BLASTOCYSTIS: INVESTIGATION OF PARASITE ADHESION AND ITS ROLE IN MEDIATING INTESTINAL BARRIER COMPROMISE

Ph.DDOCTOR OF PHILOSOPH

Intra-Subtype Variation in Enteroadhesion Accounts for Differences in Epithelial Barrier Disruption and Is Associated with Metronidazole Resistance in <i>Blastocystis</i> Subtype-7

<div><p><i>Blastocystis</i> is an extracellular, enteric pathogen that induces intestinal disorders in a range of hosts including humans. Recent studies have identified potential parasite virulence factors in and host responses to this parasite; however, little is known about <i>Blastocystis</i>-host attachment, which is crucial for colonization and virulence of luminal stages. By utilizing 7 different strains of the parasite belonging to two clinically relevant subtypes ST-4 and ST-7, we investigated <i>Blastocystis</i>-enterocyte adhesion and its association with parasite-induced epithelial barrier disruption. We also suggest that drug resistance in ST-7 strains might result in fitness cost that manifested as impairment of parasite adhesion and, consequently, virulence. ST-7 parasites were generally highly adhesive to Caco-2 cells and preferred binding to intercellular junctions. These strains also induced disruption of ZO-1 and occludin tight junction proteins as well as increased dextran-FITC flux across epithelial monolayers. Interestingly, their adhesion was correlated with metronidazole (Mz) susceptibility. Mz resistant (Mz^r) strains were found to be less pathogenic, owing to compromised adhesion. Moreover, tolerance of nitrosative stress was also reduced in the Mz^r strains. In conclusion, the findings indicate that <i>Blastocystis</i> attaches to intestinal epithelium and leads to epithelial barrier dysfunction and that drug resistance might entail a fitness cost in parasite virulence by limiting entero-adhesiveness. This is the first study of the cellular basis for strain-to-strain variation in parasite pathogenicity. Intra- and inter-subtype variability in cytopathogenicity provides a possible explanation for the diverse clinical outcomes of <i>Blastocystis</i> infections.</p></div

Inhibition of <i>Blastocystis</i> ST-7 (H) adhesion by galactose rescues <i>Blastocystis</i> ST-7 (H)–induced ZO-1 tight junction degradation.

<p>(A) Dose-dependent inhibition of galactose on <i>Blastocystis</i> ST-7 (H) adhesion to Caco-2 monolayers. <i>Blastocystis</i> ST-7 (H) were incubated with epithelial cells in the presence of different concentrations of galactose and glucose (50 and 100 mM, respectively). A value of 100% was assigned to number of binding parasites without addition of sugars as control. The numbers of attached parasites with galactose addition were normalized to control.<i>*</i>, p<0.01 vs. control. (B) Representative western blot analysis of ZO-1 level in Caco-2 epithelium. Caco-2 monolayers were infected with <i>Blastocystis</i> ST-7 (H) in the presence of saccharides galactose and glucose at 100 mM and incubated for 1 h. Monolayers were washed and prepared for western blotting. Normal culture media with no sugar addition was used as the negative control. Galactose rescued <i>Blastocystis</i>–induced ZO-1 tight junction degradation. (C) Quantification of ZO-1 levels through densitometry analysis of Western blot radiographs. Densitometric values of ZO-1 signals were quantified and expressed as the ratio to α-Tubulin. ZO-1 degradation was significantly rescued by addition of galactose. <i>*</i>, p<0.01 vs. ST-7 (H)-treated sample. Values are the means ± standard errors from data of three experiments. Error bars represent the standard errors. Glu, glucose; Gal, Galactose.</p

Device-to-device communications at the terahertz band:open challenges for realistic implementation

Abstract One of the key parameters that plays a major role in enabling the data rate requirements is spectrum or bandwidth, which is scarce and expensive. Therefore, spectrum management policies are required to optimize its usage to meet all the requirements and the promised data rate growth. Another strategy to deal with spectrum scarcity is to move toward higher frequency bands (terahertz bands), which are expected in the next 6G communication standard. It is therefore important to develop not only new techniques that enable efficient dynamic spectrum access and sharing at such bands, but also suitable channel models for the terahertz bands. Meanwhile, offloading mechanisms are very promising for cellular networks where a plethora of options have been proposed in the research arena in terms of device-to-device, licensed assisted access, or WiFi offloading, among others; but their behavior, when operated at high frequencies (terahertz band) remains unclear. Therefore, this article will tackle two technologies that will shape future networks: terahertz channel modeling/communications and offloading mechanisms

<i>Blastocystis</i> exhibits intra-subtype variation in susceptibility and resistance to Mz.

<p>Graph representing IC50s of Mz against <i>Blastocystis</i> ST-4 and ST-7 isolates tested in the study using the resazurin assay. Y axis was presented on a logarithmic scale in base 5. The IC50s of Mz against ST-7 isolates C, G and H were found to be significantly lower than those of isolates B, E within the same subtype (p<0.01). **, p<0.01 vs. ST-7 (B, E). Each point represents a mean of nine readings derived from three independent experiments, triplicate each. Error bars represent the standard errors.</p

Intra- and inter-subtype variations in protease activity in <i>Blastocystis</i>.

