Cross-species genetic exchange between visceral and cutaneous strains of in the sand fly vector

Audrey Romanoa, Ehud Inbara, Alain Debrabantb, Melanie Charmoya, Phillip Lawyera, Flavia Ribeiro-Gomesa, Mourad Barhoumia, Michael Grigga, Jahangheer Shaikc, Deborah Dobsonc, Stephen M. Beverleyc,1, and David L. Sacksa,1,2

aLaboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892; bDivision of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892; and cDepartment of Molecular Microbiology, Washington University School of Medicine, St. Louis, MO 63110

Edited by Anthony A. James, University of California, Irvine, CA, and approved October 22, 2014 (received for review August 6, 2014) Genetic exchange between strains during their the frequency of these recombination events underlies the con- development in the sand fly vector has been experimentally tinuing clonality vs. sexuality debate (17). shown. To investigate the possibility of genetic exchange between By using drug-resistance markers, the first direct demonstra- different Leishmania species, a cutaneous strain of L. major and tion of genetic exchange between different L. major strains was a visceral strain of Leishmania infantum, each bearing a different reported (18) and possibly between L. donovani lines using drug-resistant marker, were used to coinfect fluorescent markers (19). In each case, the hybrids were gener- sand flies. Eleven double–drug-resistant progeny clones, each the ated between the extracellular promastigote stages in the sand fly product of an independent mating event, were generated and vector. These and more recent studies (20, 21) critically inform submitted to genotype and phenotype analyses. The analysis of the population genetics data by formally demonstrating that multiple allelic markers across the genome suggested that each Leishmania are capable of intraspecies and even intraclonal progeny clone inherited at least one full set of chromosomes from mating. The present studies were designed to provide, to our each parent, with loss of heterozygosity at some loci, and unipa- knowledge, the first experimental demonstration of cross-species rental retention of maxicircle kinetoplast DNA. Hybrids with DNA mating and to explore the heritability of the genes controlling contents of approximately 2n, 3n, and 4n were observed. In vivo tissue-specific tropisms. By using L. major and L. infantum pa- studies revealed clear differences in the ability of the hybrids to rental lines bearing independent drug-resistance markers and produce pathology in the skin or to disseminate to and grow in the viscera, suggesting polymorphisms and differential inheritance of that reproduce in mice their respective human cutaneous and the gene(s) controlling these traits. The studies, to our knowledge, visceral tropisms, hybrid progeny were recovered from coin- represent the first experimental confirmation of cross-species mat- fected Lutzomyia longipalpis sand flies. Detailed genotyping and ing in Leishmania, opening the way toward genetic linkage anal- phenotyping of 11 progeny clones are described. ysis of important traits and providing strong evidence that genetic Results exchange is responsible for the generation of the mixed-species genotypes observed in natural populations. Generation of Hybrids. To generate hybrids between L. major and L. infantum, we used Lu. Longipalpis, a sand fly species that is Leishmania | genetic exchange | sand fly experimentally permissive to both strains (22). We tested L. major strain Friedlin clone V1 (LmjF) expressing a drug-resistance gene inetoplastid protozoan parasites of the genus Leishmania Kare transmitted by a sand fly bite into the skin of the Significance mammalian host. More than 20 different species of Leishmania are known to produce disease in humans, ranging from localized, Protozoan parasites of the genus Leishmania are transmitted self-limiting cutaneous lesions that develop at the site of in- by sand flies and produce diseases in humans ranging from oculation to visceralizing infections that are fatal in the absence of localized cutaneous lesions to fatal visceral infection. Although treatment (1). These diverse clinical outcomes have generally clear these clinical outcomes have clear parasite species associations, parasite species and strain associations, although the ability of the genes controlling these differences are not known. We provide, to our knowledge, the first experimental demonstra- visceral strains to occasionally produce cutaneous disease, and vice tion of genetic exchange in the sand fly vector between dif- versa, is also well described. The specific contribution of parasite ferent Leishmania species: a cutaneous strain of Leishmania genotype to disease outcome remains largely unknown. Studies by major and a visceral strain of Leishmania infantum. Eleven full Zhang et al. demonstrated that the introduction of Leishmania genomic hybrids were generated that displayed differences in donovani-specific genes into Leishmania major increased their their ability to grow in the skin or viscera of mice, indicating abilitytogrowinthevisceraofBALB/cmice,butinnocase that the genes controlling these traits may be polymorphic reached the level of infection produced by the visceral strains (2, 3), within the parental species and are potentially amenable to suggesting that these tissue tropisms are under multigenic control. identification by classical linkage analysis. The Leishmania genome is organized in 36 chromosomes for the Old World species, such as L. major, L. donovani, and Author contributions: A.R., E.I., P.L., S.M.B., and D.L.S. designed research; A.R., E.I., M.C., P.L., F.R.-G., J.S., D.D., and D.L.S. performed research; A.R., A.D., M.B., M.G., J.S., D.D., and Leishmania infantum, and in 35 and 34 for the New World S.M.B. contributed new reagents/analytic tools; A.R., E.I., J.S., D.D., S.M.B., and D.L.S. species and , re- analyzed data; and A.R., S.M.B., and D.L.S. wrote the paper. spectively (4, 5). Although approximately a diploid organism, The authors declare no conflict of interest. aneuploidy, including mosaic aneuploidy, is now known to be This article is a PNAS Direct Submission. – “ ” widespread (6 10); we refer to the nearly diploid state as 2n 1S.M.B. and D.L.S. contributed equally to this work. here. A series of population genetic studies have suggested that 2To whom correspondence should be addressed. Email: [email protected]. clonal lineages have experienced at least occasional bouts of This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. genetic exchange both within and between species (11–16), and 1073/pnas.1415109111/-/DCSupplemental.

