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.

Trypanosomatid parasite dynamically changes the transcriptome during infection and modifies honey bee physiology

“…They are cell-surface proteins abundantly expressed in the intracellular amastigote stages of Trypanosoma spp. and Leishmania spp 32,33 . The second highest fraction (759, 24.1%) corresponded to the genes downregulated from the middle to late stage of infection (PI 12-27) and many of them encoded proteins in the kinetoplast respiratory chain with enriched GO terms related to "ATP synthesis coupled proton transport" (Fig.…”

The Transcriptome ofLeishmania majorDevelopmental Stages in Their Natural Sand Fly Vector

“…Interestingly, Pearson correlation and principal component analysis suggested a greater similarity between MP and AM than to the other promastigote stages that would indicate a preadaptation process of the metacyclics to the mammalian host environment. For instance, many of the mRNAs that encode amastin, the main surface glycoprotein that is essential for amastigote intracellular survival (64), are strongly upregulated already in the MP stage. From the metabolic point of view, increased mitochondrial activity that is associated with amastigote survival and growth already occurs at the MP stage.…”

Wnt5a Signaling Promotes Host Defense againstLeishmania donovaniInfection

“…One of the early strategies used by the invading L. donovani parasites is to inhabit macrophages, stationing and sustaining themselves within protective vacuoles (1,2). The role of L. donovani-specific amastin in parasite survival and sustainment of infection has recently been reported (30). Parasite-induced changes in the host macrophage cytoskeleton and its associated components have also been suggested to contribute to the altered immune status that supports parasite sustainment (41)(42)(43).…”

“…L. donovani-specific amastin gene expression was used to validate L. donovani infection (Fig. 1D) (30). A decrease in Wnt5a protein upon L. donovani promastigote infection could be due to the activity of L. donovani proteases, such as GP63.…”