Amastin Knockdown in <i>Leishmania braziliensis</i> Affects Parasite-Macrophage Interaction and Results in Impaired Viability of Intracellular Amastigotes

<div><p>Leishmaniasis, a human parasitic disease with manifestations ranging from cutaneous ulcerations to fatal visceral infection, is caused by several <i>Leishmania</i> 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 <i>Trypanosoma cruzi</i> and <i>Leishmania</i> spp. Here, we showed that the genome of <i>L</i>. <i>braziliensis</i> contains 52 amastin genes belonging to all four previously described amastin subfamilies and that the expression of members of all subfamilies is upregulated in <i>L</i>. <i>braziliensis</i> 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 <i>L</i>. <i>braziliensis</i>, their essential role during infection became evident. δ-amastin knockdown parasites showed impaired growth after <i>in vitro</i> 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.</p></div

Phylogenetic tree of amino acid sequences of 52 amastins from <i>L</i>. <i>braziliensis</i>.

<p>The sequences were aligned using the MUSCLE algorithm and a neighbor joining tree was generated using the MEGA6 software. Branch lengths are drawn proportion to evolutionary change with bootstrap values shown on each node. Classification into four amastin sub-families shown on the right was based on Jackson (2010).</p

Amastin knockdown affects amastigote interactions with macrophage membranes.

<p>(A) Transmission electron microscopy of macrophages infected with WT <i>L</i>. <i>braziliensis</i> (WT) and two cloned cell lines expressing amastin siRNA, RNAi-1060 cl1 and RNAi-1060 cl5. Left side panels show amastigotes surrounded by the macrophage parasitophorous vacuole (PV) membrane. In contrast to WT parasites, that are in close contact with the PV membrane, larger distances between the membranes of intracellular amastigotes and the PV membranes are observed in macrophages infected with both clones of RNAi knockdown mutants. Right side panels show magnified images of the points of contact between parasite (arrows) and PV (arrowheads) membranes with regions where disrupted membrane structures are observed in macrophages infected with the RNAi knockdown mutants. Parasite nucleus (n), kinetoplast (k), subpellicular microtubule (m) and flagellum (f) are indicated by lower case letters. (B) Ploted ratios between the area of macrophage PV and the area of the amastigote inside each vacuole. Error bars represented the SD values obtained from measurements of the areas of twenty vacuoles and their respective parasites in each experimental group.</p

Differential expression of amastin mRNA during the <i>L</i>. <i>braziliensis</i> life cycle.

<p>Total RNA (10 μg/lane), extrated from promatigote (P) and axenic amastigote (A) forms were separated by electrophoresis, transfered to nylon membranes and probed with the ³²P-labelled sequences corresponding to an α-amastin (LbM.28.1550), β-amastin (LbM.30.0980), γ-amastin (LbrM.24.1600) and two δ-amastins (LbM.08.0300 and LbrM.20.1060). Bottom panels show hybridization of the same membranes with a fragment of the 5S rRNA.</p

Subcellular localization of distinct amastins in fusion with GFP.

<p>Promastigotes were transiently transfected with the plasmids pSP-Ama0980-GFP (A), pSP-Ama1600-GFP (B), pSP-Ama1060-GFP (C) and pSP-nGFP (D) as a control plasmid. Transfected parasites were fixed with 2% paraformaldehyde, stained with DAPI and visualized under a fluorescence microscope. Nuclear and kinetoplast DNA are shown in blue.</p

Re-expression of amastin sequences in RNAi knockdown parasites rescue infection capacity of <i>L</i>. <i>braziliensis</i>.

<p>(A) Total protein extracts from promastigotes (P) and amastigotes (A) of WT <i>L</i>. <i>braziliensis</i>, the cloned cell RNAi-1060 cl1 and two cloned cell lines derived from the RNAi-1060 parasites the were transfected with an RNAi-resistant amastin gene (RNAi-1060-R2 and RNAi-1060-R4) were analysed by western blot using an anti-HA antibody. Bands corresponding to the Ama1060 synthetic gene containing the HA epitope are shown only in the re-expressor cell lines. The same blot was incubated with anti-tubulin antibody as a loading control. (B) Stationary phase promastigotes from WT <i>L</i>. <i>braziliensis</i>, two cloned cell lines expressing amastin dsRNA, RNAi-1060 cl1 and cl5 and two cloned cell lines that express a RNAi resistant amastin sequence were used to infect BALB/c mice peritoneal macrophages at a ratio of 10:1 (parasites/cell) and the numbers of intracellular amastigotes determined 24 and 72 hours post-infection. (C) Stationary phase promastigotes (10⁷ parasites) from WT <i>L</i>. <i>braziliensis</i>, <i>L</i>. <i>braziliensis</i> transfected with the pIR1PHLEO vector containing GFP, the two cloned cell lines expressing amastin dsRNA, RNAi-1060 cl1 and cl5 and two cloned cell lines that express a RNAi resistant amastin sequence were used to infected BALB/c mice footpads. One or two weeks post-infection, parasitism was evaluated by the limiting dilution of parasites recovered from the mice footpads.</p

RNAi knockdown of amastin genes in <i>Leishmania braziliensis</i>.

