Targeted Deletion of Centrin in Leishmania braziliensis Using CRISPR-Cas9-Based Editing

Abstract

Leishmania braziliensis is the main causative agent of Tegumentary Leishmaniasis in the Americas. However, difficulties related to genome manipulation, experimental infection, and parasite growth have so far limited studies with this species. CRISPR-Cas9-based technology has made genome editing more accessible, and here we have successfully employed the LeishGEdit approach to attenuate L. braziliensis. We generated a transgenic cell line expressing Cas9 and T7 RNA polymerase, which was employed for the targeted deletion of centrin, a calcium-binding cytoskeletal protein involved in the centrosome duplication in eukaryotes. Centrin-deficient Leishmania exhibit growth arrest at the amastigote stage. Whole-genome sequencing of centrin-deficient L. braziliensis (LbCen−/−) did not indicate the presence of off-target mutations. In vitro, the growth rates of LbCen−/− and wild-type promastigotes were similar, but axenic and intracellular LbCen−/− amastigotes showed a multinucleated phenotype with impaired survival following macrophage infection. Upon inoculation into BALB/c mice, LbCen−/− were detected at an early time point but failed to induce lesion formation, contrary to control animals, infected with wild-type L. braziliensis. A significantly lower parasite burden was also observed in mice inoculated with LbCen−/−, differently from control mice. Given that centrin-deficient Leishmania sp. have become candidates for vaccine development, we propose that LbCen−/− can be further explored for the purposes of immunoprophylaxis against American Tegumentary Leishmaniasis.

Deletion of MIF gene from live attenuated LdCen−/− parasites enhances protective CD4+ T cell immunity

“…Since the LdCen −/− parasite have growth defects in amastigote forms, but not in promastigotes 23 , 24 , this deletion prevents cell division and long-term persistence in animals (mice and hamsters) or in human macrophages ex vivo 23 . The same non-virulent characteristics of Centrin gene deletion were also observed in other species, like L. major , L. mexicana and L. braziliensis 25 – 27 . In pre-clinical studies, vaccination with LdCen −/− proved to be safe, protective and in mice (BALB/c and SCID), hamsters and dogs, after challenge with virulent parasites, in addition to cross-protection in animals challenged with L. braziliensis, L. infantum and L. mexicana 14 – 21 , 28 .…”

“…The data demonstrated that live attenuated parasite induced strong and durable protection against virulent secondary challenges 103 , indicating that is a good way to achieve protective immunity against Leishmania infection by vaccination. In our previous work, vaccination with the attenuated parasite proved to be safe, protective and persistent in mice (BALB/c and SCID), hamsters and dogs, after challenge with wild forms, in addition to cross-protection in animals challenged with L. braziliensis , L. infantum 14 – 18 , 27 , 40 . In addition, LdCen −/− MIF −/− immunized mice appear to clear infection in spleen and liver sooner than those immunized with LdCen −/− parasites.…”

Next-Generation Leishmanization: Revisiting Molecular Targets for Selecting Genetically Engineered Live-Attenuated Leishmania

“…Following this discovery, more recent studies on attenuation have utilized CRISPR to generate null mutants with high efficiency. Among the deleted targets are Centrin1 [29,34,36,71], eukaryotic translation initiation factor 4E-1 (EIF4E1) [72] and the noncatalytic component of the GPImannosyltransferase (GPI-MTI) complex [73].…”

“…Several gene targets that encode proteins important for the selection of attenuated parasites have been reported, including arabino1,4-lactone oxidase (ALO) protein [32,83], cytochrome c oxidase complex component p27 [84][85][86], HSP70 type II heat shock protein [31,56,[102][103][104], centrin1 [29,[33][34][35][36]53,54,71,[117][118][119] and kharon1 [57][58][59]. Other target candidates have also shown potential, such as biopterin transporter (BT1) [37], dihydrofolate reductase/thymidylate synthase (DHFR-TS) [88] and Golgi GDP-mannose transporter (LPG2) [92,137].…”

Draft Genome Sequence of the Protozoan Parasite Leishmania braziliensis Strain BA788, Isolated from a Clinical Case in Bahia State, Brazil

“…Promastigote parasites were maintained in Schneider medium (Sigma Chemical Co.) supplemented with 100 U/mL of penicillin, 100 μg/mL of streptomycin, and 10% heat-inactivated fetal calf serum (Life Technologies). Promastigotes were grown in airtight flasks (T25 [nonvented]), incubated upright in a 25°C incubator, and passaged every 3 days ( 4 ). Genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega) following the manufacturer’s instructions.…”