Vector Biological Studies in Leishmaniasis

在利什曼原虫的自然分离株中已经多次描述了杂交基因型，并且从与利什曼原虫不同菌株或物种共感染的沙蝇中恢复的实验性杂交品种已正式证明该属的成员具有遗传交换机制。在实验上，杂交形成到目前为止仅限于在沙蝇载体中发育的promastigote阶段。由于很难直接观察苍蝇的有性繁殖，对于无性繁殖谱系发生性行为的频率、推测的减数分裂形式的身份、导致它们在载体中发育的时间、位置和条件，人们仍然知之甚少。尽管有能力培养利什曼原虫的昆虫阶段，但仍然没有报告证实在体外使用培养形式或在体外使用从苍蝇中恢复的阶段产生的杂交。我们报告的遗传交换涉及利什曼原虫的无性系培养形式的第一个示范。将两株热带乳杆菌原毛菌分别携带不同的耐药和荧光标记物，共培养可获得双耐药双荧光克隆。到目前为止，我们已经从200多个独立的体外交配事件中产生了杂交体。通过分析snp标记遗传和高分辨率全基因组测序数据，选择的后代克隆被证明是全基因组杂交。有些杂种接近2n，但大多数是多倍体，不是3n就是4n。一个二倍体杂种的全基因组测序显示，所有36条染色体的亲本贡献大致相等，这与每个二倍体亲本都经历了类似减数分裂的过程，然后是单倍体配子细胞的融合是一致的。这一发现确立了在培养中可以产生具有交配能力的利什曼原虫的原则，并消除了对载体沙蝇的需求，这是产生大量重组寄生虫用于实验分析的主要障碍，包括使用正向遗传学对重要基因进行定位克隆。

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.