Growth arrested live-attenuated Leishmania infantum KHARON1 null mutants display cytokinesis defect and protective immunity in mice

Ana Maria Murta Santi,André Luís Branco De Barros,Silvane Maria Fonseca Murta,Luiza Guimarães Tunes,Andréa Teixeira De Carvalho,Juliane Sousa Lanza,Gaétan Roy,Frédéric Frézard,Alessandra Da Silva Orfanó,Rubens Lima Do Monte-Neto,Jacqueline Araújo Fiuza

doi:10.1038/s41598-018-30076-7

Abstract

There is no safe and efficacious vaccine against human leishmaniasis available and live attenuated vaccines have been used as a prophylactic alternative against the disease. In order to obtain an attenuated Leishmania parasite for vaccine purposes, we generated L. infantum KHARON1 (KH1) null mutants (ΔLikh1). This gene was previously associated with growth defects in L. mexicana. ΔLikh1 was obtained and confirmed by PCR, qPCR and Southern blot. We also generate a KH1 complemented line with the introduction of episomal copies of KH1. Although ΔLikh1 promastigote forms exhibited a growth pattern similar to the wild-type line, they differ in morphology without affecting parasite viability. L. infantum KH1-deficient amastigotes were unable to sustain experimental infection in macrophages, forming multinucleate cells which was confirmed by in vivo attenuation phenotype. The cell cycle analysis of ΔLikh1 amastigotes showed arrested cells at G2/M phase. ΔLikh1-immunized mice presented reduced parasite burden upon challenging with virulent L. infantum, when compared to naïve mice. An effect associated with increased Li SLA-specific IgG serum levels and IL-17 production. Thus, ΔLikh1 parasites present an infective-attenuated phenotype due to a cytokinesis defect, whereas it induces immunity against visceral leishmaniasis in mouse model, being a candidate for antileishmanial vaccine purposes.

Highlights

Leishmaniasis is a group of diseases caused by protozoa belonging to Leishmania genus (Ross, 1903)
In the complemented line, KH1 probe revealed a very strong signal that is characteristic of extrachromosomal plasmid DNA migration in the gel, which corresponds to the construct pSP72αZEOα/KH1 (Fig. 1d)
In the present study we successfully disrupted KHARON1 (KH1) gene in L. infantum based on gene replacement by conventional homologous recombination

Summary

Introduction

Leishmaniasis is a group of diseases caused by protozoa belonging to Leishmania genus (Ross, 1903). The infection with live attenuated Leishmania is similar to an infection with pathogenic parasites but has the advantages of preventing overt disease while allowing the host immune system to interact with a large gamma of leishmanial essential antigens in the development of a protective immunity[15]. The immune response in mammalian hosts infected by Leishmania involves a complex biochemical network recruiting different cell types such as, chemo- and cytokines, and a thorough understanding of vaccine-induced immunity will further reveal the important mediators of a subsequent protective resp onse[5,11,12,13,14,16,17,18,19,20]. After an infection in susceptible mouse strains, such as BALB/c and Swiss, an induction of a Th2-type immune response characterized by secretion of anti-inflammatory or regulatory cytokines, IL-10, IL4, IL- 5, IL-6, can inhibit the production of IL-12 cytokine – inductor of macrophage leishmanicidal action – and down-regulate the activity of the enzyme inducible nitric oxide synthase (iNOS) so, the nitric oxide (NO) and IFN-γ production are often impaired, allowing parasites to disseminate and persist in the host tissue causing severe damage[6,22]

Methods

Results

Conclusion