Deleção direcionada de centrina em Leishmania Braziliensis utilizando CRISPR-Cas9: geração de uma linhagem atenuada para imunoprofilaxia

Abstract

Leishmaniases are neglected tropical diseases and there are no vaccines for human use. In Latin America, Leishmania braziliensis is the main causative agent of tegumentary leishmaniasis. Here, aiming at the development of an attenuated strain of L. braziliensis for immunoprophylaxis purposes, we successfully employed the CRISPR/Cas9 technology. We generated a line of transgenic parasites expressing Cas9 and T7 RNA polymerase, which was employed for the targeted deletion of centrin, a cytoskeletal calcium-binding protein involved in centrosome duplication in eukaryotes. Whole genome sequencing of centrin deficient L. braziliensis (LbCen-/-) did not indicate the presence of off-target mutations. In vitro, growth rates of LbCen-/- and wild-type promastigotes were similar, but axenic and intracellular LbCen-/- amastigotes showed a multinucleated phenotype with impaired survival in infected macrophages. After experimental inoculation in BALB/c mice, LbCen-/- failed to induce lesion development and the parasites were rapidly eliminated. Furthermore, immunization with LbCen-/- did not confer protection against subsequent challenges, unlike other centrin-deficient leishmanias. The immune response induced by inoculation with LbCen-/- was characterized by significantly higher levels of IL-4 and IL-10, not observed in mice leishmanized with LbWT. Therefore, immunization with LbCen-/- is not capable of inducing a Th1-type immune response, making mice susceptible to challenges, and LbCen-/- cannot reproduce the immunity observed in leishmanization. In conclusion, centrin deficiency in L. braziliensis causes parasite attenuation, interrupts the ability to cause disease, but otherwise cannot stimulate a protective immune response.