Fig 1.
KHARON1 localization in Leishmania mexicana amastigotes.
Scale bars, 3 μm. A. Leishmania mexicana promastigotes were stained with antibody against α-tubulin (green), with the YL1/2 antibody against tyrosinated α-tubulin (red), and DAPI (blue). The basal body, which is anterior to the kinetoplast (white arrowhead), and the tubulin cytoskeleton are labeled by YL1/2. The yellow arrowheads indicate flagella. B. Cell division stages of axenic amastigotes stained with the YL1/2 antibody (red) and DAPI (blue). The amastigote flagellar structures are labeled with YL1/2 (yellow arrowheads). The kinetoplast is shown in blue, while the nuclei are out of focus in these sections. C. Co-localization of KH1::2A (green) and YL1/2 antibody (red) in axenic amastigotes. K indicates kinetoplast DNA, and N indicates the nucleus. The white arrowhead points to the base of the flagellum, and the yellow arrowhead points to the flagellar structure. D. Immunoblot of KH1::2A expression in purified intracellular amastigotes, and negative control (wild type amastigotes). E. Immunofluorescence of purified intracellular amastigotes, expressing KH1::2A from the Kh1 locus, using antibody against 2A (green) and YL1/2 (red).
Fig 2.
Rescue of ∆kh1 mutants by genomic integration of a Kh1 allele.
A. Schematic representation of genomic region surrounding the Kh1 gene in the wild type locus (top) and in the Δkh1[intKh1] add-back line in which a Kh1::2A::Luc::BSD fusion ORF was integrated into the gene locus (bottom). EcoR1 (RI) and EcoRV (RV) restriction sites are indicated along with the expected size of the digested genomic DNA (gDNA) fragment containing the Kh1 open reading frame. B. Southern blot probed with the Kh1 ORF (top) comparing EcoRI/EcoRV digested gDNA from the Δkh1[intKh1] add-back line to the Δkh1 mutants and an add-back line carrying an episomal copy of the Kh1 ORF, Δkh1[pKh1] [7]. DNA from wild type (WT) and heterozygous knockout (Δkh1/+) lines is also shown. The blot was stripped and re-probed with the LmxGT2 ORF (GT2) as a DNA loading indicator. C. Immunoblot probed with antibody against the 2A epitope to detect the KH1::2A fusion protein. The Luc::BSD product is cleaved from the KH1::2A::Luc::BSD fusion and migrates at approximately 55 kDa (arrowhead), as demonstrated by probing the blot with antibody against luciferase (Luc). The slightly more rapidly migrating band present in each lane probably represents cross-reaction of the antibody with tubulin proteins (middle panel). A replicate blot was probed with anti-α-tubulin antibody (Tub) (bottom panel). D. Growth curve of cultured promastigotes comparing growth rate of wild type (wt), Δkh1mutants and Δkh1[intKh1] add-back parasites over a 7-day time course. Data represent the average and standard deviation of 3 independent growth curves (n = 3). Each sample from each growth curve time point was counted in triplicate and averaged. E. Quantification of THP-1 infection with wild type, Δkh1mutants, and Δkh1[intKh1] add-back promastigotes. Times post infection: h = hours, D = days. Data represent the averages and standard deviations of triplicate infections (n = 3), each counted in triplicate and averaged. These results are similar to those previously published for the Δkh1 null mutant complemented with an episomal copy of Kh1 [7], but the current experiment confirms complementation by the re-integrated Kh1 gene, the complemented line employed throughout this study.
Fig 3.
Infection of BALB/c mice with Δkh1 mutant and Δkh1[intKh1] add-back promastigotes.
