Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes

PLoS Pathogens

Wanderson Duarte daRocha¹  Normanda Souza Melo¹  Rita Marcia Cardoso de Paiva²  Rondon Pessoa Mendonça-Neto²  Santuza M. R. Teixeira²  Brenda Naemi Nakagaki²  Viviane Grazielle-Silva²  Patrícia Massara Martinelli³  Mariana Santos Cardoso⁴  Adriana Monte Cassiano Canavaci⁵  Ana Paula Fernandes⁵ 

[1] Departamento de Bioquimica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, Paraná, Brazil;Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil;Departamento de Morfologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil;Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil;Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil

关键词: Parasitic diseases;    Amastigotes;    Macrophages;    Small interfering RNAs;    Promastigotes;    RNA interference;    Leishmania;    Trypanosoma cruzi;   

DOI  :  10.1371/journal.ppat.1005296

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several Leishmania species. These protozoan parasites replicate as extracellular, flagellated promastigotes in the gut of a sandfly vector and as amastigotes inside the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several trypanosomatids and highly expressed in the intracellular amastigote stages of Trypanosoma cruzi and Leishmania spp. Here, we showed that the genome of L. braziliensis contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in L. braziliensis amastigotes. Although primary sequence alignments showed no homology to any known protein sequence, homology searches based on secondary structure predictions indicate that amastins are related to claudins, a group of proteins that are components of eukaryotic tight junction complexes. By knocking-down the expression of δ-amastins in L. braziliensis, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after in vitro infection of mouse macrophages and completely failed to produce infection when inoculated in BALB/c mice, an attenuated phenotype that was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-parasite interactions, electron microscopy analyses of macrophages infected with amastin knockdown parasites showed significant alterations in the tight contact that is normally observed between the surface of wild type amastigotes and the membrane of the parasitophorous vacuole.

【 授权许可】

CC BY