16808–16813 | PNAS | November 25, 2014 | vol. 111 | no. 47 www.pnas.org/cgi/doi/10.1073/pnas.1415109111 Downloaded by guest on September 27, 2021 for nourseothricin (SAT)(18)andL. infantum LLM-320 (LinL) expressing a drug-resistance gene for hygromycin B (HYG), each inserted into the ribosomal RNA locus located on chromosome (Chr) 27 (Materials and Methods). As shown in Fig. S1, clonal lines of each were able to establish and maintain midgut infec- tions during and after blood-meal digestion and excretion, al- though the intensity of the infection was 1.6–6.7 times greater for L. major compared with L. infantum. The Lu. longipalpis sand flies were coinfected with 4 × 106 and 8 × 106 parasites per mL of blood of L. major–SAT (Lm) and L. infantum–HYG (Li), respectively. A total of 446 sand flies from five independent coinfections were dissected at different times postinfection (p.i.; 5–11 d p.i.) to recover double–drug- resistant parasites (Table S1). Of the cultures established from the midgut homogenates of these flies, 345 were free of bacterial or fungal contamination, and of these a total of 11 yielded doubly drug-resistant parasites (3.2%) from which 11 progeny clones were generated, each representing an independent crossing event. The percent of flies yielding hybrids was less than that seen in our previous studies involving intraspecies mating of L. major [7–26% (18, 20)], which may be related to the greater genetic distance between the Lm–Li parents vs. different geo- graphic L. major isolates. Because the low frequency of mating requires selection for both parental markers, only 25% of the successful mating events involving the heterozygous parents can be recovered in 2n progeny

SNP Analysis of the Lm/Li Hybrids. Using specific primers, we confirmed for each progeny clone the presence of both parental antibiotic-resistance genes (Fig. 1A). The inheritance patterns of nine additional unlinked marker loci located on nine different chromosomes (Table S2) were determined by SNP–cleaved amplification polymorphic site (SNP-CAPS) analysis (represen- tative data shown in Fig. 1 B–E). The SNPs were identified by

comparing gene alignments determined from the genome data GENETICS bases of L. major and L. infantum. Table 1 summarizes the nu- clear markers genotyping. Ten of the 11 hybrids inherited both parental alleles at all of the loci analyzed, whereas LimH1 inherited both parental alleles at all marker loci tested except for Fig. 1. Genotype analysis of the progeny clones. (A) PCR products for pa- the marker on Chr 29, for which only the Lm parental allele was rental selectable markers SAT and HYG.(B and C) SNP-CAPS analysis for loci observed (Fig. 1B and Table 1). These genotypes were maintained on Chrs 9 and 29 (B) and for the same marker on Chr 29 (C) following re- following passage of each of the hybrid progeny through mice and covery of progeny clones from C57BL/6 mice. (D) Digestion with BsteII of the LmjF.25.2420 locus. The bar graph shows the ratio between the intensity of their subsequent serial subculture as promastigotes, except for the middle band from Lm and the uncut upper band from Li. Values shown LimH2, which became homozygous for the Lm parental allelic are the means ± SD of three independent experiments. *P < 0.002 (vs. 1:1 marker on Chr 29 (Fig. 1C). The simplest interpretation of these mix). (E) Digestion with PstI of the LmjF.35.0050 locus. The bar graph shows data is that the hybrids are full genomic hybrids, in which limited the ratio between the intensity of the upper band (519 bp) from Lm and the regions of the genome show one parental allele via mechanisms intermediate band (300 bp) from Li. Values shown are the means ± SD of leading to loss of heterozygosity, as proposed previously (20). two independent experiments. #P < 0.05 (vs. 1:1 mix). We included in our nuclear DNA genotyping an analysis of the A2 loci because the expression of these amastigote-specific genes is associated with the capacity of L. donovani and L. infantum to most of the parental chromosomes are disomic. Staining with propidium iodide does not distinguish kinetoplast (mitochondrial visceralize (23). In these species, multiple copies of the A2 genes “ ” are arranged in tandem on Chr 22, whereas in L. major, the DNA) and nuclear DNA, which are both included as total corresponding sequences have been found functionally expressed DNA. By this standard, five hybrids showed profiles for 2n DNA as a single copy gene (24). Amplification of these sequences in Li content, whereas five bore 3n and one bore 4n DNA content. The and Lm confirmed a full-length and truncated copy of the A2 DNA content of each of the 3n and 4n hybrids appeared stable gene(s), respectively, and revealed that each of the hybrids had after their isolation as tissue amastigotes from infected mice and inherited at least one A2 locus from each parent (Fig. S2). The their subsequent serial subculture as promastigotes. hybrids were also comparable in the stress-induced expression of To identify the origin of the extra chromosomes, we de- the A2 genes following exposure of the promastigotes to elevated termined the relative intensity of SNP-CAPS digestion products temperature, as described (25). for two loci in the 3n progeny. Analysis of a SNP in gene LmjF.25.2420 results in the presence of a BstEII restriction site Hybrids Show 2n, 3n, and 4n DNA Contents. The DNA contents of in the Lm allele, but not in the Li allele. The ratio between the all of the progeny clones were measured relative to those of the intensity of the middle band from Lm and the uncut upper band parental lines at the cell population level by flow cytometry (Fig. from Li are shown for selected hybrids on the bar graph in Fig. S3 and Table S3). Both the parental Lm and Li lines showed 1D and in comparison with controls generated by mixing the similar profiles; here we refer to the major G1 peak as 2n, be- respective parental DNA Lm:Li at 1:1; 2:1, and 1:2 ratios. A cause despite the occurrence of aneuploidy in the two parents, similar analysis was carried out on a SNP in LmjF.35.0050 that

