Viability Screen of LOPAC1280 Reveals Phosphorylation Inhibitor Auranofin as a Potent Inhibitor of Blastocystis Subtype 1, 4, and 7 Isolates.

Blastocystis is an enteric parasite with extensive global prevalence. Studies have linked infection with this protist with a variety of gastrointestinal disorders, including irritable bowel syndrome. Due to the polymorphic nature of Blastocystis, studies on the parasite could be complicated, as results can be easily misinterpreted. Metronidazole is the commonly prescribed drug for Blastocystis infection, although there have been increasing reports of drug resistance. Hence, there is a need to identify alternative drugs to eliminate Blastocystis infection. In this study, LOPAC1280 was screened and drugs that can decrease the viability of three Blastocystis isolates in cultures were identified. Using apoptosis assay and imaging flow cytometry, phenotypic changes in Blastocystis cells after treatment were also analyzed to obtain insights into the possible mechanism of action of these drugs. Three drugs-diphenyleneiodonium chloride, auranofin, and BIX 01294 trihydrochloride hydrate-were effective against all three isolates tested. Repurposing of these drugs for Blastocystis treatment could be a way of combating metronidazole resistance relatively quickly and at a lower cost.

Blastocystis Isolate B Exhibits Multiple Modes of Resistance against Antimicrobial Peptide LL-37.

Blastocystis is one of the most common eukaryotic organisms found in humans and many types of animals. Several reports have identified its role in gastrointestinal disorders, although its pathogenicity is yet to be clarified. Blastocystis is transmitted via the fecal-to-oral route and colonizes the large intestines. Epithelial cells lining the intestine secrete antimicrobial peptides (AMPs), including beta-defensins and cathelicidin, as a response to infection. This study explores the effects of host colonic antimicrobial peptides, particularly LL-37, a fragment of cathelicidin, on different Blastocystis subtypes. Blastocystis is composed of several subtypes that have genetic, metabolic, and biological differences. These subtypes also have various outcomes in terms of drug treatment and immune response. In this study, Blastocystis isolates from three different subtypes were found to induce intestinal epithelial cells to secrete LL-37. We also show that among the antimicrobial peptides tested, only LL-37 has broad activity on all the subtypes. LL-37 causes membrane disruption and causes Blastocystis to change shape. Blastocystis subtype 7 (ST7), however, showed relative resistance to LL-37. An isolate, ST7 isolate B (ST7-B), from this subtype releases proteases that can degrade the peptide. It also makes the environment acidic, which causes attenuation of LL-37 activity. The Blastocystis ST7-B isolate was also observed to have a thicker surface coat, which may protect the parasite from direct killing by LL-37. This study determined the effects of LL-37 on different Blastocystis isolates and indicates that AMPs have significant roles in Blastocystis infections.

Characterisation of novel functionality within the Blastocystis tryptophanase gene.

In recent years, the human gut microbiome has been recognised to play a pivotal role in the health of the host. Intestinal homeostasis relies on this intricate and complex relationship between the gut microbiota and the human host. While much effort and attention has been placed on the characterization of the organisms that inhabit the gut microbiome, the complex molecular cross-talk between the microbiota could also exert an effect on gastrointestinal conditions. Blastocystis is a single-cell eukaryotic parasite of emerging interest, as its beneficial or pathogenic role in the microbiota has been a subject of contention even to-date. In this study, we assessed the function of the Blastocystis tryptophanase gene (BhTnaA), which was acquired by horizontal gene transfer and likely to be of bacterial origin within Blastocystis. Bioinformatic analysis and phylogenetic reconstruction revealed distinct divergence of BhTnaA versus known bacterial homologs. Despite sharing high homology with the E. coli tryptophanase gene, we show that Blastocystis does not readily convert tryptophan into indole. Instead, BhTnaA preferentially catalyzes the conversion of indole to tryptophan. We also show a direct link between E. coli and Blastocystis tryptophan metabolism: In the presence of E. coli, Blastocystis ST7 is less able to metabolise indole to tryptophan. This study examines the potential for functional variation in horizontally-acquired genes relative to their canonical counterparts, and identifies Blastocystis as a possible producer of tryptophan within the gut.

Interactions between a pathogenic Blastocystis subtype and gut microbiota: in vitro and in vivo studies.

