rd
23 Congress of the International Union for Biochemistry and Molecular Biology
th
44 Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology
th
th
Foz do Iguaçu, PR, Brazil, August 24 to 28 , 2015
RNAi KNOCK DOWN OF AMASTIN GENES AFFECTS INTERACTION OF
LEISHMANIA BRAZILIENSIS AMASTIGOTES WITH MACROPHAGE MEMBRANES
Cardoso de Paiva, R.M.1; Santos-Cardoso, M.2; Nakagaki, B.N.1; Mendonça-Neto,
R.P.1; Martins, A.M.C.3; Souza-Melo, N.4; Martinelli, P.M.5; Fernandes, A.P.3; daRocha,
W.D.4 and Teixeira, S.M.R.1
1
Departamento de Bioquímica e Imunologia, Universidade Federal de Minas Gerais,
Belo Horizonte, MG, Brazil; 2Departamento de Parasitologia, Universidade Federal de
Minas Gerais, Belo Horizonte, MG, Brazil; 3Faculdade de Farmácia, Universidade
Federal de Minas Gerais, Belo Horizonte, MG, Brazil; 4Departamento de Bioquimica e
Biologia Molecular, Universidade Federal do Paraná, Curitiba, PR, Brazil;
5
Departamento de Morfologia, Universidade Federal de Minas Gerais, Belo Horizonte,
MG, Brazil.
Introduction and objectives: 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 various Leishmania
species. These intracellular parasites are responsible for diseases that affect large
numbers of people in the tropical world and for which new methods for disease control
are urgently needed. Materials and methods: We perform sequence analyses of 52
amastin genes present in the genome of L. braziliensis, determine the mRNA levels of
these genes as well as cellular protein localization using parasite expressing GFP
fusions of different amastin genes. We have also knocked-down the expression of a
group of delta-amastin genes and examined the effect of reduced amastin expression in
macrophage infection. Results and Conclusion: Most members of all four amastin
subfamilies present in the L. braziliensis are upregulated in amastigotes and encoded
proteins that are localized in the parasite surface. 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 a group
of proteins that are components of eukaryotic tight junction complexes named claudins.
By knocking-down the expression of amastins in L. braziliensis, their essential role
during infection became evident. 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, which might involve the formation of “claudinlike” structures, electron microscopy analyses of macrophages infected with amastin
knockdown parasites showed drastic alterations in the tight contact that is established
between the surface of wild type amastigotes and the membrane of the parasitophorous
vacuole.
Acknowledgements and Key Words
Brazilian Society for Biochemistry and
Molecular Biology (SBBq)