Romano et al. PNAS | November 25, 2014 | vol. 111 | no. 47 | 16809 Downloaded by guest on September 27, 2021 Table 1. Summary of nuclear markers genotyping by SNP-CAPS fit hybrids for survival in P. duboscqi, suggesting a gene- Chromosome no. dosage effect. Cutaneous in BALB/c and C57BL/6 mice. Infection of Parasite 579212225293135BALB/c mice with L. major in the footpad represents a classical experimental model of nonhealing . As Lm MMMM M M M M Mshown in Fig. 3A, footpad inoculation of 106 metacyclics of the Li IIII I I I I ILm parental line produced rapidly progressing lesions, whereas LimH1 H H H H H H M H H the Li parent produced no footpad swelling, although low LimH2 H H H H H H H/M* H H numbers of viable organisms could be recovered from the in- LimH3 H H H H H H H H H oculation site at even late time points. All of the hybrids behaved LimH4 H H H H H H H H H identically to the Li parent and failed to produce any lesions for LimH5 H H H H H H H H H up to 9 wk p.i. Thus, the inability to grow or to produce cuta- LimH6 H H H H H H H H H neous pathology in BALB/c mice is inherited from the Li parent LimH7 H H H H H H H H H as a fully dominant trait. LimH9 H H H H H H H H H In contrast to the nonhealing infections in BALB/c mice, LimH10 H H H H H H H H H L. major infection in C57BL/6 mice results in self-limiting LimH11 H H H H H H H H H lesions, and the ear dermis has become a favored inoculation site LimH12 H H H H H H H H H to study cutaneous leishmaniasis in this model. Fig. 3B shows the H, heterozygous; I, Li;M,Lm. development of large, chronic lesions in the ear dermis p.i. with *Genotype pre-/postpassage in mice. the Lm parent and the minimal and transient cutaneous pa- thology produced by Li. The progeny clones in this case did not behave uniformly like the Li parent, with both 3n hybrids with results in a PstI restriction site in both the Li and Lm alleles (Fig. the extra chromosomes from Lm and two of the 2n hybrids 1E). From both analyses, we conclude that LimH1 and LimH5 (LimH2 and LimH7) producing larger and persisting lesions. By inherited the extra trisomic Chrs 25 and 35 from Lm, whereas for contrast, the 3n hybrids with the extra chromosomes from Li and LimH9 and LimH12, the extra trisomic chromosome genome is the 2n LimH11 and LimH4 hybrids produced minimal pathology, from Li. The differences in the Lm/Li ratios particularly likely similar to the Li parent. The 3n LimH3 hybrid also produced reflect mosaic aneuploidy in the progeny, where trisomy would minimal pathology in the skin. be present in a variable proportion of each cell population. Note in C57BL/6 mice. Inoculation of mice in the ear that discordant results for the two markers were obtained for dermis with a high dose of L. infantum metacyclic promastigotes LimH3, suggesting that both parents were able to transmit an has been introduced as an experimental model of visceral extra chromosome to this hybrid. leishmaniasis that better reflects the conditions of natural in- fection compared with i.v. inoculation (27). Fig. 3C shows the Maxicircle Inheritance. We then assessed the inheritance of the parasite loads in the liver and spleen 6 wk p.i. with 2 × 106 kinetoplast maxicircle DNA by sequence and SNP-CAPS anal- metacyclics in the ear dermis. The Li parental line established ysis of SNPs identified within three kinetoplast DNA (kDNA) low but reproducible infections in the spleen (102) and liver (103) genes: 12s rRNA, CYTB, and ND5 (Table S3 and Fig. S4). All of following intradermal (i.d.) inoculation, whereas the Lm parent the hybrid clones were homozygous for each kDNA gene ana- was minimally detectable in these organs (<10). The 3n hybrids lyzed, with three having inherited their kDNA only from the Lm bearing the extra chromosomes from Lm behaved like the Lm parent and eight only from the Li parent, suggesting uniparental parent, with virtually no parasites detectable in the liver or retention of maxicircle kDNA. A summary of the ploidy and spleen, whereas the two 2n hybrids (LimH2 and LimH7) that maxicircle inheritance patterns of the 11 progeny clones, and produced the greatest cutaneous pathology were minimally their associations with the phenotypes described below, is pre- detected in the spleen and were significantly different from both sented in Table 2.