<p>(A) The plR1-Phleo plasmid containing two opposite amastin fragments with a stem-loop stuffer fragment (black box), Phleomycin resistance (gene PHLEO) and the rRNA promoter (P rRNA) is shown integrated into the SSU rRNA locus of <i>Leishmania</i> (gray box). (B) Northern blot analyses of RNA isolated from (P) promastigote and (A) axenic amastigotes from wild type <i>L</i>. <i>braziliensis</i> (WT) and two cloned cell lines of <i>L</i>. <i>braziliensis</i> transfected with a construct that generates δ-amastin dsRNA named RNAi-1060-cl1 and cl5. The blots were probed with a ³²P-labelled DNA fragment corresponding to the LbrM.08.1060 amastin gene. (C) Low-molecular-weight RNAs isolated from promastigotes and amastigotes from WT <i>L</i>. <i>braziliensis</i> as well as from promastigotes from RNAi-1060 cl1 and amastigotes from the two cloned cell line transfected with amastin dsRNA constructs (RNAi-1060 cl1 and cl5) were fractionated on a 15% polyacrylamide gel and probed with a mixture of ³²P-labelled oligonucleotide probes corresponding to the full length LbrM.08.1060 amastin gene. siRNA indicates the position of small interfering RNA bands that hybridized with δ-amastin oligonucleotide probes, which co-migrate with a 26 nt DNA molecular weight marker. Hybridization of the same blot with a probe corresponding to the <i>L</i>. <i>braziliensis</i> Glu-tRNA is also shown as a loading control. (D) Total protein extracts from the cloned cell RNAi-1060 cl1 was analyzed by western blot using an antibody generated against the recombinant Ama1060. The same blot was incubated with anti-α-tubulin as a loading control.</p

Homology-based 3D modeling of α, β, γ and δ amastins.

<p>Structural predictions were done using PHYRE web server and the predicted structures of α, β, γ and δ amastins, were imaged using the UCFS Chimera program. α-amastin is shown in green, β-amastin is shown in gray, γ-amastin is shown in red, δ-amastin is shown in yellow and the superimposed mouse claudin 15 model is shown in blue.</p

Infection of mouse macrophages and BALB/c mice footpads with WT <i>L</i>. <i>braziliensis</i> and RNAi-1060 cell lines.

<p>(A) Intraperitoneal macrophages from BALB/c mice were incubated for 24 hours at 34°C with stationary phase promastigotes from WT <i>L</i>. <i>braziliensis</i> cultures and the two cloned cell lines RNAi-Ama1060 cl1 and cl5 at a ratio of 10: 1 parasites/cell. After washing non-internalized promastigotes macrophages were incubated for 24, 48 or 72 hours before the cells were stained with DAPI and the numbers of intracellular amastigotes, visualized by fluorescence microscopy, were determined. (B) BALB/c mice were infected in the footpads with 10⁷ stationary phase promastigotes from WT <i>L</i>. <i>braziliensis</i>, <i>L</i>. <i>braziliensis</i> transfected with the pIR1PHLEO vector containing GFP, and two cloned cell lines expressing δ-amastin siRNA, RNAi-1060cl1 and cl5. Two, four and nine weeks after infection, parasitism was evaluated by the limiting dilution method.</p

Video_5_High-dimensional intravital microscopy reveals major changes in splenic immune system during postnatal development.mp4

Spleen is a key organ for immunologic surveillance, acting as a firewall for antigens and parasites that spread through the blood. However, how spleen leukocytes evolve across the developmental phase, and how they spatially organize and interact in vivo is still poorly understood. Using a novel combination of selected antibodies and fluorophores to image in vivo the spleen immune environment, we described for the first time the dynamics of immune development across postnatal period. We found that spleens from adults and infants had similar numbers and arrangement of lymphoid cells. In contrast, splenic immune environment in newborns is sharply different from adults in almost all parameters analysed. Using this in vivo approach, B cells were the most frequent subtype throughout the development. Also, we revealed how infections – using a model of malaria - can change the spleen immune profile in adults and infants, which could become the key to understanding different severity grades of infection. Our new imaging solutions can be extremely useful for different groups in all areas of biological investigation, paving a way for new intravital approaches and advances.</p