Ishemgulova Aygul, Hlaváčová Jana, Majerová Karolina, Butenko Anzhelika, Lukeš Julius, Votýpka Jan, Volf Petr, Yurchenko Vyacheslav
Life Science Research Centre, Faculty of Science, University of Ostrava, Ostrava, Czech Republic.
Biology Centre, Institute of Parasitology, Czech Academy of Sciences, České Budejovice (Budweis), Czech Republic.
PLoS One. 2018 Feb 13;13(2):e0192723. doi: 10.1371/journal.pone.0192723. eCollection 2018.
Leishmania parasites cause human cutaneous, mucocutaneous and visceral leishmaniasis. Several studies proposed involvement of certain genes in infectivity of these parasites based on differential mRNA expression data. Due to unusual gene expression mechanism, functions of such genes must be further validated experimentally. Here, we investigated a role of one of the putative virulence factors, LmxM.22.0010-encoded BTN1 (a protein involved in Batten disease in humans), in L. mexicana infectivity. Due to the incredible plasticity of the L. mexicana genome, we failed to obtain a complete knock-out of LmxM.22.0010 using conventional recombination-based approach even after ablating four alleles of this gene. To overcome this, we established a modified CRISPR-Cas9 system with genomic expression of Cas9 nuclease and gRNA. Application of this system allowed us to establish a complete BTN1 KO strain of L. mexicana. The mutant strain did not show any difference in growth kinetics and differentiation in vitro, as well as in the infectivity for insect vectors and mice hosts. Based on the whole-transcriptome profiling, LmxM.22.0010-encoded BTN1 was considered a putative factor of virulence in Leishmania. Our study suggests that ablation of LmxM.22.0010 does not influence L. mexicana infectivity and further illustrates importance of experimental validation of in silico-predicted virulence factors. Here we also describe the whole genome sequencing of the widely used model isolate L. mexicana M379 and report a modified CRISPR/Cas9 system suitable for complete KO of multi-copy genes in organisms with flexible genomes.
利什曼原虫寄生虫可导致人类皮肤利什曼病、黏膜皮肤利什曼病和内脏利什曼病。多项研究基于差异mRNA表达数据提出某些基因参与了这些寄生虫的感染性。由于基因表达机制异常，此类基因的功能必须通过实验进一步验证。在此，我们研究了一种假定的毒力因子，即LmxM.22.0010编码的BTN1（一种与人类巴滕病相关的蛋白质）在墨西哥利什曼原虫感染性中的作用。由于墨西哥利什曼原虫基因组具有惊人的可塑性，即使在敲除该基因的四个等位基因后，我们仍未能使用传统的基于重组的方法获得LmxM.22.0010的完全敲除。为克服这一问题，我们建立了一种改良的CRISPR-Cas9系统，该系统可在基因组中表达Cas9核酸酶和gRNA。应用该系统使我们能够建立墨西哥利什曼原虫的完全BTN1敲除菌株。该突变菌株在体外生长动力学和分化方面，以及对昆虫载体和小鼠宿主的感染性方面均未显示出任何差异。基于全转录组分析，LmxM.22.0010编码的BTN1被认为是利什曼原虫中的一种假定毒力因子。我们的研究表明，敲除LmxM.22.0010并不影响墨西哥利什曼原虫的感染性，并进一步说明了对计算机预测的毒力因子进行实验验证的重要性。在此，我们还描述了广泛使用的模式分离株墨西哥利什曼原虫M379的全基因组测序，并报告了一种改良的CRISPR/Cas9系统，该系统适用于在基因组具有灵活性的生物体中对多拷贝基因进行完全敲除。