Phenotype Analysis of the Lm/Li Hybrids. Table 2. Summary of hybrid genotypes and phenotypes in mice Infection in . duboscqi P. duboscqi is a natural vector of Genotype Phenotype L. major transmission in west Africa, and both in nature and † ‡ ‡ ‡ ‡ experimentally, its vector competence is restricted to L. major Parasite Ploidy CAPS kDNA s.c.* i.d. i.d. spl i.d.liv i.v. spl i.v. liv (26). Survival of Leishmania in the sand fly midgut is tightly Lm 2n 1:1 M M M M M M M controlled by the polymorphic structure of the surface lipo- Li 2n 1:1 I I I I I I I phosphoglycan (LPG) that mediates promastigote adhesion to LimH2 2n 1:1 I I M M Int Int Int midgut epithelial cells and prevents their elimination during LimH4 2n 1:1 I I I I I I I excretion of the digested blood meal. Thus, Li promastigotes, β LimH6 2n 1:1 M I I M I Int I synthesizing LPG lacking in 1,3-galactosyl side chains recog- LimH7 2n 1:1 I I M M M Int Int nized by the monoclonal antibody (mAb) WIC79.3 (Fig. 2A), LimH11 2n 1:1 I I I I I I I grow adequately in the blood-fed midgut of P. duboscqi during LimH1 3n M > II IM M M M M the first 2 d p.i., but are eliminated from the midgut along with LimH5 3n M > IM I M M M IntInt the blood meal remnants by day 5 (Fig. 2B and Fig. S5). By LimH3 3n n.d. I I I M M M Int contrast, Lm promastigotes expressing a poly-galactosylated LPG LimH9 3n M < IM I I I I I I and strongly agglutinated by WIC79.3 are retained in the midgut LimH12 3n M < II I I I I I I after blood meal excretion. The Li/Lm hybrids expressed in- termediate levels of galactosylated LPG and accordingly dis- n.d., not determined. *M (Lm), progressive footpad lesion in BALB/c mice; I (Li), no lesion. † played a variable, although generally intermediate, phenotype I, transient ear lesion in C57BL/6 mice; M, persistent lesion >5 wk. ‡ with respect to the percentage of flies retaining infections after I, parasite load significantly different from Lm; M, parasite load significantly blood meal excretion. The 3n hybrids for which the extra chro- different from Li; Int, intermediate parasite load significantly different from mosomes were transmitted by the Li parent were the least Li and Lm.

16810 | www.pnas.org/cgi/doi/10.1073/pnas.1415109111 Romano et al. Downloaded by guest on September 27, 2021 Fig. 2. LPG expression and P. duboscqi fitness phe- notypes of parents and progeny clones. (A)WIC79.3- mediated agglutination profiles of parents (Left), diploid (Center), and triploid (Right) progeny clones. (B) P. duboscqi were infected with 4 × 106 parasites per milliliter of mouse blood. The infection was monitored at 2 d (Left)and5d(Right) postblood meal (PBM), corresponding to the times before and just after passage of the digested blood. The percentage of flies (10–12 flies per group) harboring viable midgut promastigotes is shown. White bars represent the parental lines, black bars the 2n hybrids, and stippled bars the 3n hybrids. Values shown are the means ± SD of three independent experiments. #P < 0.05 (vs. Lm); *P < 0.05 (vs. Li).

parental phenotypes in the liver. The two 3n hybrids with the extra set of chromosomes could be contributed by either parent. extra chromosomes from Li, and the 2n LimH11 and LmH4 Furthermore, the 3n and 4n DNA contents were maintained in hybrids that produced minimal pathology in the skin, behaved the hybrids throughout numerous in vitro passages as promasti- comparably to the Li parent in both the liver and spleen. LimH3 gotes and after recovery of tissue amastigotes from infected failed to disseminate to and/or grow in the viscera, but because it mice, suggesting that the polyploidy is not intrinsically unstable. also grew poorly in the sand fly midgut and produced minimal As with all of the Leishmania intraspecies hybrids described pathology in the skin, this hybrid appears to have a generalized previously (18, 20), analysis of markers on the maxicircle DNA growth defect that is unrelated to developmental stage or tissue indicates that kDNA inheritance was uniparental and that either preference. parent could contribute kDNA to the progeny. Although a sim- The visceral dissemination and growth phenotypes of the ilar conclusion was accorded the inheritance of maxicircle poly-

