Dados do Trabalho

Título

Development of paratransgenic Lutzomyia longipalpis by engineered bacteria driving refractoriness to Leishmania infantum infection.

Introdução

The parasite Leishmania infantum is the etiologic agent of American visceral leishmaniasis (AVL), a neglected disease transmitted by the sandfly Lutzomyia longipalpis. The use of insecticides to control vector populations is one of the main strategies to limit the spread of AVL. However, this approach has limitations, including drug resistance and growing socioecological impact due to the need for more potent compounds. The current scenario of territorial expansion with increasing incidence and lethality of AVL highlights the need for new strategies to control the spread of this disease. Paratransgenesis is a viable approach with biotechnological potential, in which symbiotic bacteria of insect vectors are engineered to drive antiparasitic effector molecules and reduce their vectorial capacity.

Objetivo (s)

 The aim of the present work is to select and test molecules with anti-leishmanial activity to produce a paratransgenic sandfly to block L. infantum transmission.

Material e Métodos

To select an effector molecule, we used the MTT assay to test the cytotoxic effect of 6 antimicrobial peptides (AMPs). Artificial infection experiments were performed to assess the AMP effect on parasite load and sandfly fitness. To establish a delivery system for the AMP in the vector, bacteria from the gut of female L. longipalpis were isolated after blood feeding, identified by sequencing the 16S rRNA, and tested for hemolytic activity and resistance to AMP.  In addition, we constructed a plasmid for constitutive expression and signal peptide-mediated secretion of AMP. This plasmid was electroporated into bacteria from gut microbiota and AMP secretion and their activity was tested through Dot Blot and MTT assay.

Resultados e Conclusão

We determined that 16µM of the AMP-3 was toxic to L. infantum in axenic cultures and artificially infected L. longipalpis females, but had no deleterious effects on the vector or bacterial symbionts from their gut.  We selected an Escherichia sp. non-sensitive to this AMP and genetically modified it to constitutively express and secrete it. We observed that the conditioned media from the transgenic bacteria was able to reduce the L. infantum axenic culture viability. Our next steps include testing this transgenic bacteria effects on parasite viability during their development in L. longipalpis. In this way, we propose the development of a paratransgenic sandfly with reduced vectorial capacity to transmit the L. infantum.    

Palavras Chave

Lutzomyia longipalpis; Leishmania infantum; Paratransgenesis; leishmaniose