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，是对这种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。
本研究结合PacBio测序读段（PacBio reads）、Illumina测序读段（Illumina reads）数据以及与墨西哥利什曼原虫（Leishmania mexicana）基因组的同线性（synteny）信息，完成了亚马逊利什曼原虫PH8菌株的测序与组装。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。研究鉴定了多个编码毒力因子的多基因家族，如A2抗原、无鞭毛体蛋白（amastins）、金属蛋白酶GP63（metalloprotease GP63）以及半胱氨酸蛋白酶，并与其他利什曼原虫物种的基因组注释结果进行了比对。
亚马逊利什曼原虫PH8菌株基因组中，存在27个编码无鞭毛体蛋白全部四个亚类（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，本研究在该菌株中鉴定到了更多的基因拷贝数，并构建了更为完整的亚马逊利什曼原虫毒力因子基因库。
鉴于这类因子近期被证实为疾病建立与感染进展所必需的毒力因子，本研究还鉴定了14个编码参与寄生虫铁与血红素代谢蛋白的基因，并将其与其他锥虫科（Trypanosomatids）物种的同类基因库进行了比较。
为通过遗传学方法跟进上述研究，以直接阐明这些毒力因子的功能，我们测试了两种CRISPR-Cas9系统方案以构建亚马逊利什曼原虫基因敲除细胞系。以米替福辛转运蛋白（Miltefosine Transporter, MT）基因为概念验证靶点，我们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，我们将重组的金黄色葡萄球菌（Staphylococcus aureus）Cas9与sgRNA形成复合物后转染前鞭毛体。两种策略均实现了MT基因的高效敲除，经聚合酶链式反应（PCR）、限制性酶切分析以及米替福辛抗性寄生虫的获得均可证实这一点。