hybrids were largely replicated by using the more conventional morphisms in hybrid progeny of , subsequent GENETICS visceral leishmaniasis infection model involving high-dose i.v. analyses at an earlier stage of hybrid generation indicated that inoculation (Fig. 3D). The 3n hybrids with the extra Lm chro- kDNA inheritance was biparental, with segregation of max- mosomes again showed the least growth in both organs, whereas icircles during subsequent mitotic divisions (28). 2n LimH2 and LimH7 hybrids also displayed reduced growth in Our attempts to cross-hybridize Leishmania species was de- both organs compared with the Li parent, although significantly liberately explored by using L. infantum and L. major because greater than the Lm parent. The 2n LimH6 hybrid showed sig- the biology of these species is so clearly distinguishable in both nificantly reduced growth only in the spleen. By contrast, the 3n their invertebrate and vertebrate hosts. In the sand fly, the nat- hybrids with the extra chromosomes from Li and the 2n LimH11 ural as well as experimental vector competence of P. duboscqi is Li and LimH4 hybrids again behaved most like the parent, with restricted to L. major, whereas infections with other Leishmania comparable growth in both the liver and spleen. Together, these species, including L. infantum, are lost during excretion of the results indicate that the origin of the extra chromosomes in the blood-meal remnants (26). The hybrids displayed variable, but 3n progeny correlates to the phenotype, whereas for the 2n more or less intermediate, phenotypes compared with either progeny, a number of mechanisms, including differential allelic parent, both in the expression of poly-galactosylated LPG that inheritance, may control their tissue tropisms. mediates midgut attachment and their persistence in the midgut Discussion after blood-meal excretion. The increased fitness of some of the This study, to our knowledge, represents the first experimental hybrids compared with the Li parent for potential transmission confirmation of cross-species mating in Leishmania. Eleven hy- by a normally refractory vector replicates the findings reported brid progeny were generated between L. major and L. infantum for natural genetic hybrids between L. infantum and L. major in Lu. longipalpis. The genetic analysis of multiple allelic markers (29). The experimental hybrids validate that these genotypes can distributed across the genome indicates that every progeny is arise via genetic exchange. likely a full genomic hybrid, with occasional loss of heterozy- The phenotype analysis of the Lm/Li hybrids for their tissue gosity at some loci. The conclusion that the apparent uniparental tropisms in the mammalian host was of obvious interest. Using inheritance of a marker locus in two of the progeny clones was in the classical experimental model to study nonhealing forms of fact the result of a postmating process leading to the loss of cutaneous leishmaniasis, we unexpectedly found that all of the heterozygosity is supported by the sequential genotyping of one hybrids, including the 3n hybrids bearing the extra genome from of the hybrids (LimH2) showing the loss of one the parental Lm, phenocopied the Li parent in producing no footpad lesions alleles after mouse passage. DNA content analysis of the hybrid at all. A similar phenotype was previously observed by using clones revealed that the 11 independent crosses produced five cosmid-transfected L. major capable of expressing multiple copies 2n, five 3n, and a single 4n progeny. Triploid progeny have also of the L. donovani A2 gene (30). Because the development of been observed for many of the L. major intraspecies hybrids nonhealing L. major lesions in BALB/c mice is largely dependent described (18, 20). Based on the relative intensities of SNP- on a parasite-driven Th2 response, the effect of the Li genes, CAPS digestion products, we concluded that at least part of the including A2, is likely due to an inhibition of this response.

