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

Rohit Sharma,Larissa Pinheiro Marques,Daniella C Bartholomeu,João Luís Reis-Cunha,Claudio P Figueira,Camila I De Oliveira,Francys Avendaño Rangel,Pedro B Borba,Tom Beneke,Sayonara M Viana

doi:10.3389/fcimb.2021.790418

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.

Highlights

Human leishmaniasis results in mortality and morbidity worldwide, contributing to ~1 million new cases each year and placing 350 million individuals at risk of infection (Burza et al, 2018)
A BLASTP search in the TriTrypDB database revealed the presence of a putative centrin gene in L. braziliensis (LbrM.22.1290) showing homology to previously characterized L. donovani centrin [GenBank—AF406767; (Selvapandiyan et al, 2001)]
These in silico analyses suggest that the putative centrin gene in L. braziliensis is a structural homolog of T. brucei centrin 4 and centrin 4 is highly conserved across Leishmania and Trypanosoma spp

Summary

Introduction

Human leishmaniasis results in mortality and morbidity worldwide, contributing to ~1 million new cases each year and placing 350 million individuals at risk of infection (Burza et al, 2018). Infection with live parasites generates immunity, no vaccine against human leishmaniasis is yet available (Sundar and Singh, 2014). To this end, numerous attempts have been undertaken including but not limited to immunization with (i) live whole parasites, (ii) killed whole parasites, (iii) Leishmaniaspecific antigen and (iv) parasites attenuated by genetic modification [reviewed in (Zutshi et al, 2019)]. Since the recent introduction of CRISPR-Cas9-based methodologies, a significant advance has been made in this field: CRISPR-Cas-9 enabled the deletion of hundreds of genes, including, BTN1 (Ishemgulova et al, 2018) and LeishIF4E-3 (Shrivastava et al, 2019), genes encoding protein kinases (Baker et al, 2021) and flagellar proteins (Beneke et al, 2019) in L. mexicana, as well as LPG2 in L. infantum (Jesus-Santos et al, 2020), RAD51 in L. major (Damasceno et al, 2020) and Ros in L. braziliensis (Espada et al, 2021). The advantages of the LeishGEdit approach are that cloning procedures, PCR purifications or in vitro transcription before transfection are unnecessary with the advantage that it can be used for high throughput screening of mutants (Beneke et al, 2019; Damianou et al, 2020; Baker et al, 2021)

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Results

Conclusion