Levels of Genetic Variants Among Symptomatic Blastocystis Subtypes and their Relationship to Mucosal Immune Surveillance in the Precancerous Colons of Experimentally Infected Rats

Acta Parasitologica https://doi.org/10.1007/s11686-022-00628-z ORIGINAL PAPER Levels of Genetic Variants Among Symptomatic Blastocystis Subtypes and their Relationship to Mucosal Immune Surveillance in the Precancerous Colons of Experimentally Infected Rats Eman M. Hussein1 · Muhammad A. A. Muhammad2 · Abdalla M. Hussein3 · Sherine M. Elzagawy1 · Wafaa M. Zaki1 · Ashraf G. Temsah4 · Mohamed S. Badr5 · Maha M. Alabbassy1 Received: 25 March 2022 / Accepted: 3 October 2022 © The Author(s) 2022 Abstract Purpose The relationship between the genetic diversity of Blastocystis and immune surveillance in precancerous colons with blastocystosis is still under investigation. This study aimed to identify the genetic Blastocystis variants among 54 symptomatic human isolates and their relationship to mucosal immune surveillance in the precancerous polyps of experimentally infected rats. Methods Polymerase chain reaction and high-resolution melting (PCR/HRM) curves discriminated human symptomatic Blastocystis isolates into subtypes (STs)/intrasubtypes, which were orally administered to rats to induce experimental infection. Then, the mucosal immune responses of the infected colons were evaluated in relation to polyp formation through immunostaining to identify mucus MUC2 and determine mucosal immune cell (goblet, lymphocyte and mast) counts, secretory IgA levels and parasitic intestinal invasion. Results ST1, ST3, and ST4 were found in 18.5% (10/54), 54.7% (29/54), and 27.8% (15/54) of the samples, respectively. Then, the HRM curve discriminated ST3 into the wild, mutant, and heterozygous [17/54 (31.5%), 5/54 (9.3%), and 7/54 (12.9%)] intrasubtypes. ST1 and ST4 had no genetic variations. Precancerous polyps were detected in the colons of 40.5% of the infected rats. ST1 constituted 14.7% of these cases, while the wild, mutant, and heterozygous intrasubtypes of ST3 showed polyps in 12.9%, 5.5%, and 5.5% of cases, respectively. Only 1.9% of the polyps were related to ST4. MUC2 showed weak immunostaining in 44.5% of the infected colons, and 38.9% were polyp inducers. Low goblet cell numbers and high interepithelial lymphocyte counts were significantly associated with polyp formation, particularly with ST1 and wild ST3. Among the polyp inducers, high numbers of mast cells were detected in wild ST3 and ST4, while a low number was found with heterozygous ST3. The level of secretory IgA was low in polyp-inducing STs. Most of the results were statistically significant. Conclusion Immunosurveillance showed a potential relationship between ST1 and the ST3 intrasubtypes and precancerous polyps. This relationship may provide insight into the prevention and/or development of new immunotherapeutic strategies to combat colorectal cancer. Keywords Blastocystis · Intrasubtype · Immune surveillance · MUC2 · Precancerous · Polyps Introduction Blastocystis, the most common eukaryotic organism worldwide, inhabits the intestinal tract of approximately two billion humans and a wide range of animals [60]. * Eman M. Hussein ; Extended author information available on the last page of the article Indeed, Blastocystis presents in both patients and healthy individuals, so its role in disease development has yet to be established [20]. Although up to 25 genetic subtypes (STs) of Blastocystis have been found in birds and mammals based on the small-subunit (SSU) rRNA gene sequences, only STs 1–9 and 12 have been detected in humans [42, 65]. STs 1–4 constitute 90% of human Blastocystis [4]. Recently, phylogenetic and sequencing studies added intrasubtype and intersubtype variants, providing wide genetic diversity of Blastocystis [64]. High-resolution 13 Vol.:(0123456789) Acta Parasitologica melting (HRM) curves are a novel post-polymerase chain reaction (PCR) method that analyses genetic variations via single nucleotide polymorphisms, mutations and methylations in PCR amplicons based on the GC/AT ratio, length, and distribution [27]. The PCR/HRM curve has differentiated Blastocystis STs into wild, mutant, and heterozygous variants [70]. These intra- and intersubtype variabilities exhibit remarkable differences in the outcome of Blastocystis infection [73]. The pathogenicity of Blastocystis depends on the induction of intestinal epithelial cell apoptosis [65]. In addition, Blastocystis modulates host immune responses by upregulating or downregulating certain inflammatory cytokines [52]. Moreover, Blastocystis symptomatic isolates exhibit high cysteine protease levels that facilitate the downregulation of epithelial antiparasitic nitric oxide formation [16, 44]. Interestingly, solubilized antigens of Blastocystis induce the in vitro proliferation of HCT116 human colorectal carcinoma cells [14, 15]. Nevertheless, Chen et al. [17] showed that 17.3% of Blastocystis-infected patients had large intestinal polyps (colorectal adenoma). Likewise, the blastocystosis prevalence rate is 34% among patients with colorectal adenoma [63]. Colorectal adenoma carries a high risk of developing into colorectal cancer (CRC) as a premalignant lesion [10]. Recently, Blastocystis parasites were identified in 12.15% of European CRC patients versus 2.42% of controls [67]. Notably, these precancerous polyps have been found in rats experimentally infected with Blastocystis ST1 and ST3 [1, 28]. In addition, Blastocystis obtained from CRC patients had different tissue proliferation and invasion capabilities in experimentally infected mice [2]. Up to 20% of cancer cases worldwide are associated with infection (a major driver of chronic inflammation), which plays an important role as a tumor promoter during immunosurveillance [23]. Mucus is excreted by goblet and epithelial cells and is an important part of the gastrointestinal tract (GIT) immunity [30]. Goblet cells synthesize MUC2, which is the most protective mucin in the GIT [49]. Patients with CRC have shown weak expression of MUC2 [35]. In addition to goblet cells and their mucins, cytotoxic intraepithelial lymphocytes (IELs) are also involved in mucosal immunosurveillance and have significant impacts in CRC [21]. Additionally, mast cells are an integral feature of the tumor microenvironment [29]. ST1 and ST3 facilitate mast cell activation during blastocystosis [11, 41]. Furthermore, fecal secretory immune globulin A (sIgA), a potential marker for CRC screening and early detection [13], is predominant during blastocystosis [39, 59]. However, the immunological changes that occur during blastocystosis that lead to carcinogenesis are not well established. Therefore, in the present study, PCR/HRM curve analysis was used to explore the levels of intrasubtype Blastocystis variants from Egyptian human symptomatic isolates and their relationship 13 to immune surveillance of the precancerous colon in rats experimentally infected with Blastocystis. Materials and Methods The Source of the Blastocystis Isolates Stool specimens from 350 patients with gastrointestinal symptoms attending the (...truncated)