Romano et al. PNAS | November 25, 2014 | vol. 111 | no. 47 | 16811 Downloaded by guest on September 27, 2021 observed. A possible effect of increasing gene dosage due to poly- ploidy was clearly evident, because the triploid hybrids displayed distinct skin or viscera tropisms depending on the parental origin of the extra chromosomes, although differences in allelic inheritance might still be influencing the behavior of these clones. Although there is strong evidence that A2 genes influence the ability of Leishmania to disseminate to and/or grow in the viscera (23), there were no apparent differences between the hybrids in A2 gene inheritance or inducible expression in vitro (Fig. S1). At least three additional L. donovani-specific orthologs of L. infantum genes were found to promote L. major survival in the viscera (2, 3). Quantitative trait loci mapping of the genes con- trolling these traits in a larger series of Lm/Li hybrids should advance our understanding of these basic characters beyond what has so far been possible using reverse genetic approaches. Our formal demonstration that experimental hybridization between different Leishmania species can occur could be viewed as a challenge to the conventional species definition of these eukaryotic microbes, which in Leishmania and other microbes has largely devolved toward molecular distance criteria (33). However, it is important to note that the fertility of the offspring themselves has not yet been addressed, an important criterion because interspecies hybrids in metazoans are typically infertile, the classic example being the mule. Additionally, it is well known that taxa can differ in their ability to undergo hybridization over evolutionary distances (34). Thus, the ability of these Lm/Li hybrids to cross among themselves or with either parent—and the consequences of this information to understanding of Leishmania species boundaries and biology—is an important area for future study. Based on their high degree of synteny and low number of species-specific genes (35), it may not be sur- prising that these species can experimentally cross-hybridize, presented the opportunity. As a tool for experimental analysis, Fig. 3. Progeny virulence in mouse models of cutaneous and visceral leish- cross-species mating offers a powerful genetic approach to ad- maniasis. (A) Two million metacyclic promastigotes were inoculated s.c. in the vance our understanding of the molecules controlling the re- footpad of BALB/c mice. Footpad width (millimeters) was measured weekly. Results shown are means ± SD of three mice per group. The experiment was markable biological diversity of the genus. repeated once with identical results. (B) Four million metacyclic promastigotes were inoculated i.d. in the ear pinnae of C57BL/6 mice. Ear lesion diameter Materials and Methods (millimeters) was measured weekly. Results shown are means ± SD of the pool Parasite Strains. L. major FV1 (SAT), containing a heterozygous nourseo- of two independent experiments (n = 5 per group per experiment). (C) Par- thricin-resistance marker integrated into one allele of the 18S rRNA locus asite load in the spleen (Left) and liver (Right) of C57BL/6 mice 6 wk p.i. with located on Chr 27 (5), was derived from NIH Friedlin clone V1 (MHOM/IL/80/ 2 × 106 metacyclic promastigotes in the ear. Results correspond to geometric FN) as described (18). L. infantum (HYG) was derived from L. infantum mean + 95% confidence interval of the pool of two independent experiments (MHOM/ES/92/LLM-320; isoenzyme typed MON-1) (27), provided by Diane (n = 4 per group per experiment). (D) C57BL/6 mice were infected i.v. with 3 × MacMahon-Pratt (Yale School of Public Health, New Haven, CT). Wild-type 106 metacyclic promastigotes, and the parasite loads in the spleen (Left)and L. infantum promastigotes were transfected with the RFP/Hyg expression the liver (Right) were determined 5 wk p.i. Results correspond to geometric cassette and selected on hygromycin B-containing agar plates as described mean + 95% confidence interval of the pool of two independent experiments (36). The integration of the expression cassette into the 18S rRNA locus in (n = 3 per group per experiment). #P < 0.05 (vs. Lm); *P < 0.05 (vs. Li); **P < selected drug-resistant clones was verified by PCR. LinL–HYG clone 7 also 0.05 (vs. groups 4, 11, 9, and 12). showed good survival in Lu. longipalpis and was chosen for the studies shown in this work. All parental and progeny lines were cultured in vitro at 26 °C in complete medium 199 (CM199) supplemented as described in Finally, we used i.d. inoculation of the hybrids in C57BL/6 SI Materials and Methods. Infective-stage metacyclic promastigotes were mice to explore the heritability of the genes controlling the de- isolated from stationary cultures (4–6 d old) by centrifugation through velopment of pathology associated with healing forms of cutaneous a Ficoll-step gradient as described (37). LPG-mediated agglutination assay of parents and hybrids is described in SI Materials and Methods. leishmaniasis and, more critically, the ability to disseminate from the skin and establish growth in the deep organs. The diploid Infection of Sand Flies and Hybrid Recovery. Lu. longipalpis and P. duboscqi hybrids appeared to differentially segregate the skin and viscera were obtained from field specimens collected in Brazil and Mali, re- tropisms of the parents, with two hybrids producing only transient spectively. Two- to 4-d-old female sand flies were infected by artificial pathology in the skin but disseminating to and growing in the liver feeding through a chick-skin membrane containing heparinized, heat-inac- and/or spleen equivalent to the Li parent, and two behaving more tivated mouse blood and parasites. For the generation of hybrids in Lu. like the Lm parent in producing stronger pathology in the skin but longipalpis, the blood was seeded with a mixture of 4 × 106 L. major (SAT) growing poorly in the viscera. Thus, even these few 2n progeny and 8 × 106 L. infantum (HYG) logarithmic phase promastigotes per milliliter appear to have differentially inherited the genes controlling the of blood; relatively high concentrations were used to compensate for the respective tissue tropisms of their parents, suggesting that one or suboptimal growth of these lines in the colonized flies. Infected flies were dissected at different times p.i., and midgut homogenates were prepared both of the parents are heterozygous for these genes(s). Alterna- for selection of doubly drug-resistant hybrids as described in SI Materials and tively, or in concert with specific inheritance patterns, epigenetic Methods. For the phenotyping of hybrid progeny in P. duboscqi, the flies mechanisms involved in regulating transcription initiation or ter- were infected with 4 × 106 per milliliter logarithmic phase promastigotes, mination of the relevant genes (31, 32), or changes in gene dosage and at different times p.i. midgut homogenates were prepared and de- due to aneuploidy, might contribute to the phenotypic differences posited on a hemocytometer to count the numbers of parasites per midgut.

