Fig 1.
Comparison of the Rag pathway and sequence between humans and Leishmania.
Left: The Rag sensing pathway upstream of mTORC1 signaling based on human studies. Cytosolic amino acid sensors responding to levels of Leucine, Arginine or Methionine are shown on top and inhibit Rag activation repressors or activate activators. Multi-protein complexes labeled in bold on segmented circles. Lysosomal amino acid sensors (vATP-ase and SLC38A9) and Rag heterodimer anchoring complex (Ragulator) shown on the lysosome. The Rag complex integrates signals from nutrient sensors in addition to growth factors (FLCN/FNIP1) resulting in modifications to its GTP/GDP bound state. Active state RagA-RagC bind Raptor effectively recruiting TORC1. Note: The Rag protein dimer are overlapping on the human diagram as they perform the same function. This dimer consists either of RagA with RagC or of RagB with RagD. Right: Multiple sequence alignment of human, wildtype and cutaneous Leishmania RagC proteins with domain structure labeled on the human RagC sequence with GTPase domain (blue) and C-terminal Roadblock domain (green). The R231C polymorphism identified in the cutaneous isolate is highlighted in red. Sequence identity is marked with (*), sequence similarity is marked with (:).
Table 1.
List of Leishmania donovani proteins homologous to humans in the RagC axis of the TOR pathway.
Proteins part of the RagC arm of the TOR pathway in humans and their parent complex names are listed along with UniProt accession numbers used for sequence retrieval. Matching L. donovani proteins resulting from a BLASTP search are listed when available along with their respective matching sequence length, percent identity, BLAST score and E-values. Homologues were marked as ABSENT when no protein was found. The 5th column indicates the presence or absence of homologous proteins in L. infantum (I), L. major (Mj), L. tropica (Tr), L. mexicana (Mx) or L. Braziliensis (Br). When matching proteins were found in species other than L. donovani only, the BLASTP metrics are reflective of the top scoring hit in the indicated species.
Fig 2.
Generation of the RagC single amino acid substitution (R231C) mutant and RagC disruption mutant by CRISPR/Cas9.
A. Strategy used to generate the RagC R231C mutant and RagC gene disrupted strains. A gRNA was designed to target a site (green) close to the R231C polymorphism (red) identified in the RagC gene of the Sri Lankan cutaneous L. donovani isolate. L. donovani 1S2D promastigotes were transfected with a CRISPR vector (pLdCNld366140) expressing this RagC specific gRNA, followed by transfection of the donor repair template which contained either: the targeted point mutation (C/T, red) and an additional six nucleotides resulting in silent mutations (purple) to protect the repaired genome from subsequent Cas9 cleavage, or a bleomycin selection marker (black). Genomic DNA from these L. donovani cells clones was subjected to PCR and sequencing analysis. B. Partial sequence of the oligo donor repair induced mutations resulting in a single amino acid substitution in RagC protein (R231C, shown in red) and inactivation of the gRNA targeting site (shown in green, interspaced with disrupting silent mutations in purple). C. Direct sequencing of a PCR product amplified from a L. donovani clone showing both alleles of the RagC gene have been edited to the sequence of the oligo donor (see A & B) repair template. D. PCR analysis of RagC double allele gene disrupted mutants. PCR analysis of three phleomycin resistant clones with primers F2 and R2 show the Bleomycin resistance gene has been inserted into the target site as expected resulting in a 987 bp band, and no 447 bp WT F2R2 band was detected in these RagC disruption mutants.
Fig 3.
Biological effects of a Rag C single amino acid change (R231C) and disruption of the RagC gene.
A. Proliferation of RagC WT (red), gene disrupted (-/-) (blue) and gene edited (R231C) strains (light blue), and RagC WT addback transfections in gene disrupted (orange) and gene edited (pink) strains. Data was measured in quadruplicate and statistical significance calculated using 2way ANOVA with multiple comparisons using RagC WT as the control group and marked for significance if consistent for every time point past day 2. This is the representative data of four repeat experiments. B. Effects of a single amino acid change (R231C) and disruption of RagC on L. donovani 1S2D infection in mice. BALB/c mice were infected by tail vein injection (1x108 pro/mouse) with L. donovani WT, gene edited RagC R231C, gene disrupted RagC (RagC-/-), and their corresponding RagC WT addback strains. Mice were examined for liver parasite burden four weeks after infection. Data was measured using 4 mice per group and statistical significance calculated using 2way ANOVA with multiple comparisons using RagC WT as the control group.
Fig 4.
Incompatibility between the co-expression of wildtype and mutant RagC due to dominant negative effect.
A. Immunoblotting of FLAG-tagged RagC wildtype and R231C mutant transfected in L. donovani 1S2D (Ld 1S2D) and cutaneous L. donovani from Sri Lanka (Cutaneous SL-Ld) strains followed over a period of 30 days. B. Immunoblotting of FLAG-tagged RagC wildtype and R231C transfected in L. donovani 1S2D cell following disruption of the endogenous RagC gene. The results are representative of three independent experiments.
Fig 5.
Leishmania RagA and RagC proteins form a heterodimer complex.
A. Heterodimer formation between wildtype RagC and RagA. Top Panel: RagC was immunoprecipitated with FLAG antibodies and immunoblotting with HA antibodies shows the presence of HA-tagged RagA (Lanes 9) specifically in RagC co-transfected cells. Western blot analysis of input lanes 1–5 are also shown. Bottom Panel: Co-immunoprecipitation showing RagC is captured following immunoprecipitation of RagA. HA-tagged RagA was immunoprecipitated followed by immunoblotting with FLAG antibodies shows the presence of RagC (Lane 9) specifically in Rag A co-transfected cells. Western blot analysis of input lanes 1–5 are also shown. B. Heterodimer formation between mutant R231C RagC and RagA. Top panel: RagC R231C was immunoprecipitated using FLAG antibodies followed by immunoblotting with HA antibodies shows the presence of HA-tagged RagA in RagC R231C co-transfected cells (Lane 10 and positive control Lane 12). Western blot analysis of input lanes 1–6 are shown. 5B Bottom Panel: HA-tagged RagA was immunoprecipitated using HA antibodies followed by immunoblotting with FLAG antibodies shows the presence of FLAG-tagged RagC (Lane 10 and positive control Lane 12). Western blot analysis of input lanes 1–6 are shown. The results are representative of three independent experiments.
Fig 6.
RagA is essential for Leishmania.
A. Strategy used to generate the single allele or double allele L. donovani RagA disruption mutants. A gRNA was designed to target the first half of the RagA coding sequence. L. donovani 1S2D cells were transfected with a CRISPR vector (pLdCNld131620) expressing this RagA specific gRNA, followed by transfection of the bleomycin selection marker donor with 25 nt flanking sequences to integrate into the Cas9 cut site. B. PCR analysis of the surviving phleomycin resistant clones showing the bleomycin resistance gene was inserted into the target site of one RagA allele as expected, but the 1118 bp WT RagA band was still detected in all these phleomycin resistant clones. Note: the middle band is the annealing product during PCR between the 1118 bp WT RagA band and the 1658 bp disruption band. C. Microscope images showing the disruption of both RagA alleles is lethal for L. donovani. The RagA+/- partial mutant cells expressing the RagA targeting gRNA were cloned in a 96 well plate and cell growth was monitored by microscopy. The image for RagA +/- cells was taken one week after cloning; The image for RagA-/- cells was taken four weeks after cloning.