THE ROLE OF COLONIC ANTIMICROBIAL PEPTIDES IN HUMAN BLASTOCYSTIS INFECTIONS

Ph.DDOCTOR OF PHILOSOPH

Membrane Surface Features of Blastocystis Subtypes

Blastocystis is a common intestinal protistan parasite with global distribution. Blastocystis is a species complex composed of several isolates with biological and morphological differences. The surface coats of Blastocystis from three different isolates representing three subtypes were analyzed using scanning electron microscopy. This structure contains carbohydrate components that are also present in surface glycoconjugates in other parasitic protozoa. Electron micrographs show variations in the surface coats from the three Blastocystis isolates. These differences could be associated with the differences in the pathogenic potential of Blastocystis subtypes. Apart from the surface coat, a plasma membrane-associated surface antigen has been described for Blastocystis ST7 and is associated with programmed cell death features of the parasite

Seeing the Whole Elephant: Imaging Flow Cytometry Reveals Extensive Morphological Diversity within <i>Blastocystis</i> Isolates

<div><p><i>Blastocystis</i> is a common protist isolated in humans and many animals. The parasite is a species complex composed of 19 subtypes, 9 of which have been found in humans. There are biological and molecular differences between <i>Blastocystis</i> subtypes although microscopy alone is unable to distinguish between these subtypes. <i>Blastocystis</i> isolates also display various morphological forms. Several of these forms, however, have not been properly evaluated on whether or not these play significant functions in the organism's biology. In this study, we used imaging flow cytometry to analyze morphological features of <i>Blastocystis</i> isolates representing 3 subtypes (ST1, ST4 and ST7). We also employed fluorescence dyes to discover new cellular features. The profiles from each of the subtypes exhibit considerable differences with the others in terms of shape, size and granularity. We confirmed that the classical vacuolar form comprises the majority in all three subtypes. We have also evaluated other morphotypes on whether these represent distinct life stages in the parasite. Irregularly-shaped cells were identified but all of them were found to be dying cells in one isolate. Granular forms were present as a continuum in both viable and non-viable populations, with non-viable forms displaying higher granularity. By analyzing the images, rare morphotypes such as multinucleated cells could be easily observed and quantified. These cells had low granularity and lower DNA content. Small structures containing nucleic acid were also identified. We discuss the possible biological implications of these unusual forms.</p></div

Multinucleated <i>Blastocystis</i> cells do not display higher granularity compared to uninucleated cells.

<p>The spot wizard of the IDEAS software was used to group cells based on number of nuclei. This was done by counting the ‘spots’ stained with Hoechst. The images were then visually analysed to determine the precise spot values that correspond to the number of nuclei (A). Dot-plots were generated for each ST to analyse granularity in multinucleated cells. These plots show that cells with high granularity are found in the populations of uninucleated cells.</p

Viable <i>Blastocystis</i> cells with high granularity have higher DNA content.

<p>Dot-plot shows the populations of <i>Blastocystis</i> cells plotted according to circularity and Hoechst staining.</p

Gating for round and irregular shapes of <i>Blastocystis</i>.

<p>The shapes of <i>Blastocystis</i> were selected based on aspect ratios from the brightfield channel (M03) and CFSE staining (M02). Viable and non-viable cells were plotted separately. The above graphs shows the gating for <i>Blastocystis</i> ST1-NUH9 isolate (A). The average proportion of round and irregular shapes fond in cultures of <i>Blastocystis</i> ST1-NUH9, ST4-WR1 and ST7-B were then plotted in a graph (B). These are based on three separate batches of cultures and independent runs in ImageStream. Error bars signify standard error values. <i>p</i>-values comparing the proportion of round cells between viable and non-viable populations are 0.06, 0.47 and 0.31 for ST1-NUH9, ST4-WR1 and ST7-B isolates, respectively.</p

Image gallery of rare <i>Blastocystis</i> multinucleated cells.

<p>The nuclei is visualized by Hoechst staining. The nuclei number more than three and some appear to be concentrated in the center and not along the edges (A). Some of these cells also appear to show nuclear condensation. (B) Image gallery showing small structures which contains DNA.</p

Image gallery of <i>Blastocystis</i> showing classical morphological forms at varying sizes in the viable populations.

<p>Images were acquired using EDF setting of the imaging flow cytometer. Each cell is represented by two images: Hoechst-CFSE-staining composite and brightfield image. The nuclei were stained with Hoechst and the vacuole with CFSE. These round forms show a single nuclei located at the edge of the cell. These forms comprise more than half of the population in all the subtypes studied.</p

Analysis of <i>Blastocystis</i> based on granularity.

<p><i>Blastocystis</i> populations were plotted according to mean pixel intensities of both side scatter channel and PI staining to determine the cells' granularity and viability, respectively (A). Cells with higher PI staining have more granularity compared to cells with lower PI staining. This observation is common to all subtypes used in this study as shown in a graph (B); *, <i>p</i> < 0.05. Sample images showing cells with high granularity among viable and non-viable populations (C). These cells may represent true granular cells and degenerating cells, respectively.</p

Viable <i>Blastocystis</i> size profiles indicated by cell diameter range and average diameter.

<p>Viable <i>Blastocystis</i> size profiles indicated by cell diameter range and average diameter.</p