Gutiérrez-Corbo Camino, Domínguez-Asenjo Bárbara, Pérez-Pertejo Yolanda, García-Estrada Carlos, Bello Felio J, Balaña-Fouce Rafael, Reguera Rosa M
Departamento de Ciencias Biomédicas, Facultad de Veterinaria, Universidad de León, León, Spain.
Facultad de Ciencias Agropecuarias, Programa de Medicina Veterinaria, Universidad de la Salle, Bogotá, Colombia.
PLoS Negl Trop Dis. 2022 Feb 9;16(2):e0010170. doi: 10.1371/journal.pntd.0010170. eCollection 2022 Feb.
Diseases caused by trypanosomatids are serious public health concerns in low-income endemic countries. Leishmaniasis is presented in two main clinical forms, visceral leishmaniasis-caused by L. infantum and L. donovani-and cutaneous leishmaniasis-caused by many species, including L. major, L. tropica and L. braziliensis. As for certain other trypanosomatids, sexual reproduction has been confirmed in these parasites, and formation of hybrids can contribute to virulence, drug resistance or adaptation to the host immune system. In the present work, the capability of intraclonal and interspecies genetic exchange has been investigated using three parental strains: L. donovani, L. tropica and L. major, which have been engineered to express different fluorescent proteins and antibiotic resistance markers in order to facilitate the phenotypic selection of hybrid parasites after mating events. Stationary and exponential-phase promastigotes of each species were used, in in vitro experiments, some of them containing LULO cells (an embryonic cell line derived from Lutzomyia longipalpis). Several intraclonal hybrids were obtained with L. tropica as crossing progenitor, but not with L. donovani or L. major. In interspecies crossings, three L. donovani x L. major hybrids and two L. donovani x L. tropica hybrids were isolated, thereby demonstrating the feasibility to obtain in vitro hybrids of parental lines causing different tropism of leishmaniasis. Ploidy analysis revealed an increase in DNA content in all hybrids compared to the parental strains, and nuclear analysis showed that interspecies hybrids are complete hybrids, i.e. each of them showing at least one chromosomal set from each parental. Regarding kDNA inheritance, discrepancies were observed between maxi and minicircle heritage. Finally, phenotypic studies showed either intermediate phenotypes in terms of growth profiles, or a decreased in vitro infection capacity compared to the parental cells. To the best of our knowledge, this is the first time that in vitro interspecies outcrossing has been demonstrated between Leishmania species with different tropism, thus contributing to shed light on the mechanisms underlying sexual reproduction in these parasites.
锥虫病在低收入流行国家是严重的公共卫生问题。利什曼病主要有两种临床形式，即由婴儿利什曼原虫和杜氏利什曼原虫引起的内脏利什曼病，以及由多种物种引起的皮肤利什曼病，包括硕大利什曼原虫、热带利什曼原虫和巴西利什曼原虫。至于某些其他锥虫，已证实这些寄生虫存在有性繁殖，杂种的形成可导致毒力、耐药性或对宿主免疫系统的适应性。在本研究中，使用了三个亲本菌株来研究克隆内和种间基因交换的能力：杜氏利什曼原虫、热带利什曼原虫和硕大利什曼原虫，这些菌株经过改造以表达不同的荧光蛋白和抗生素抗性标记，以便在交配事件后便于对杂种寄生虫进行表型选择。在体外实验中使用了每个物种的静止期和指数期前鞭毛体，其中一些含有LULO细胞（一种源自长须罗蛉的胚胎细胞系）。以热带利什曼原虫作为杂交亲本获得了几个克隆内杂种，但杜氏利什曼原虫或硕大利什曼原虫没有。在种间杂交中，分离出了三个杜氏利什曼原虫×硕大利什曼原虫杂种和两个杜氏利什曼原虫×热带利什曼原虫杂种，从而证明了获得引起不同利什曼病嗜性的亲本系体外杂种的可行性。倍性分析显示，与亲本菌株相比，所有杂种的DNA含量均增加，核分析表明种间杂种是完全杂种，即它们中的每一个都显示出来自每个亲本的至少一组染色体。关于线粒体DNA遗传，在大、小环遗传方面观察到差异。最后，表型研究表明，在生长曲线方面表现为中间表型，或者与亲本细胞相比体外感染能力下降。据我们所知，这是首次证明具有不同嗜性的利什曼原虫物种之间在体外进行种间杂交，从而有助于阐明这些寄生虫有性繁殖的潜在机制。