<p>Protease activity of <i>Blastocystis</i> ST-4 and ST-7 isolates was determined by azocasein assays. Except isolate G, most of ST-7 strains exhibited significantly higher protease activities than the two ST-4 isolates (p<0.01). PBS as a background control showed activity that was significantly lower when compared to protease activities of all the isolates (p<0.01). Note that cysteine protease inhibitor iodoacetamide (IA) abolished protease activity of all the isolates, which is comparable to PBS. ##, p<0.01 vs. ST-4; ‡, p<0.01 vs. PBS control. Each value represents mean of six samples, taken from three independent experiments. Error bars represent the standard errors.</p

<i>Blastocystis</i> exhibits intra- and inter-subtype variation in attachment to Caco-2 cells.

<p>(A) Representative confocal micrographs illustrating intra- and inter-subtype variation in <i>Blastocystis</i> attachment to Caco-2 cells. Caco-2 monolayers were grown to confluency on glass converslips and were then co-incubated with the same number of parasites of different strains of <i>Blastocystis</i> pre-stained with CFSE (green). Normal culture media was used as a negative control. After co-incubation, the non-attached parasites were washed away. The Caco-2 monolayers were then stained with DAPI and then were viewed using confocal microscope (Olympus Fluoview FV1000; Olympus, Japan). More green in the field represents more parasites attached to the monolayer. Both ST-4 strains adhere with a negligible number. An intra-subtype variation in the number of attachment within ST-7 is obvious. Isolates C, G, H appeared to attach at a much higher level than B and E to Caco-2 cells. Scale bar = 100 µm. (B) Graph representing number of <i>Blastocystis</i> parasites attached to host cells. ST-7 strains C, G and H exhibited a significantly higher number of attached parasites than ST-4 strains and ST-7 isolates B and E. *, p<0.05 vs. ST-7 (B, E); **, p<0.01 vs. ST-7 (B, E); ##, p<0.01 vs. ST-4. Each value represents a mean of six readings derived from 3 independent experiments. Error bar represents standard error. (C) Relationship between attachment and permeability increase by <i>Blastocystis</i> ST-4 and ST-7 parasites. The data points indicate individual strains. <i>x</i> and <i>y</i> error bars indicate the standard error for the respective measurements (n = 3). The R² for the trend line shown is 0.8506, and the p value is 0.0031. There is a positive correlation between the level of attachment and permeability increase (R = 0.9223).</p

Strain-Dependent Induction of Human Enterocyte Apoptosis by Blastocystis Disrupts Epithelial Barrier and ZO-1 Organization in a Caspase 3- and 9-Dependent Manner

Blastocystis is an emerging protistan parasite colonizing the human intestine. It is frequently reported to cause general intestinal symptoms of vomiting, diarrhea, and abdominal pain. We recently demonstrated that Blastocystis rearranged cytoskeletal proteins and induced intestinal epithelial barrier compromise. The effect of Blastocystis on enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and increased epithelial permeability has yet to be determined. The aim of this study is to assess if Blastocystis induces human enterocyte apoptosis and whether this effect influences human intestinal epithelial barrier function. Monolayers of polarized human colonic epithelial cell-line Caco-2 were incubated with Blastocystis subtype 7 and subtype 4. Assays for both early and late markers of apoptosis, phosphatidylserine externalization, and nuclear fragmentation, respectively, showed that Blastocystis ST-7, but not ST-4, significantly increased apoptosis in enterocytes, suggesting that Blastocystis exhibits host specificity and strain-to-strain variation in pathogenicity. ST-7 also activated Caco-2 caspases 3 and 9 but not 8. ST-7 induced changes in epithelial resistance, permeability, and tight junction (ZO-1) localization. Pretreatment of Caco-2 monolayers with a pan-caspase inhibitor z-VAD-fmk significantly inhibited these changes. This suggests a role for enterocyte apoptosis in Blastocystis-mediated epithelial barrier compromise in the human intestine

IC50s of Mz and nitric oxide donors against <i>Blastocystis</i> ST-7 isolates (µg/ml).

a<p>See Reference<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002885#pntd.0002885-Mirza5" target="_blank">[49]</a>.</p>b<p>See Reference<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002885#pntd.0002885-Mirza3" target="_blank">[30]</a>.</p

Strain-Dependent Induction of Human Enterocyte Apoptosis by Blastocystis Disrupts Epithelial Barrier and ZO-1 Organization in a Caspase 3- and 9-Dependent Manner

Blastocystis is an emerging protistan parasite colonizing the human intestine. It is frequently reported to cause general intestinal symptoms of vomiting, diarrhea, and abdominal pain. We recently demonstrated that Blastocystis rearranged cytoskeletal proteins and induced intestinal epithelial barrier compromise. The effect of Blastocystis on enterocyte apoptosis is unknown, and a possible link between microbially induced enterocyte apoptosis and increased epithelial permeability has yet to be determined. The aim of this study is to assess if Blastocystis induces human enterocyte apoptosis and whether this effect influences human intestinal epithelial barrier function. Monolayers of polarized human colonic epithelial cell-line Caco-2 were incubated with Blastocystis subtype 7 and subtype 4. Assays for both early and late markers of apoptosis, phosphatidylserine externalization, and nuclear fragmentation, respectively, showed that Blastocystis ST-7, but not ST-4, significantly increased apoptosis in enterocytes, suggesting that Blastocystis exhibits host specificity and strain-to-strain variation in pathogenicity. ST-7 also activated Caco-2 caspases 3 and 9 but not 8. ST-7 induced changes in epithelial resistance, permeability, and tight junction (ZO-1) localization. Pretreatment of Caco-2 monolayers with a pan-caspase inhibitor z-VAD-fmk significantly inhibited these changes. This suggests a role for enterocyte apoptosis in Blastocystis-mediated epithelial barrier compromise in the human intestine