Vector Biological Studies in Leishmaniasis

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Hybrid genotypes have been repeatedly described among natural isolates of Leishmania, and the recovery of experimental hybrids from sand flies co-infected with different strains or species of Leishmania has formally demonstrated that members of the genus possess the machinery for genetic exchange. Experimentally, hybrid formation is so far confined to promastigote stages developing within the sand fly vector. Due to the difficulty in making direct observations of sexual reproduction in the fly, there remains little understanding regarding the frequency with which clonally reproducing lineages engage in sex, the identity of the putative meiotic forms, the timing, location, and conditions that lead to their development in the vector. Despite the ability to culture the insect stages of Leishmania, there remains no report of confirmed hybrids generated in vitro using culture forms or ex vivo using stages recovered from the fly. We report the first demonstration of genetic exchange involving axenic culture forms of Leishmania. By co-culturing the promastigotes of two strains of L. tropica, each carrying a different drug resistance and fluorescence marker, we could obtain double drug resistant and double-fluorescent clones. So far, we have generated hybrids from over 200 independent mating events in vitro. By analyzing SNPs marker inheritance and high-resolution, whole genome sequencing data, selected progeny clones were shown to be full genomic hybrids. Some of the hybrids were close to 2n, although the majority were polyploid, either 3n or 4n. Whole genome sequencing of a diploid hybrid revealed roughly equal parental contributions to all 36 chromosomes, consistent with each of the diploid parents having undergone a meiotic-like process followed by fusion of haploid, gametic cells. The findings establish the principle that mating competent forms of Leishmania promastigotes can be generated in culture, and remove the requirement for vector sand flies as the major impediment to generate large numbers of recombinant parasites for experimental analysis, including the use of forward genetics for positional cloning of important genes. Importantly, the frequency of hybrid formation in vitro was substantially less compared to flies, indicating that one or more components of the sexual cycle are optimally triggered by conditions that are unique to the microenvironment of the vector midgut. Nonetheless, a great advantage of the in vitro mating system is the high yield of cultured cells and the relative ease with which the axenic culture conditions can be manipulated in attempts to promote the sexual cycle. In particular, we observed that addition of hydrogen peroxide, a source of oxidative stress, as well as exposure of the parasites to gamma-irradiation, both led to an increased frequency of hybrid generation in vitro. The treatments allowed us to generate in vitro intraspecies hybrids involving other Leishmania species, including L. donovani, L. infantum, and L. braziliensis, as well as interspecies hybrids between L. tropica and L. infantum. Both treatments are known to induce DNA double strand breaks, suggesting a link between DNA damage and induction of sexual reproduction that we are currently investigating. Interestingly, hydrogen peroxide and gamma-irradiation both also lead to a higher proportion of cells expressing Hap2, a protein described in other lower eukaryotes as essential for the fusion of gametic cells. We also provide direct evidence for self-mating in Leishmania and present the first high resolution genotype analysis of self-mating progeny using whole genome sequencing data. By singly introducing two different drug resistance markers into the same L. major strain, we could select for double drug resistant, self-mating progeny in co-infected sand flies. Genomic comparisons of the parental sub-clones and hybrids revealed that self-mating generated more frequent changes in somy and ploidy, which were shown to directly affect transcript abundance. Virulence tests in mice revealed that self-mating yielded some progeny with greater virulence than either parent, and could reconstitute virulence that was lost during clonal growth of the parasite in the sand fly or in culture. Virulence acquisition could not be explained by allelic inheritance or gene dosage, but was strongly associated with mRNA abundance of histones and the presence of S-phase parasites among the arresting metacyclic promastigotes. Conversely, loss of virulence was associated with increased expression of small nucleolar (sno) RNAs, important in ribosome biogenesis and chromatin remodeling. Overall, the data suggest that sex in Leishmania can reset development programs essential for the establishment of virulence in the mammalian host, which in the context of self-mating, can potentially occur in every naturally infected sand fly. On the Indian sub-continent, kala-azar or visceral leishmaniasis (VL) is a fatal form of leishmaniasis caused by Leishmania donovani, and transmitted by the bites of the vector sand fly, Phlebotomus argentipes. To achieve and sustain elimination of VL, the transmission potential of L. donovani exposed individuals from across the infection spectrum needs to be urgently addressed. We conducted the most extensive xenodiagnostic studies to date in order to evaluate the relative infectiousness of VL and PKDL patients pre- and post-treatment, as well as asymptomatic subjects, to the sand fly vector. We performed direct xenodiagnosis using colonized female P. argentipes sand flies on active VL patients (n=77), Post Kala-azar Dermal Leishmaniasis (PKDL) patients (n=26) at pre-treatment and after successful therapy, and on asymptomatic subjects (n=184). At 60 -72 hours post- blood meal, flies were dissected and evaluated for L.donovani infection by microscopy as well as by quantitative polymerase chain reaction (qPCR). Transmission of infection correlated with severity of disease in active VL patients. None of the drug cured VL patients were found xenodiagnosis positive by microscopy at 30 days post-treatment, although 7.7 % (6/77) were still positive by qPCR. Both nodular and macular PKDL patients were infectious to sand flies, with enhanced transmission when the flies were fed on nodular lesions. Importantly, none of the 184 asymptomatic subjects were infectious to sand flies, affording a 97.5% confidence that there is less than a 2% probability that an asymptomatic subject would infect a fly. These findings confirm that active VL and PKDL patients transmit L. donovani to the vector, but that early diagnosis and treatment will effectively remove these individuals as infection reservoirs. An important role for asymptomatic individuals in the maintenance of the transmission cycle is not supported.