16812 | www.pnas.org/cgi/doi/10.1073/pnas.1415109111 Romano et al. Downloaded by guest on September 27, 2021 Hybrids Genotyping. Total DNA contents were determined by flow cytometry To compare the virulence of the parental and progeny clones in estab- as described (18) (SI Materials and Methods). For genotype analysis, total lished models of cutaneous leishmaniasis, 2 × 106 metacyclic promastigotes DNA was extracted by using the Wizard genomic DNA purification Kit. PCR were injected s.c. in one hind footpad of BALB/c mice or i.d. in one ear of amplifications were performed in 20-μL final volume, using 20 ng of DNA C57BL/6 mice. Lesion development was monitored weekly by measuring and 2× GeneAmp PCR Master Mix (Applied Biosystems) and 25 pmol of each footpad width or the diameter of the induration in the ear by using primer specific for the marker genes listed in Table S1.DNAproductswere a direct-reading vernier caliper. To study the dissemination to and verified by electrophoresis on a 1.5% (wt/vol) agarose gel and visualized by growth in the deep organs, C57BL/6 mice infected in the ear dermis with ethidium bromide staining. PCR primers for SNP-CAPS analysis were described 2 × 106 metacyclic promastigotes were killed at 6 wk p.i., and the (8) and are summarized in Table S1. PCR products were cleaned with Wizard infected ear, spleen, and liver were harvested. Alternatively, C57BL/6 SV Gel and PCR Clean-Up System (Promega), and 6 μL of product was digested mice were infected i.v. with 3 × 106 metacyclic promastigotes and 5 wk with 10 U of restriction enzyme (Fermentas) for 16 h, electrophoresed on 1.5% p.i. were euthanized, and spleen and liver were harvested. For quanti- agarose gel, and visualized by ethidium staining. The relative intensity of the fication of parasite loads, the spleen and liver were cut with tweezers and band corresponding to the digestion products was analyzed with ImageJ homogenized with a syringe plunger, and the cell suspension was filtered software (Version 1.46). PCR primers for gene sequencing are summarized in through a 70-μm strainer. Red blood cells were lysed with ammonium- Table S1. PCR products were cleaned with ExoSAP-IT kit (USB), and sequences chloride-potassium lysing buffer for 5 min at room temperature, and cells were confirmed with forward and/or reverse reads by Rocky Mountain Labo- from each tissue were resuspended in CM199. Ear tissue was prepared ratory Genomics Unit DNA Sequencing Center, Division of Intramural Research and parasite loads were determined as described (39). (Hamilton, MT). The sequences were analyzed by using Lasergene software. Statistical Methods. Parasite loads were compared by using an exact In Vivo Infections. The 7- to 9-wk-old female BALB/c and C57BL/6 mice were stratified Wilcoxon rank sum test, stratified by experiment to allow purchased from Taconic Laboratories. All mice were maintained in the Na- pooling of experiments. Comparisons in which the data represented tional Institute of Allergy and Infectious Diseases (NIAID) animal care facility replicate samples were carried out by using t tests. All P values are under specific pathogen-free conditions and used under a study protocol two-sided. approved by the NIAID animal care and use committee (protocol no. LPD 68E). All aspects of the use of animals in this research were monitored for com- ACKNOWLEDGMENTS. We thank Kim Beacht for assistance with the pliance with the Animal Welfare Act; the Public Health Service Policy; the US animal studies. This work was supported in part by the Intramural Government Principles for the Utilization and Care of Vertebrate Animals Research Program of the National Institute of Allergy and Infectious Used in Testing, Research, and Training; and the NIH Guide for the Care and Diseases, National Institutes of Health (NIH); and by NIH Grant AI-R01- Use of Laboratory Animals (38). 29646 (to S.M.B.).

1. Banuls~ AL, Hide M, Tibayrenc M (1999) Molecular epidemiology and evolutionary 19. Sadlova J, et al. (2011) Visualisation of fluorescent hybrids genetics of Leischmania parasites. Int J Parasitol 29(8):1137–1147. during early stage development in the sand fly vector. PLoS ONE 6(5):e19851. 2. Zhang WW, Chan KF, Song Z, Matlashewski G (2011) Expression of a Leishmaniado- 20. Inbar E, et al. (2013) The mating competence of geographically diverse Leishmania novani nucleotide sugar transporter in Leishmaniamajor enhances survival in visceral major strains in their natural and unnatural sand fly vectors. PLoS Genet 9(7):e1003672. organs. Exp Parasitol 129(4):337–345. 21. Calvo-Álvarez E, et al. (2014) First evidence of intraclonal genetic exchange in try- 3. Zhang WW, Matlashewski G (2010) Screening Leishmania donovani-specific genes panosomatids using two Leishmania infantum fluorescent transgenic clones. PLoS required for visceral infection. Mol Microbiol 77(2):505–517. Negl Trop Dis 8(9):e3075. 4. Britto C, et al. (1998) Conserved linkage groups associated with large-scale chromo- 22. Walters LL, Irons KP, Chaplin G, Tesh RB (1993) Life cycle of Leishmania major

