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.

Pleiotropic effects of Blastocystis spp. Subtypes 4 and 7 on ligand-specific toll-like receptor signaling and NF-κB activation in a human monocyte cell line.

Blastocystis spp. is a common enteric stramenopile parasite that colonizes the colon of hosts of a diverse array of species, including humans. It has been shown to compromise intestinal epithelial cell barrier integrity and mediate the production of pro-inflammatory cytokines and chemokines. Mucosal epithelial surfaces, including the intestinal epithelium, are increasingly recognized to perform a vital surveillance role in the context of innate immunity, through the expression of pathogen recognition receptors, such as Toll-like receptors (TLRs). In this study, we use the human TLR reporter monocytic cell line, THP1-Blue, which expresses all human TLRs, to investigate effects of Blastocystis on TLR activation, more specifically the activation of TLR-2, -4 and -5. We have observed that live Blastocystis spp. parasites and whole cell lysate (WCL) alone do not activate TLRs in THP1-Blue. Live ST4-WR1 parasites inhibited LPS-mediated NF-κB activation in THP1-Blue. In contrast, ST7-B WCL and ST4-WR1 WCL induced pleiotropic modulation of ligand-specific TLR-2 and TLR-4 activation, with no significant effects on flagellin-mediated TLR-5 activation. Real time-qPCR analysis on SEAP reporter gene confirmed the augmenting effect of ST7-B on LPS-mediated NF-κB activation in THP1-Blue. Taken together, this is the first study to characterize interactions between Blastocystis spp. and host TLR activation using an in vitro reporter model.

Statin pleiotropy prevents rho kinase-mediated intestinal epithelial barrier compromise induced by Blastocystis cysteine proteases.

Blastocystis is an enteric parasite that causes acute and chronic intestinal infections, often non-responsive to conventional antibiotics. The effects of Blastocystis infections on human epithelial permeability are not known, and molecular mechanisms of Blastocystis-induced intestinal pathology remain unclear. This study was conducted to determine whether Blastocystis species alters human intestinal epithelial permeability, to assess whether these abnormalities are rho kinase (ROCK)-dependent, and to investigate the therapeutic potential of the HMG-CoA reductase inhibitor Simvastatin in altered intestinal epithelial barrier function. The effect of metronidazole resistant (Mz(r)) Blastocystis isolated from a symptomatic patient on human colonic epithelial monolayers (Caco-2) was assessed. Modulation of enterocyte myosin light chain phosphorylation, transepithelial fluorescein isothiocyanate-dextran fluxes, transepithelial resistance, cytoskeletal F-actin and tight junctional zonula occludens-1 (ZO-1) by parasite cysteine proteases were measured in the presence or absence of HMG-CoA reductase and ROCK inhibition. Blastocystis significantly decreased transepithelial resistance, increased epithelial permeability, phosphorylated myosin light chain and reorganized epithelial actin cytoskeleton and ZO-1. These alterations were abolished by inhibition of enterocyte ROCK, HMG-CoA reductase and parasite cysteine protease. Our findings suggest that cysteine proteases of Mz(r) Blastocystis induce ROCK-dependent disruption of intestinal epithelial barrier function and correlates with reorganization of cytoskeletal F-actin and tight junctional ZO-1. Simvastatin prevented parasite-induced barrier-compromise, suggesting a therapeutic potential of statins in intestinal infections.

A rapid, high-throughput viability assay for Blastocystis spp. reveals metronidazole resistance and extensive subtype-dependent variations in drug susceptibilities.

Blastocystis is an emerging protistan parasite of controversial pathogenesis. Although metronidazole (Mz) is standard therapy for Blastocystis infections, there have been accumulating reports of treatment failure, suggesting the existence of drug-resistant isolates. Furthermore, very little is known about Blastocystis susceptibility to standard antimicrobials. In the present study, we established resazurin and XTT viability microassays for Blastocystis spp. belonging to subtypes 4 and 7, both of which have been suggested to represent pathogenic zoonotic subtypes. The optimized resazurin assay was used to screen a total of 19 compounds against both subtypes. Interestingly, subtype 7 parasites were resistant to Mz, a 1-position-substituted 5-nitroimidazole (5-NI), while subtype 4 parasites were sensitive. Some cross-resistance was observed to tinidazole, another 1-position 5-NI. Conversely, subtype 4 parasites were resistant to emetine, while subtype 7 parasites were sensitive. Position 2 5-NIs were effective against both subtypes, as were ornidazole, nitazoxanide, furazolidone, mefloquine, quinicrine, quinine, cotrimoxazole (trimethoprim-sulfamethoxazole), and iodoacetamide. Both subtypes were resistant to chloroquine, doxycycline, paromomycin, ampicillin, and pyrimethamine. This is the first study to report extensive variations in drug sensitivities among two clinically important subtypes. Our study highlights the need to reevaluate established treatment regimens for Blastocystis infections and offers clear new treatment options for Mz treatment failures.

Current Views on the Clinical Relevance of Blastocystis spp.

Blastocystis is an enteric protistan parasite of uncertain clinical relevance. Recent studies indicate that the parasite is a species complex and humans are potentially hosts to nine Blastocystis subtypes, most of which are zoonotic. Subtype 3 is the most common in prevalence studies, followed by subtype 1. Laboratory diagnosis is challenging; the currently recommended diagnostic approach is trichrome staining of direct smears coupled with stool culture. Polymerase chain reaction testing from stools or culture is useful for determining Blastocystis subtype information. The controversial pathogenesis of Blastocystis is attributed to subtype variations in virulence; although current studies seem to support this idea, evidence suggests other factors also contribute to the clinical outcome of the infection. Clinical signs and symptoms of blastocystosis include abdominal pain, diarrhea, bloating, and flatulence. Extraintestinal manifestations, predominantly cutaneous, also were reported. In vitro and animal studies shed new light on the pathobiology of Blastocystis.