Leishmania braziliensis strain:M2904 Raw sequence reads

Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by more than 20 Leishmania species. These protozoan parasites replicate in the gut of a sandfly vector as extracellular, flagellated promastigotes as well as non flagellated amastigotes in the parasitophorous vacuole of vertebrate host macrophages. Amastins are surface glycoproteins encoded by large gene families present in the genomes of several Leishmania species as well as other trypanosomatids and known to be 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 sub-families is up-regulated in L. braziliensis amastigotes. Although primary sequence alignments show no homology to any known protein sequence, homology based on secondary structure predictions indicates that amastins are related to a group of proteins that are components of tight junction complexes named claudins. By knocking-down the expression of amastins in L. braziliensis their essential role during infection became evident, since amastin knock-down parasites showed impaired growth after in vitro infection of mouse macrophages and completely fail to produce infection when inoculated in BALB/c mice footpads. This attenuated phenotype was reverted by the re-expression of an RNAi-resistant amastin gene. Further highlighting their essential role in host-cell interaction, electron microscopy analyses of macrophages infected with parasites in which amastin genes have been knocked-down showed drastic alterations in the tight contact stablished between the surface of amastigotes and the parasitophorous vacuole membrane.