A Metronidazole-Resistant Isolate of Blastocystis spp. Is Susceptible to Nitric Oxide and Downregulates Intestinal Epithelial Inducible Nitric Oxide Synthase by a Novel Parasite Survival Mechanism

Abstract

Blastocystis, one of the most common parasites colonizing the human intestine, is an extracellular, noninvasive, luminal protozoan with controversial pathogenesis. Blastocystis infections can be asymptomatic or cause intestinal symptoms of vomiting, diarrhea, and abdominal pain. Although chronic infections are frequently reported, Blastocystis infections have also been reported to be self-limiting in immunocompetent patients. Characterizing the host innate response to Blastocystis would lead to a better understanding of the parasite's pathogenesis. Intestinal epithelial cells produce nitric oxide (NO), primarily on the apical side, in order to target luminal pathogens. In this study, we show that NO production by intestinal cells may be a host defense mechanism against Blastocystis. Two clinically relevant isolates of Blastocystis, ST-7 (B) and ST-4 (WR-1), were found to be susceptible to a range of NO donors. ST-7 (B), a metronidazole-resistant isolate, was found to be more sensitive to nitrosative stress. Using the Caco-2 model of human intestinal epithelium, Blastocystis ST-7 (B) but not ST-4 (WR-1) exhibited dose-dependent inhibition of Caco-2 NO production, and this was associated with downregulation of inducible nitric oxide synthase (iNOS). Despite its higher susceptibility to NO, Blastocystis ST-7 (B) may have evolved unique strategies to evade this potential host defense by depressing host NO production. This is the first study to highlight a strain-to-strain variation in the ability of Blastocystis to evade the host antiparasitic NO response.Blastocystis is an extracellular and noninvasive unicellular enteric parasite with zoonotic potential (28,41,42). It is among the most common parasites found in the human intestine, with prevalence ranging between 10% of the population in developed countries and 50% in developing countries (42). It is a species complex belonging to the Stramenopile group. According to a recent classification, it is divided into 11 subtypes, 9 of which are known to infect humans. Other common hosts are chickens, rats, pigs, reptiles, and insects (41). Despite recent advances in our understanding of the parasite's classification and pathobiology, several questions concerning the parasite's biology remain unanswered. The parasite's pathogenicity is also controversial owing to asymptomatic carriage, coinfection with other pathogens, and nonresponsiveness to conventional chemotherapeutic agents.Blastocystis infections or blastocystosis present with common intestinal symptoms, such as abdominal pain, vomiting, and bloating, as well as mucous and watery diarrhea (42). Dermatological disorders (15,45) and irritable bowel syndrome (39) are also frequently associated with Blastocystis infections. It is transmitted through the feco-oral route, and metronidazole is the treatment of choice for Blastocystis infections. The parasite has a high prevalence in immunocompromised individuals (17,29,43). In immunocompetent individuals, Blastocystis infections are often asymptomatic (7, 39) or self-limi...

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

“…For example, Blastocystis was found to induce nitric oxide production in epithelial cells as part of the host defense (14). In addition, Blastocystis could also induce epithelial cells to express antimicrobial peptides as part of the host's innate immune response.…”

“…On the other hand, ST7-B is relatively resistant to LL-37 and is also rare in terms of being detected in human populations. This ST is also resistant to a number of drugs, including metronidazole (13,48), and is capable of downregulating the expression of nitric oxide synthase (14), which is an essential element in the innate immune response. Isolates of ST7 have also been found to cause epithelial barrier disruption and have more adhesive properties in cell cultures (49), thereby making this ST potentially more pathogenic than the rest of the STs.…”

“…There are also variations in drug sensitivities (13,14). Some researchers have suggested that symptomatology is dictated by ST identity (15).…”

Differential Regulation of Proinflammatory Cytokine Expression by Mitogen-Activated Protein Kinases in Macrophages in Response to Intestinal Parasite Infection

“…This is supported by studies revealing variation of cysteine protease activity between subtypes (23). Additionally, while ST-7 (B) was shown to be more sensitive to nitrosative stress than ST-4 (WR-1), it appeared to possess unique evasion strategies, with its ability to inhibit epithelial nitric oxide (NO) production by downregulating epithelial inducible nitric oxide synthase (iNOS) expression (24). These studies suggesting subtype-dependent variation show the need to further investigate differences in pathogenic potential across different subtypes of Blastocystis.…”

“…classification system (36). The parasites were cultured axenically, as described previously (23,24). In short, cultures of each isolate were maintained in 10 ml of prereduced Iscove's modified Dulbecco's medium (IMDM) containing 10% inactivated horse serum (Gibco) and incubated at 37°C in an anaerobic jar (Oxoid, United Kingdom) with an AnaeroGen gas pack (Oxoid, United Kingdom).…”