somal rearrangements between Old World and New World Leishmania genomes. (: Trypanosomatidae) in the neotropical sand fly Lutzomyia longipalpis GENETICS Gene 222(1):107–117. (Diptera: Psychodidae). J Med Entomol 30(4):699–718. 5. Wincker P, et al. (1996) The Leishmania genome comprises 36 chromosomes con- 23. McCall LI, Zhang WW, Matlashewski G (2013) Determinants for the development of served across widely divergent human pathogenic species. Nucleic Acids Res 24(9): visceral leishmaniasis disease. PLoS Pathog 9(1):e1003053. 1688–1694. 24. Garin YJ, et al. (2005) A2 gene of Old World cutaneous Leishmania is a single highly 6. Cruz AK, Titus R, Beverley SM (1993) Plasticity in chromosome number and testing of conserved functional gene. BMC Infect Dis 5:18. essential genes in Leishmania by targeting. Proc Natl Acad Sci USA 90(4):1599–1603. 25. McCall LI, Matlashewski G (2012) Involvement of the Leishmania donovani virulence 7. Downing T, et al. (2011) Whole genome sequencing of multiple Leishmania donovani factor A2 in protection against heat and oxidative stress. Exp Parasitol 132(2): – clinical isolates provides insights into population structure and mechanisms of drug 109 115. resistance. Genome Res 21(12):2143–2156. 26. Svárovská A, et al. (2010) Leishmania major glycosylation mutants require phospho- 8. Lachaud L, et al. (2014) Constitutive mosaic aneuploidy is a unique genetic feature glycans (lpg2-) but not lipophosphoglycan (lpg1-) for survival in permissive sand fly vectors. PLoS Negl Trop Dis 4(1):e580. widespread in the Leishmania genus. Microbes Infection 16(1):61–66. 27. Ahmed S, et al. (2003) Intradermal infection model for pathogenesis and vaccine 9. Rogers MB, et al. (2011) Chromosome and gene copy number variation allow major studies of murine visceral leishmaniasis. Infect Immun 71(1):401–410. structural change between species and strains of Leishmania. Genome Res 21(12): 28. Gibson W (2001) Sex and evolution in trypanosomes. Int J Parasitol 31(5-6):643–647. 2129–2142. 29. Volf P, et al. (2007) Increased transmission potential of Leishmania major/Leishmania 10. Sterkers Y, Lachaud L, Crobu L, Bastien P, Pagès M (2011) FISH analysis reveals infantum hybrids. Int J Parasitol 37(6):589–593. aneuploidy and continual generation of chromosomal mosaicism in Leishmania ma- 30. Zhang WW, et al. (2003) Comparison of the A2 gene locus in Leishmania donovani jor. Cell Microbiol 13(2):274–283. and Leishmania major and its control over cutaneous infection. J Biol Chem 278(37): 11. Bañuls AL, et al. (1997) Evidence for hybridization by multilocus enzyme electro- 35508–35515. phoresis and random amplified polymorphic DNA between Leishmania braziliensis 31. Anderson BA, et al. (2013) Kinetoplastid-specific histone variant functions are con- and Leishmania panamensis/guyanensis in Ecuador. J Eukaryot Microbiol 44(5): served in Leishmania major. Mol Biochem Parasitol 191(2):53–57. – 408 411. 32. van Luenen HG, et al. (2012) Glucosylated hydroxymethyluracil, DNA base J, prevents 12. Chargui N, et al. (2009) Population structure of Tunisian Leishmania infantum and transcriptional readthrough in Leishmania. Cell 150(5):909–921. evidence for the existence of hybrids and gene flow between genetically different 33. Schönian G, Mauricio I, Cupolillo E (2010) Is it time to revise the nomenclature of – populations. Int J Parasitol 39(7):801 811. Leishmania? Trends Parasitol 26(10):466–469. 13. Nolder D, Roncal N, Davies CR, Llanos-Cuentas A, Miles MA (2007) Multiple hybrid 34. Wilson AC, Maxson LR, Sarich VM (1974) Two types of molecular evolution. Evidence genotypes of Leishmania (viannia) in a focus of mucocutaneous Leishmaniasis. Am J from studies of interspecific hybridization. Proc Natl Acad Sci USA 71(7):2843–2847. – Trop Med Hyg 76(3):573 578. 35. Peacock CS, et al. (2007) Comparative genomic analysis of three Leishmania species 14. Ravel C, et al. (2006) First report of genetic hybrids between two very divergent that cause diverse human disease. Nat Genet 39(7):839–847. Leishmania species: Leishmania infantum and Leishmania major. Int J Parasitol 36(13): 36. Chagas AC, et al. (2014) Lundep, a sand fly salivary endonuclease increases Leishmania 1383–1388. parasite survival in neutrophils and inhibits XIIa contact activation in human plasma. 15. Rogers MB, et al. (2014) Genomic confirmation of hybridisation and recent inbreeding PLoS Pathog 10(2):e1003923. in a vector-isolated Leishmania population. PLoS Genet 10(1):e1004092. 37. Späth GF, Beverley SM (2001) A lipophosphoglycan-independent method for isolation 16. Rougeron V, et al. (2011) Reproductive strategies and population structure in of infective Leishmania metacyclic promastigotes by density gradient centrifugation. Leishmania: Substantial amount of sex in Leishmania Viannia guyanensis. Mol Ecol Exp Parasitol 99(2):97–103. 20(15):3116–3127. 38. Committee on Care and Use of Laboratory Animals (1996) Guide for the Care and Use 17. Tibayrenc M, Ayala FJ (2013) How clonal are Trypanosoma and Leishmania? Trends of Laboratory Animals (Natl Inst Health, Bethesda), DHHS Publ No (NIH) 85-23. Parasitol 29(6):264–269. 39. Belkaid Y, et al. (1998) Development of a natural model of cutaneous leishmaniasis: 18. Akopyants NS, et al. (2009) Demonstration of genetic exchange during cyclical de- Powerful effects of vector saliva and saliva preexposure on the long-term outcome of velopment of Leishmania in the sand fly vector. Science 324(5924):265–268. Leishmania major infection in the mouse ear dermis. J Exp Med 188(10):1941–1953.

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