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

Abstract

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. Blastocystis is an intestinal protistan parasite commonly detected in humans and many types of animals (1, 2). This organism is classified under the stramenopiles, although it lacks chloroplasts or structures for locomotion, which are common features of many members of this group. Blastocystis is widely distributed throughout the world. Parasitological surveys usually place Blastocystis as the most common eukaryotic parasite detected (3,4). This organism has a global distribution and has numerous animal hosts. There is little host specificity for this organism, and there are reports indicating the zoonotic potential of Blastocystis (5). This organism, in its cyst form, is transmitted via the fecal-tooral route. It then colonizes the large intestine and is excysted to its various forms. These forms may appear vacuolar, multivacuolar, avacuolar, or granular under a light microscope. Blastocystis has been implicated in a number of intestinal disorders, although its pathogenesis is yet to be elucidated. There are few reports associating it with gastrointestinal disease, the most common symptoms of which are diarrhea, vomiting, nausea, and urticaria (1, 5-7). Blastocystis is a species complex that is comprised of up to 19 subtypes (STs), with ST1 to ST9 having been isolated from humans (8). Recently, a report found that ST12 and another possible novel ST also in...

Membrane Active Peptides and Their Biophysical Characterization

“…Imaging flow cytometry (IFC) combines the features of FC that enables objective statistical analysis of high cell counts and the features of fluorescent microscopy that provides high-resolution images of cells [ 269 ]. Different from FC, IFC allows analysis of cellular events on the basis of both fluorescent and morphological parameters upon interaction with the peptide [ 270 , 271 ].…”

Viability Screen of LOPAC 1280 Reveals Phosphorylation Inhibitor Auranofin as a Potent Inhibitor of Blastocystis Subtype 1, 4, and 7 Isolates

“…In a study comparing the genome sequences of isolates from the three STs mentioned, the data showed differences in DNA base composition, genome size, number of genes, and number of introns (43). These differences then could contribute to the variations in drug susceptibilities (7,44), as well as effects on the host (2,45) and host immune responses (9,(46)(47)(48). Geographical variations of Blastocystis incidence also occur.…”

Membrane Surface Features of Blastocystis Subtypes

“…Different Blastocystis isolate exhibit variations of a layer outside the cell membrane called the surface coat. We have hypothesized that this coat could protect the organism from innate host immune response as well as contribute to greater adhesion during colonization [ 13 , 16 ]. One of the earliest studies on the ultrastructure of Blastocystis describing a surface coat structure was made by Tan and Zierdt [ 17 ].…”

“…In a study on the susceptibility of Blastocystis to a colonic antimicrobial peptide (AMP) LL-37, we also hypothesized that the parasite’s surface coat could act as a barrier preventing this AMP from reaching the membrane. The AMP then will not be able to exert its pore-forming effect on the organism [ 16 ]. Indeed, using fluorescent-labeled antibodies against LL-37, we found that Blastocystis ST7-B isolate could soak this AMP into its surface, affecting its viability to a lesser degree compared with an ST4-WR1 isolate.…”

“…We also obtained scanning electron micrographs (SEM’s) to analyze the surface coats of the three axenized Blastocystis isolates. The method and preparation were done as described previously [ 16 ]. Blastocystis ST1 surface appears more uniform compared with the other isolates ( Figure 2 A,B), while the surface of ST4 cells were uneven in some areas of the cells ( Figure 2 C,D).…”