A. Average lesion size caused by wild type (n = 5 mice), Δkh1mutants (n = 5), and Δkh1[intKh1] add-back (n = 4) L. mexicana parasites. One mouse (out of 5) inoculated with Δkh1[intKh1] add-back parasites did not form a detectable lesion, possibly due to failed injection, and therefore was excluded from this analysis. The slight difference in lesion kinetics between wild type and add-back parasites may be due to the fact that the add-back line encompasses only one, rather than two, copies of the Kh1 gene. The data in the graph represent the average and standard deviations of measurements for all the footpads (n = 4 or 5) representing each strain. B. Estimated parasite load from limiting dilution experiments. Footpads were removed from 5 mice for the WT and Δkh1 strains and for 4 mice from the Δkh1[intKh1] strain at week 8 post infection. Eight limiting dilution replicates were done for each footpad, and the results were averaged to obtain a number for each footpad. The line in each graph represents the average estimated parasite load for all footpads representing that strain (2.5x1012 parasites per footpad for wild type and 1.3x1012 parasites per footpad for the Δkh1[intKh1] strain). These parasite burdens are similar to those observed for BALB/c mice infected with L. amazonensis under conditions that generated lesions of similar thickness [31]. The numbers and horizontal bars indicate the p values for comparisons of the data for relevant strains.
Fig 4.
Characterization of Δkh1 axenic amastigotes.
A. Immunoblot of wild type (wt) and Δkh1 mutant probed with anti-P4 and anti-α-tubulin antibodies as a loading control. The positions of molecular weight markers are indicated. B. Immunofluorescence of the P4 amastigote-specific antigen. Left, axenic amastigotes were stained with anti-P4 antibody (P4, green), DAPI (blue), and anti-α-tubulin antibody (Tub, red). The 100% shown in the P4 panel represents the percentage of cells analyzed that are positive for P4 (wt, n = 212; Δkh1, n = 240). Right, promastigotes were stained for the same marker as a negative control for the P4 antibody. Scale bar, 3 μm. C. Growth curve of axenic amastigotes comparing growth rate of wild type, Δkh1mutants, and Δkh1[intKh1] add-back lines over a 16-day time course. Numbers represent the averages and ranges from two independent growth curves (n = 2). Each sample from each growth curve time point was counted in triplicate and the values averaged. D. Quantification of infection of THP-1 macrophages with wild type, Δkh1mutants, and Δkh1[intKh1] add-back axenic amastigotes. Times post infection: h = hours, D = days. Data represent the averages and standard deviations of triplicate infections (n = 3), each counted in triplicate and averaged. This experiment is similar to that shown in Fig 2E, except that axenic amastigotes, rather than promastigotes, were employed to initiate infection.
Fig 5.
Evidence of multi-nucleated intracellular amastigotes.
A. Images of THP-1 cells infected with wild type, Δkh1mutants, and Δkh1[intKh1] add-back axenic amastigotes at 3 days post infection. For this experiment, a 10:1 ratio of axenic amastigotes to macrophage was used. Multinucleate parasites are indicated by arrowheads (2N) and chevrons (>2N). Bottom, close-up of cells with single or multiple nuclei. N = Nuclei. Scale bars, 3 μm. B. Quantification by microscopy of multi-nucleated intracellular amastigotes. For all time points, the number of intracellular amastigotes examined for each line was >300. Percentages of parasites with 1, 2, and >2 nuclei are indicated as designated by the graph legend at the right. The p-values were calculated for the number of cells with more than two nuclei (>2N) between the different cell lines at Day 5: wt vs. Δkh1 mutants, p<<0.01; Δkh1 mutants vs. Δkh1[intKh1], p<<0.01; wt vs. Δkh1[intKh1], p = 0.8. The p-value calculated for the number of cells with more than two nuclei (>2N) between the different cell lines at Day 5 were as follows: wild type versus Δkh1 mutants, p<0.01; Δkh1 mutants versus Δkh1[intKh1], p<0.01; wild type versus Δkh1[intKh1], p = 0.8.
Fig 6.
Ultra-structural studies of intracellular amastigotes.
All scale bars represent 200 nm. A. Morphology of wild type and Δkh1 amastigotes 1-day post-infection. Arrowheads indicate the tip of the flagellum making contact with the parasitophorous vacuole. B. Multinucleate and multiflagellated Δkh1 amastigotes inside infected THP-1 macrophages. Nucleus, N; flagellum, F; kinetoplast, K. C. Cellular integrity of wild type and Δkh1 amastigotes on days 1 and 3 post-infection.