Date Published: December 7, 2015
Publisher: Public Library of Science
Author(s): Rita Marcia Cardoso de Paiva, Viviane Grazielle-Silva, Mariana Santos Cardoso, Brenda Naemi Nakagaki, Rondon Pessoa Mendonça-Neto, Adriana Monte Cassiano Canavaci, Normanda Souza Melo, Patrícia Massara Martinelli, Ana Paula Fernandes, Wanderson Duarte daRocha, Santuza M. R. Teixeira, Stephen M. Beverley.
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.
More than 20 species of the genus Leishmania cause leishmaniasis, a human illness with a large spectrum of clinical manifestations that range from self-resolving skin lesions to life-threatening visceral diseases. Endemic in eighty-eight countries from tropical and subtropical areas of the world, Leishmaniasis has an estimated prevalence of 12 million cases with annual mortality rate of 60,000 people (www.who.int/topics/leishmaniasis/en/) for which there is no vaccine or adequate treatment. Thus, studies of various Leishmania species including the complete genome sequences of Leishmania major, Leishmania infantum and Leishmania braziliensis [1, 2] have been directed towards the identification of virulence factors used by the parasite to infect and survive within mammalian host cells as well as towards the development of new forms of treatment and disease prevention. Although comparative studies showed that L. major, L. braziliensis and L. infantum have very similar genomes regarding gene content and organization, the presence of specific sequences and pathways, such as retrotransposons and an active RNAi machinery found in L. braziliensis [3, 4], indicate a greater than expected diversity within this species. The differences regarding the presence of RNAi machinery also imply that different approaches must be used for functional genomic studies with these parasites.
Expansion of families encoding surface proteins is one of the main characteristics revealed with the complete genome sequences of different trypanosomatid parasites. Several of these proteins appear to be involved with host parasite interactions and are likely to play a role in the parasite evasion of host immune responses. In the case of T. cruzi and Leishmania, gene families encoding surface proteins may be directly responsible for the ability of these parasites to invade and multiply within mammalian host cells. However, because they are encoded by multiple genes that are usually dispersed in the genome, knockout experiments to determine their functions may not be applied and, in the case of T. cruzi and most Leishmania species, the strategy of gene silencing has also been hampered by the absence of functional RNAi pathways in these parasites.