Figure 1.
Heterogeneity in the distribution of average DNA content is similar in different axenic isolates of Entamoeba histolytica.
Cells from E. histolytica isolates (A) HM-1:IMSS, (B) 200:NIH, (C) KCG:0687:03, (D) 2592100 and (E) DS4-868 were fixed 48 h after sub-culture, stained with PI and the cellular DNA content (x-axis) was analyzed by flow cytometry. The y-axis represents cell count.
Figure 2.
Distribution of nuclear DNA content in different axenic isolates of E. histolytica.
Cells from 48 h cultures of (A) E. histolytica HM-1:IMSS; 200:NIH; KCG:0687:03 and E. invadens IP-1 and (B) E. histolytica HM-1:IMSS, 2592100 and DS4-868 were fixed and stained with DAPI to analyse the DNA content of individual nuclei. The DAPI fluorescence values for each isolate were drawn as individual histograms. The x-axis shows fluorescence values, representing the nuclear DNA content on a linear scale and the y-axis represents the number of nuclei as a fraction of the highest number of nuclei obtained in any sub-class of each scan.
Figure 3.
Nuclear DNA content of E. histolytica trophozoites is higher during axenic growth.
(A) Cells from xenic and axenic cultures of 2592100 and DS4-868 were fixed and stained with DAPI to analyze the DNA content of individual nuclei. The x-axis is fluorescence plotted on a logarithmic scale to demonstrate the great difference in DNA content between xenic and axenic nuclei. The y-axis is as in fig. 2. (B) Nuclear contour area was measured as an indicator of differences in nuclear size or volume in cells growing under xenic and axenic conditions. The y-axis is as above.
Table 1.
Percentage of uni-nucleated, bi-nucleated and multi-nucleated cells in different isolates of E. histolytica.
Figure 4.
Cell division of E. histolytica in axenic cultures.
CFSE labeled E. histolytica HM-1:IMSS cells were monitored for cell division and proliferation over time. CFSE labeled cells were harvested and fixed at 12 h intervals. CFSE fluorescence of these samples is plotted as an overlay diagram. PI fluorescence (cellular DNA content) from a parallel culture is also plotted as an overlay. Auto-fluorescence (AF) was determined at 0 h. y-axis represents cell count. The data is representative from 3 independent replicates.
Table 2.
Cell number and percentage of uni-nucleated, bi-nucleated and multi-nucleated cells during cell division of E. histolytica in axenic cultures.
Figure 5.
E. histolytica trophozoites show heterogeneity in nuclear number during intestinal tissue invasion.
Intestinal tissue sections were stained with polyclonal anti-Eh lectin antibody to identify amoeba cells followed by hematoxylin staining of the nuclei. (A) Two representative images are shown where amoeba cells (indicated by arrowheads) contain different number of nuclei (indicated by arrows). Scale bar is 10 µm. (The image of each amoeba cell was verified at high magnification to be of a single intact cell and the size of host cell nuclei was found to be double that of the amoeba nuclei- data not shown). (B) Intestinal tissue sections (n = 3) were counted for the presence of uni- (1), bi- (2) and multi- (>2) nucleated amoeba cells.
Figure 6.
Re-xenisation leads to reduction in DNA content of axenic trophozoites.
Axenic cultures of E. histolytica 2592100 were re-xenized by inoculation into xenic culture medium containing the original bacterial flora. Cells were fixed for analysis of nuclear DNA content after the third (Sc3) and seventh (Sc7) subcultures. DNA content (x-axis) of re-xenized samples as well as the xenic and axenic cells of the same isolate were analyzed by scanning cytometry and are represented as histograms. y-axis is described above. The three panels A B and C depict independent re-xenisation experiments.
Figure 7.
E. invadens IP-1 trophozoites contain 40 fold higher nuclear DNA content than cysts.
The DAPI fluorescence values of E. invadens cyst and trophozoite nuclei are compared on a logarithmic scale (x-axis). y-axis represents the number of nuclei as a fraction of the highest number of nuclei obtained in any one sub-class of each scan.
Figure 8.
Relative abundance of a representative set of target sequences in E. histolytica and E. invadens.
(A) qPCR (protein coding sequences and rDNA) and semi-quantative PCR (tRNA genes) was performed on genomic DNA isolated from fixed cells of xenic and axenic E. histolytica 2592100 using specific primer sets described in Table S1. Relative abundance of target sequences was calculated as described in the methods section. Values greater than 1 on the y-axis indicate greater abundance in axenic cells and values less than 1 on the y-axis indicate greater abundance in xenic cells. (B) qPCR (protein coding sequences) and semi-qPCR (tRNA genes) was performed on genomic DNA isolated from E. invadens IP-1 trophozoites and cysts using specific primer sets described in Table S2. Relative abundance was calculated as described in the methods section. Values greater than 1 on the y-axis indicate greater abundance in trophozoites and values less than 1 on the y-axis indicate greater abundance in cysts.