Fig 1.
Genomic organization of the amastin genes in L. braziliensis.
The schematic representation of 52 amastin gene copies located on nine different chromosomes was based on the complete genome sequence of L. braziliensis MHOM/BR/75/M2904 obtained from the Tritryp database (www.tritrypdb.org). The numbers below the boxes correspond to the given names of each amastin L.braziliensis gene homolog in GeneDB. Boxes with the same background correspond to amastin gene copies belonging to the same subfamily. Eight tuzin genes, shown as gray boxes, are associated only with δ-amastins.
Fig 2.
Phylogenetic tree of amino acid sequences of 52 amastins from L. braziliensis.
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).
Fig 3.
Homology-based 3D modeling of α, β, γ and δ amastins.
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.
Fig 4.
Differential expression of amastin mRNA during the L. braziliensis life cycle.
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.
Fig 5.
Subcellular localization of distinct amastins in fusion with GFP.
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.
Fig 6.
RNAi knockdown of amastin genes in Leishmania braziliensis.
(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 Leishmania (gray box). (B) Northern blot analyses of RNA isolated from (P) promastigote and (A) axenic amastigotes from wild type L. braziliensis (WT) and two cloned cell lines of L. braziliensis 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 L. braziliensis 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 L. braziliensis 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.
Fig 7.
Infection of mouse macrophages and BALB/c mice footpads with WT L. braziliensis and RNAi-1060 cell lines.
(A) Intraperitoneal macrophages from BALB/c mice were incubated for 24 hours at 34°C with stationary phase promastigotes from WT L. braziliensis 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 L. braziliensis, L. braziliensis 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.
Fig 8.
Re-expression of amastin sequences in RNAi knockdown parasites rescue infection capacity of L. braziliensis.
(A) Total protein extracts from promastigotes (P) and amastigotes (A) of WT L. braziliensis, 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 L. braziliensis, 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 L. braziliensis, L. braziliensis 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.
Fig 9.
Amastin knockdown affects amastigote interactions with macrophage membranes.
(A) Transmission electron microscopy of macrophages infected with WT L. braziliensis (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.