polyploid hybrids. By single-cell RNA-seq analysis, we could identify transcriptionally unique populations of promastigotes whose numbers were drastically increased in each parental line following exposure to γ-radiation, and for which specific meiotic gene homologues, including the ancestral gamete fusogen HAP2, were found to be upregulated. By generating reporter constructs for HAP2, we could select for promastigotes that could hybridize or not in vitro.

Our findings add to the list of facultatively sexual eukaryotes, including yeast and green alga, that exhibit more sexual events under stress conditions. As each of the stress conditions that we imposed are known to produce DNA damage, the results also support studies in varied model eukaryotes, suggesting that DNA repair is a central adaptive function of meiotic recombination (Bernstein et al., 2018; Ram and Hadany, 2016; Schoustra et al., 2010). Homologous chromosome pairing during meiosis provides an undamaged, template chromosome for DNA repair. The generation and repair of double-strand breaks is evidenced in our in vitro hybrids by the substantial number of chromosomes for which there were changes within the chromosome in the relative contribution of the parental alleles. A role for SPO11 protein in the induction of programmed DNA double-strand breaks is argued to be a universal feature of meiotic crossover events in eukaryotes (Keeney et al., 1997). However, meiotic recombination induced by X-irradiation in S. cerevisiae and in C. elegans was shown to occur even when SPO11 was absent or nonfunctional (Dernburg et al., 1998; Thorne and Byers, 1993). In our studies, expression of the homologous SPO11 gene in L. tropica was not upregulated in the irradiated, hybridization-competent cells.

In order to identify hybridization-competent cells present in Leishmania cultures, we performed single-cell RNA sequencing of L747 and MA37 cultured cells exposed or not to γ-radiation. To our knowledge, this is the first application of single-cell transcriptomics in Leishmania, although it has been applied to other kinetoplastid pathogens to reveal the heterogeneity of the parasite populations arising in their mammalian hosts or insect vectors (reviewed in Briggs et al., 2021). Our analyses revealed transcriptomic heterogeneity within and between the parental L. tropica lines in the promastigote cultures. The heterogeneity likely reflects cells in different phases of the cell cycle, as well as differences in the developmental stage progression of the cultured promastigotes. The extensive differences in the transcriptomes of cells comparing the parental lines, apparent in both the PCA and UMAP visualizations, are surprising given the genetic similarity between these L. tropica strains (Iantorno et al., 2017) and likely reflect their different culture histories.

More meaningful to the current studies is the cluster heterogeneity comparing the untreated versus irradiated cells, which for each parental line revealed the emergence of a transcriptionally unique population in the irradiated cultures. Probing the clusters for enriched transcripts related to meiotic processes identified homologues of HAP2, GEX1, and RAD51, involved respectively in cell fusion, nuclear fusion, and DNA repair, that were largely confined to the unique clusters that emerged in the irradiated cultures. Even though a large fraction of the transcriptional shift is likely explained as part of a more global cellular mechanism to recover from stress caused by the irradiation, the expansion of cells expressing HAP2 transcripts in treated samples, the co-regulation of other meiotic genes (a HAP2-like FusM homologue, GEX1, and RAD51), and validation in vitro using HAP2 reporters, all strongly suggest that these cells are associated with the enhanced hybridization frequency observed post-treatment. The possible role in genetic exchange of other genes coordinately expressed with HAP2 but without any described meiotic function will be investigated in future studies. Of note, RNA-binding proteins, such as RBP5 and ZC3H11, were found to be upregulated in these cells and could have novel regulatory functions during Leishmania mating. Critically, we did not find significant upregulation of other meiosis-specific markers, including SPO11, DMC1, and MND1. This suggests that cell fusion might be a response to DNA stress that is independent of a meiotic program, especially as the meiotic reduction steps were for the most part absent in the stress-induced hybrids. Nevertheless, a similar finding was recently reported in an scRNA-seq analysis of T. brucei stages in the tsetse fly salivary glands, in which the gamete cell cluster was defined based solely on the upregulation of HOP1 and HAP2 (Hutchinson et al., 2021). It is possible that some of these markers of gametogenesis are expressed within a narrow time frame, and by analyzing single-cell gene expression at a single time point post-irradiation, we may have missed their expression peak in what is likely a rare cell population. In addition, expression of several genes may not have been detected due to ‘dropout’ events or excessive zeros, which has often been described in this type of approach due to a lower sequencing depth when compared to bulk RNA-seq (Choi et al., 2020).

HAP2 is a type I transmembrane protein that displays the same three-dimensional fold as class II viral fusion proteins. It plays a key role in the membrane fusion of gametes or mating cells that was first identified in plants, and is broadly conserved from protists to invertebrates (Cole et al., 2014; Fédry et al., 2017; Johnson et al., 2004; Liu et al., 2008; Mori et al., 2006). HAP2 expression was recently reported in T. brucei, both in gametes and in meiotic intermediates (Peacock et al., 2021). In the current work, we generated reporter constructs for HAP2 in L747 and MA37 and could show enhanced expression or altered expression kinetics in the stressed cultures. By sorting the cells into mNG⁺ and mNG^- populations, we could confirm that HAP2 expression is a marker for hybridization-competent cells since hybridization failed unless the co-cultures included mNG⁺ selected cells from one or both of the parents. To the extent that HAP2 is itself required for hybridization, then the apparent requirement for HAP2 expression on just one of the fusing cells is consistent with a number of studies showing that it can function unilaterally; either on the mating type minus gamete in Chlamydomonas algae (Fédry et al., 2017; Liu et al., 2008) or on the male gamete in Plasmodium (Liu et al., 2008), the ciliated protozoan Tetrahymena thermophila (Cole et al., 2014), or the plant Arabidopsis thaliana (Johnson et al., 2004; Mori et al., 2006). It is interesting that the MA37 parent showed a relatively high frequency of HAP2-expressing cells even absent exposure to the exogenous stress conditions. This may explain why this L. tropica strain hybridizes with such high efficiency in flies (Inbar et al., 2019), and why we are able to obtain some in vitro hybrids even without exogenous stress so long as MA37 is a fusion partner.

