title: Amastin Knockdown in Leishmania braziliensis Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes.
abstract: 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.
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Rita Marcia Cardoso
authors.initials: RM
authors.lastname: de Paiva
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Viviane
authors.initials: V
authors.lastname: Grazielle-Silva
authors.affiliation: Departamento de Parasitologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Mariana Santos
authors.initials: MS
authors.lastname: Cardoso
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Brenda Naemi
authors.initials: BN
authors.lastname: Nakagaki
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Rondon Pessoa
authors.initials: RP
authors.lastname: Mendonça-Neto
authors.affiliation: Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Adriana Monte Cassiano
authors.initials: AM
authors.lastname: Canavaci
authors.affiliation: Departamento de Bioquimica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, Paraná, Brazil.
authors.forename: Normanda
authors.initials: N
authors.lastname: Souza Melo
authors.affiliation: Departamento de Morfologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Patrícia Massara
authors.initials: PM
authors.lastname: Martinelli
authors.affiliation: Faculdade de Farmácia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Ana Paula
authors.initials: AP
authors.lastname: Fernandes
authors.affiliation: Departamento de Bioquimica e Biologia Molecular, Universidade Federal do Paraná, Curitiba, Paraná, Brazil.
authors.forename: Wanderson Duarte
authors.initials: WD
authors.lastname: daRocha
authors.affiliation: Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil.
authors.forename: Santuza M R
authors.initials: SM
authors.lastname: Teixeira
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D004195:Disease Models, Animal; D006790:Host-Parasite Interactions / Q000502:physiology*; D007892:Leishmania braziliensis / Q000235:genetics / Q000472:pathogenicity*; D016773:Leishmaniasis, Cutaneous / Q000235:genetics*; D008264:Macrophages / Q000469:parasitology*; D008562:Membrane Glycoproteins / Q000235:genetics; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D015800:Protozoan Proteins / Q000235:genetics*; D014162:Transfection; D014774:Virulence
chemical_list: D008562:Membrane Glycoproteins; D015800:Protozoan Proteins