Figure 1.
CFAS expression in L. infantum.
(A) Quantitative analysis (RT-qPCR) of CFAS RNA expression in mid-log promastigotes (Pro) and amastigotes (Ama). RQ, relative quantity with reference to EF1α control; error bars represent standard error of mean. (B) Immunoblotting analysis of L. infantum promastigotes expressing C-terminal myc-tagged CFAS (CFAS-myc) harvested in early (EL), mid (ML) and late (LL) log phase growth and in early (ES) and late (LS) stationary phase growth. CFAS-myc is detected by anti-myc; the metacyclic protein HASPB is a marker for in vitro differentiation; the ER marker BiP is constitutively expressed during the Leishmania growth cycle. (C) Left: immunoblotting of wild type (WT) and early log phase L. infantum CFAS-HA promastigote total lysates. Right: fractionation of CFAS-HA total lysate (TL) into cytosolic (soluble, S) or membrane (pellet, P) fractions prior to immunoblotting. GP63, membrane-specific marker; BiP, loading control.
Figure 2.
Subcellular localization of CFAS-HA in L. infantum.
(A) Confocal images of L. infantum promastigotes expressing CFAS-HA. Cells were co-stained with anti-HA (red) and anti-BiP (green) and mounted with Vectorshield containing DAPI (blue, DNA). Higher magnification images of a co-stained parasite are shown on the right of the Figure. (B) Confocal images of CFAS-HA expression in intra-macrophage amastigotes at 72 hr post-infection. Macrophages infected with wild type (WT) L. infantum (left panel) and L. infantum expressing CFAS-HA (right panel). DAPI (blue), macrophage and parasite DNA; anti-HA (green), CFAS-HA.
Figure 3.
Generation of CFAS mutant parasites and in vitro growth analysis.
(A) Region of chromosome 8 containing the single L. infantum CFAS locus and the constructs used for targeted gene deletion. (B) Representative Southern blot of L. infantum and L. major wild type (wt) and CFAS mutant DNAs hybridised with a CFAS-specific probe (see A above and Materials and Methods). Two independent L. infantum CFAS null clones (1 and 2, −/−) are shown; the single allele deletion prior to generation of null clone 2 (+/−) and a complemented add-back clone from that line (−/−/+, CLN-C2, Table 1) are included. One of the clones of L. major transgenic for luciferase and CFAS (+CFAS, CLN-4, Table 1) is also shown. A labelled β-tubulin specific DNA probe was used as a loading control. (C) In vitro growth of L. infantum cell lines. L. infantum wild type and CFAS null and complemented lines (as analysed in (B)) were grown over 5 days in HOMEM/20% FCS at 26°C and parasites counted as described (Materials and Methods). Mean values derived from triplicate culture populations for each cell line are plotted. (D) In vitro growth of L. major cell lines. L. major wild type containing an integrated luciferase gene (L. major LUC) and the same line expressing CFAS (analysed in B) were grown over 7 days in either M199/20% FCS (top) or HOMEM/20% FCS (bottom) and parasites counted as described in (C).
Table 1.
C19Δ fatty acid content of wild type and CFAS mutant L. infantum and L. major promastigotes.
Figure 4.
Fatty acid analysis of wild type and CFAS mutant Leishmania by gas chromatography–mass spectrometry.
(A) Total ion chromatogram of derivatised fatty acid extracts from L. infantum wild type (Wt), CFAS null mutant (CFAS−/−) and CFAS complemented (−/−/+; Table 1, CLN-C2) cell lines. A spectral peak with a retention time of ∼45 min and corresponding to C19 cyclopropanated fatty acid (C19Δ) is present in wild type and complemented parasites but absent from the CFAS−/− null. (B) Total ion chromatogram of derivatised fatty acid extracts from L. major wild type (Wt) and CFAS expressing (+CFAS; Table 1, CLN-4) parasites. The spectral peak corresponding to C19Δ, with a retention time of ∼45 min, is absent in L. major wild type but present in extracts from L. major parasites expressing CFAS. The identity of C19Δ FAME was confirmed by comparison with bacterial C17Δ and C19Δ FAME standards.
Figure 5.
Sub-cellular localisation of cyclopropanated fatty acids in L. infantum.
(A) Immunoblot analysis of sucrose gradient-separated sub-cellular fractions of wild type L. infantum promastigotes. Fractions 16–24 are shown, probed with antibodies specific for BiP, Rab1 and gp63. (B) GC-MS was used to determine the cyclopropanated fatty acid content of the fractions analysed in A, in comparison with wild type parasites. (C) Total ion chromatogram of derivatised fatty acid extracts from fraction 23. The spectral peak corresponding to the C19 cyclopropanated fatty acid (C19Δ) has a retention time of ∼42 min in this analysis.
Figure 6.
Phenotypic analysis of L. infantum CFAS mutants in vitro.
(A) Bone marrow-derived macrophages were infected with late stationery phase wild type, CFAS null and complemented CLN-D12 L. infantum, at a macrophage to parasite ratio of 1∶10. Numbers of infected and un-infected macrophages were counted (at least 200 macrophages per cell line at each time point) and the percentage infectivity calculated. (B) Proline uptake assay. The wild type, null and complemented L. infantum lines used in (A) were incubated with 3H-labelled L-proline and the internalized radiolabel quantified by liquid scintillation counting. Assays were performed in triplicate for each cell line. The wild type, null and complemented L. infantum used in (A) were cultured in M199 medium/20% FCS (C) or in the same medium supplemented with 300 µM hydrogen peroxide (D). Parasite growth rate over 72 hr (C) or 96 hr (D) was monitored by counting parasite numbers at each time point. Statistical differences was determined using the unpaired Student’s t-test with a value of P<0.05 considered significant. The same histogram shading, as shown in (A), is used in all panels of this figure to designate the different parasite lines.
Figure 7.
Survival of L. infantum CFAS mutants following infection in vivo.
Groups of 5 BALB/c mice were infected intravenously with 2×107 L. infantum wild type (Wt), CFAS null (CFAS−/−) and CFAS complemented CLN-D12 parasites. Parasite numbers were determined in the liver (A) and the spleen (B) at 14 and 28 days post-infection (as described in Materials and Methods).
Figure 8.
CFAS expression attenuates L. major dermal infection in vivo.
BALB/c mice were infected intradermally with 1×106 L. major LUC (closed squares) or L. major LUC+CFAS (CLN-4, open triangles) and parasites were visualised by bioluminescence imaging using an IVIS over the course of the infection. Luciferase activity (photons/second) is expressed as (A) a percentage change over time and (B) as a fold increase from day 3 post-infection. Lesion progression was monitored by measurement of lesion diameter (C) and thickness (D) from week 4 onward. * p<0.05, ** p<0.01, *** p<0.001, by unpaired Student’s t-test (n = 5).
Figure 9.
The expression of CFAS does not enhance viscerotropism of L. major.
BALB/c mice were infected with L. major LUC (black bars) or L. major LUC+CFAS (white bars) and (A) hepatomegaly and splenomegaly and (B) parasite burdens were determined at Day 70 post-infection. Parasite clones were those used in Figure 8. p = 0.07 by unpaired Student’s t-test (n = 5).