Contrary to the in vitro LMA hybrids that were previously generated, which were diploid, triploid, and tetraploid (Louradour et al., 2020), the irradiation-facilitated LMA hybrids, as well as the other intra- and interspecies hybrids described in this report, are in their great majority tetraploid. These results strongly suggest fusion between the two diploid parental cells, and that the stress conditions that promoted the fusogenic potential of the cells may have produced unusual features leading to unreduced gamete formation, which is a common mechanism underlying polyploidization in plants (De Storme and Geelen, 2013). Attempts to condition the tetraploid hybrids to complete a putative meiotic program by passing them through flies or by re-exposure to stress conditions failed to reduce their ploidy, showing that their polyploid state is relatively stable. As tetraploid progeny are occasional products of hybridization events in flies, it is also possible that tetraploid intermediates, as described for some fungi (Heitman, 2010; Roman et al., 1955), are a normal part of the mating process in Leishmania, with the ploidy reduction steps somehow lacking in the in vitro protocol. Since in the related protist T. brucei, haploid gametic cells have been identified prior to fusion in cells localized to the salivary gland of the tsetse fly vector (Peacock et al., 2014), we favor a comparable sexual process can operate during Leishmania development in sand flies. The fact that a few triploid hybrids, also observed in flies, were generated by the in vitro protocol could mean that a haploid gamete was produced by at least one of the parents and may encourage additional manipulations of the culture conditions to promote a more complete and uniform sexual cycle in vitro.

The absence of random segregation and genome-wide recombinations associated with a reductional meiotic program will undermine the utility of the hybrids for use in SNP association studies. It is interesting, nonetheless, that the hybrids seem to have retained an ability for homologue pairing and recombination after fusion, as evidenced by the breaks within chromosomes in the relative contributions of the parental alleles. Since the maintenance of the tetraploid state (two genome complements from each parent) would be expected to mask recombinations generated by reciprocal crossover events typically associated with meiosis I, the data suggest noncrossovers or gene conversion as a mechanism of DNA repair that would result in gene exchange in only one of the homologs. The asymmetric parental contributions observed both within and between different chromosomes could also have arisen by chromosome loss or gain at the level of individual somy, which is well described for Leishmania genomes during vegetative growth (Dujardin et al., 2007; Sterkers et al., 2011). Aneuploidy, including mosaic aneuploidy, is a constitutive feature of the Leishmania genome (Sterkers et al., 2012). Since gene dosage can directly control the levels of gene expression in Leishmania and impact phenotypes (Iantorno et al., 2017; Prieto Barja et al., 2017), the chromosome-specific patterns of aneuploidy and recombination in the hybrids can be used for linkage studies. It is notable that the progeny that were recovered are in almost every case distinct from the other progeny clones generated in the same experimental cross with respect to the combinations of the relative parental contributions to individual chromosomes that they possess. The distinct haplotypes observed in each of the 17 hybrids generated between the L. donovani strains from India and Sri Lanka are a case in point. As these parental strains produce, respectively, visceral and cutaneous forms of disease in humans and in mice (Kariyawasam et al., 2018; Karunaweera et al., 2003), it may be possible to map the genes controlling the behavior of the progeny clones in mice by genome-wide association studies. At a minimum, the admixture of these genomes should permit assessment as to whether or not the cutaneous or visceral tropisms are expressed as a dominant trait.

The stress-facilitated, in vitro hybridization protocol described in this report removes the requirement for sand flies as the main constraint to generating large numbers of genomic hybrids between Leishmania species and strains, and offers a transformative approach for genetic analysis to understand the extraordinary phenotypic diversity of the genus.

The raw sequence data containing reads from the 51 WGS samples and 8 scRNA-seq samples sequenced are deposited in the SRA database with Accession numbers PRJNA756557 and PRJNA756571, respectively. The source data files for Figures 1 and 6 are provided. Summary statistics on the sequencing data are available in Supplementary files 6 and 7.

1. 1. Sacks D

   (2022) NCBI BioProject

   ID PRJNA756557. raw sequence data containing reads from the 51 WGS samples.

   https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA756557
2. 1. Sacks D

   (2022) NCBI BioProject

   ID PRJNA756571. scRNA-seq samples sequenced.

   https://www.ncbi.nlm.nih.gov/bioproject/?term=PRJNA756571

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Author details

1. Isabelle Louradour

   Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, United States

   Present address

   Department of Parasites and Insect Vectors, Institut Pasteur/INSERM U1201, Paris, France

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Tiago Rodrigues Ferreira

   Competing interests

   No competing interests declared

2. Tiago Rodrigues Ferreira

   Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, United States

   Contribution

   Conceptualization, Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Isabelle Louradour

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-4894-1515

3. Emma Duge

   Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, United States

   Contribution

   Formal analysis, Investigation, Methodology

   Competing interests

   No competing interests declared

4. Nadira Karunaweera

   Department of Parasitology, Faculty of Medicine, University of Colombo, Colombo, Sri Lanka

   Contribution

   Conceptualization, Resources

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-3985-1817

5. Andrea Paun

   Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, United States

   Contribution

   Investigation

   Competing interests

   No competing interests declared

6. David Sacks

   Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, United States

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Supervision, Writing – original draft, Writing – review and editing

   For correspondence

   dsacks@nih.gov

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7557-3124

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