Complete assembly, annotation of virulence genes and CRISPR editing of the genome of Leishmania amazonensis PH8 strain

<em>Leishmania amazonensis</em> is one of the etiological agents of tegumentary and visceral leishmaniasis. Genome studies focusing on genes encoding virulence factors that play crucial roles in the establishment of the infection constitute an essential step towards the molecular characterization of a protozoan parasite that is being increasingly recognized as a significant human pathogen. Here, we report the sequencing and assembly of the <em>L</em><em>. amazonensis </em>PH8 strain combining data from long PacBio reads, short Illumina reads and synteny with the <em>Leishmania mexicana</em> genome. The final assembly, composed of 34 chromosomes, represents a genome of ~32 Mb with 8317 annotated genes. Several multigene families encoding virulence factors, such as A2, amastins, metalloproteins GP63 and cysteine proteases, were identified and compared to their annotation in the genomes of other <em>Leishmania</em> species. The <em>L. amazonensis </em>PH8 strain genome has 27 genes encoding all four sub-classes of amastins (α, β, γ and δ), 5 genes encoding A2 antigens, 9 genes encoding the metalloprotease GP63 and 76 genes encoding cysteine proteases. For all gene families analysed, we identified a larger number of copies and presented a more complete gene repertoire associated with <em>L. amazonensis </em>virulence factors, compared to previously published genomes. As they have been recently recognized as virulence factors essential for disease establishment and progression of the infection, we have also identified 14 genes encoding proteins involved in the parasite iron and heme metabolism and compared them to this gene repertoire in other Trypanosomatids. To follow these studies with a genetic approach that would allow us to directly address the role of these virulence factors, we tested two CRISPR-Cas9 protocols to generate <em>L. amazonensis</em> knockout cell lines. Using the Miltefosine Transporter (MT) gene as a proof of concept, we transfected promastigotes expressing the <em>Streptococcus pyogenes</em> Cas9 with <em>in vitro</em> transcribed sgRNA targeting this gene. As an alternative protocol, promastigotes were transfected with recombinant <em>Staphylococcus aureus </em>Cas9 complexed with sgRNAs. With both strategies, we were able to disrupt the MT gene with high efficiency, as shown by PCR and restriction digestion analysis as well as the development of miltefosine-resistant parasites.

亚马逊利什曼原虫（Leishmania amazonensis）是皮肤利什曼病与内脏利什曼病的病原体之一。针对编码毒力因子的基因开展基因组研究，这类因子在感染建立过程中发挥关键作用，是对这一日益被视为重要人类病原体的原生动物寄生虫进行分子鉴定的核心步骤。本研究报道了结合长读长PacBio测序数据、短读长Illumina测序数据，以及与墨西哥利什曼原虫（Leishmania mexicana）基因组的共线性信息，对L. amazonensis PH8菌株进行测序与组装的成果。最终组装结果包含34条染色体，对应约32 Mb的基因组，注释得到8317个基因。研究人员鉴定出多个编码毒力因子的多基因家族，如A2、无鞭毛体蛋白（amastins）、金属蛋白酶GP63以及半胱氨酸蛋白酶，并将其与其他利什曼原虫物种基因组中的注释信息进行了对比。L. amazonensis PH8菌株基因组中，存在27个编码无鞭毛体蛋白全部四个亚型（α、β、γ、δ）的基因、5个编码A2抗原的基因、9个编码金属蛋白酶GP63的基因，以及76个编码半胱氨酸蛋白酶的基因。相较于已发表的其他基因组，本研究针对所有分析的基因家族，鉴定出了更多的基因拷贝数，并呈现了更完整的与L. amazonensis毒力因子相关的基因谱。鉴于这些因子近期被认定为疾病发生与感染进展不可或缺的毒力因子，本研究还鉴定出14个编码参与寄生虫铁与血红素代谢的蛋白质的基因，并将其与其他锥虫科物种的该类基因谱进行了对比。为通过遗传学方法跟进此类研究，以直接阐明这些毒力因子的功能，本研究测试了两种CRISPR-Cas9方案以构建L. amazonensis的敲除细胞系。以米替福林转运蛋白（Miltefosine Transporter, MT）基因为概念验证靶点，研究人员将体外转录的靶向该基因的sgRNA转染至表达化脓性链球菌（Streptococcus pyogenes）Cas9的前鞭毛体中。作为替代方案，研究人员将重组的金黄色葡萄球菌（Staphylococcus aureus）Cas9与sgRNA形成的复合物转染至前鞭毛体中。两种策略均实现了对MT基因的高效敲除，经PCR、限制性酶切分析以及米替福林抗性寄生虫的生长验证均可证实这一点。