BACKGROUND: Blastocystis is a common gut eukaryote detected in humans and animals. It has been associated with gastrointestinal disease in the past although recent metagenomic studies also suggest that it is a member of normal microbiota. This study investigates interactions between pathogenic human isolates belonging to Blastocystis subtype 7 (ST7) and bacterial representatives of the gut microbiota. RESULTS: Generally, Blastocystis ST7 exerts a positive effect on the viability of representative gut bacteria except on Bifidobacterium longum. Gene expression analysis and flow cytometry indicate that the bacterium may be undergoing oxidative stress in the presence of Blastocystis. In vitro assays demonstrate that Blastocystis-induced host responses are able to decrease Bifidobacterium counts. Mice infected with Blastocystis also reveal a decrease in beneficial bacteria Bifidobacterium and Lactobacillus. CONCLUSIONS: This study shows that particular isolates of Blastocystis ST7 cause changes in microbiota populations and potentially lead to an imbalance of the gut microbiota. This study suggests that certain isolates of Blastocystis exert their pathogenic effects through disruption of the gut microbiota and provides a counterpoint to the increasing reports indicating the commensal nature of this ubiquitous parasite.

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 Blastocystis Isolates.

Blastocystis 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 Blastocystis subtypes although microscopy alone is unable to distinguish between these subtypes. Blastocystis 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 Blastocystis 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.

Entamoeba histolytica and E. dispar infections in captive macaques (Macaca fascicularis) in the Philippines.

Entamoeba histolytica is a protozoan parasite that infects man and animals. This parasite has a global distribution and the disease it causes is usually characterized by diarrhea. In order to detect the parasite, it is necessary to differentiate it from Entamoeba dispar. E. dispar appears morphologically similar to E. histolytica but does not cause disease and tissue invasion. This study reports on the prevalence of E. histolytica and E. dispar among captive macaques in a primate facility in the Philippines. PCR was used to correctly identify both Entamoeba species. Indirect fluorescent antibody test (IFAT) was also performed to determine the seroprevalence of amebiasis in the captive macaques. Based on PCR targeting of the peroxiredoxin gene, of the 96 stool samples collected, 23 (24%) contained E. histolytica while 32 (33%) contained E. dispar. IFAT revealed 26 (27%) serum samples positive for antibodies against E. histolytica. Sequence analysis of the 18S rRNA gene showed that the 23 E. histolytica isolates were identical to human E. histolytica isolates deposited in the GenBank and not Entamoeba nuttalli as found in macaques in other recent reports. The Philippines is a major exporter of monkeys for biomedical research purposes, so screening animals before transporting them to other locations lessens the risk of spreading zoonoses to a wider area. This is the first report of the molecular detection of E. histolytica and E. dispar among macaques in the Philippines. This study complements the limited information available on the animal hosts of E. histolytica in the Philippines.

Genotyping of Giardia duodenalis isolates among residents of slum area in Manila, Philippines.

Giardia duodenalis is a flagellated protist that causes gastrointestinal disease throughout the world. In the Philippines, study on G. duodenalis is limited. It is also believed that prevalence rates of this organism in the country are underestimated. In this study, stool samples from residents living in a slum area in Manila were collected. These were examined under microscopy for identification of common helminthic and protistan parasites. Results showed that 22.05% of 2,354 stool samples collected contained Giardia cysts. A fraction of samples (n = 133) positive for Giardia cysts were set aside. Genomic DNA was extracted from these samples and a polymerase chain reaction-restriction fragment length polymorphism procedure based on the organism's triose phosphate isomerase gene was utilized. This particular procedure is capable of distinguishing assemblages or genotypes within G. duodenalis. The highest identified assemblage was Assemblage B (86.47%). The two genotypes of Assemblage A were also detected. This is the first report on the identification of genotypes of G. duodenalis in the Philippines. The results of this study can serve as basis for future control and prevention of giardiasis and parasitism in the country.

Serological detection of cryptosporidiosis among Filipino cancer patients.

Information on cryptosporidiosis in the Philippines is limited. To date, the disease is not routinely diagnosed in the country's medical institutions. To this end, a total of 53 Filipino cancer patients were surveyed for cryptosporidiosis using an indirect immunofluorescent antibody test. Fifteen patients (28.3%) were found to be positive for antibodies against Cryptosporidium. This study contributes to a better understanding of the incidence of cryptosporidiosis in the country.