Fatemeh Darzi, Ali Khamesipour, Minoo Tasbihi, Maryam Bahraminasab, Mahmoud Nateghi-Rostami
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引用次数: 0
Abstract
We employed a CRISPR/Cas9 technique in Leishmania major to evaluate its efficiency in editing a kDNA-associated gene, the universal minicircle sequence binding protein (UMSBP), which is involved in mitochondrial respiration and kinetoplast division. Using this toolkit, we generated UMSBP mNG-tagged and single knockout L. major (LmUMSBP+/-) parasites, which were confirmed by PCR, confocal microscopy and Western blot analyses. The growth rate of promastigotes in culture and their infectivity in macrophages were analysed in vitro. Mice were immunized with the LmUMSBP+/- mutant strain, and lesion size and parasite burden were measured upon challenge with wild-type (WT) L. major. Cytokines were quantified in supernatants of lymph node cell cultures. The results suggested successful expression and localization of the UMSBP mNG-tagged protein within the kinetoplast in both promastigote and intracellular amastigote forms, confirming the consistency of fluorescence tagging throughout various stages of the Leishmania life cycle. Attenuated LmUMSBP+/- parasites showed significantly reduced growth in culture (P < 0.05), increased apoptosis (P < 0.05) and downregulation of tryparedoxin peroxidase (TXNPx) and trypanothione synthetase (TryS) gene expression compared to WT L. major. LmUMSBP+/- mutant strains did not cause lesions in a susceptible BALB/c mouse model. Furthermore, immunization with LmUMSBP+/- parasites elicited a Th1 immune response, characterized by significantly higher IFN-γ and lower IL-4 production in cell culture (P < 0.001), which was associated with partial protection against WT L. major challenge, as evidenced by reduced parasite burden and lesion development in BALB/c mice. In this study, we successfully validated a practical CRISPR/Cas9 toolkit in L. major, targeting the kinetoplast-associated gene UMSBP. Our findings suggest that the UMSBP single-allele knockout mutant holds promise as a valuable tool for studying the role of the kinetoplast in Leishmania biology and as a potential candidate for further investigation as a live-attenuated vaccine against Leishmania infection.
我们在利什曼原虫中使用CRISPR/Cas9技术来评估其编辑kdna相关基因的效率,通用微环序列结合蛋白(UMSBP)参与线粒体呼吸和着丝体分裂。使用该工具包,我们生成了UMSBP mng标记和单敲除L. major (LmUMSBP+/-)寄生虫,并通过PCR、共聚焦显微镜和Western blot分析进行了证实。分析了体外培养中原毛菌的生长速度和对巨噬细胞的感染性。用LmUMSBP+/-突变株免疫小鼠,用野生型(WT) L. major攻毒后测量病变大小和寄生虫负荷。在淋巴结细胞培养的上清液中定量细胞因子。结果表明,在promastigote和胞内amastigote形式中,UMSBP mg标记蛋白在动质体内成功表达和定位,证实了荧光标记在利什曼原虫生命周期各个阶段的一致性。减毒的LmUMSBP+/-寄生虫在培养物中显著降低生长(P +/-突变株在易感BALB/c小鼠模型中不引起病变)。此外,LmUMSBP+/-寄生虫免疫引起Th1免疫应答,其特征是细胞培养中IFN-γ显著升高,IL-4产生显著降低(P
期刊介绍:
Medical Microbiology and Immunology (MMIM) publishes key findings on all aspects of the interrelationship between infectious agents and the immune system of their hosts. The journal´s main focus is original research work on intrinsic, innate or adaptive immune responses to viral, bacterial, fungal and parasitic (protozoan and helminthic) infections and on the virulence of the respective infectious pathogens.
MMIM covers basic, translational as well as clinical research in infectious diseases and infectious disease immunology. Basic research using cell cultures, organoid, and animal models are welcome, provided that the models have a clinical correlate and address a relevant medical question.
The journal also considers manuscripts on the epidemiology of infectious diseases, including the emergence and epidemic spreading of pathogens and the development of resistance to anti-infective therapies, and on novel vaccines and other innovative measurements of prevention.
The following categories of manuscripts will not be considered for publication in MMIM:
submissions of preliminary work, of merely descriptive data sets without investigation of mechanisms or of limited global interest,
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manuscripts on existing or modified vaccines, unless they report on experimental or clinical efficacy studies or provide new immunological information on their mode of action,
manuscripts on the diagnostics of infectious diseases, unless they offer a novel concept to solve a pending diagnostic problem,
case reports or case series, unless they are embedded in a study that focuses on the anti-infectious immune response and/or on the virulence of a pathogen.