ABSTRACT

Objective

<em>Blastocystis</em> sp. is an intestinal Stramenopile that can infect both humans and animals. Genetic variability of <em>Blastocystis</em> has been investigated with a variety of molecular methods and different subtypes (ST) have been identified. The present study aimed to characterize microsatellite loci and population structure for <em>Blastocystis</em> sp. ST3, the most common ST in human faecal samples.

Methods

The genome of <em>Blastocystis</em> sp. ST3 in GenBank was analyzed for the presence of microsatellites containing at least eight repeat units. Specific primers were designed for each locus and polymorphisms were identified using bioinformatics tools. The population structure was determined, and microsatellite typing was conducted on 18 <em>Blastocystis</em> sp. ST3 genomic DNA samples from the routine laboratory at Aydın Adnan Menderes University Hospital.

Results

The whole-genome scan of <em>Blastocystis</em> sp. ST3 revealed 12 microsatellite loci with at least eight repeats. All loci were successfully amplified with the designed primers and eight of them were selected for genotyping. Microsatellite polymorphism analysis showed that each isolate had a unique profile (18 isolates, 18 different microsatellite types). Furthermore, the isolates were grouped into two distinct population clusters.

Conclusion

<em>Blastocystis</em> sp. ST3 isolates exhibited significant variability in their microsatellite repeats. The polymorphic microsatellite loci offer a novel approach to study the detailed genetic diversity and population structure of <em>Blastocystis</em> sp. ST 3.

INTRODUCTION

Blastocystis is one of the most common protozoa in the human gastrointestinal system. Most researchers have agreed that Blastocystis transmission occurs via the faecal-oral route, primarily through the ingestion of cyst forms in water or food (1). Zoonotic transmission between a variety of animals and humans may also be possible (2). Many subtypes (STs) have been identified to date mostly relying on 18S rRNA coding gene polymorphisms, ST1-4 accounts for the great majority of human samples in many studies (3-5). The genetic diversity of Blastocystis is among the most interesting topics in recent years because it has a great contribution to host specificity, pathogenicity, experimental studies and also microbiota (5, 6). Amplification of specific parts of rRNA genes and sequencing is the most common method for detecting genotypes. Moreover, Matrix-assisted laser desorption/ionisation mass spectrometry (MALDI MS) has been used for genotype determination (7).

Microsatellites are 2-9 bp length genetic markers that can be found abundantly in the genomes of eukaryotic organisms. They allow the identification of the causative agents precisely in populations of different genotypes because they contain high amounts of genetic diversity (8). Microsatellite markers are generally regarded as neutral alleles, which makes them ideal markers for determining the history of populations. In addition, up to today, MS markers have been used in several parasitic protozoa including Leishmania tropica, Trypanosoma sp., Plasmodium sp., and Toxoplasma gondii (9-12).

The development and application of alternative genotyping methods may contribute to the understanding of controversial issues in Blastocystis sp. pathogenicity such as pathogenicity. In the present study, we aimed to characterize the microsatellite loci for Blastocystis sp. ST3 for the first time in the literature and analyse the genetic structure of Blastocystis sp. ST3 isolates.

METHODS

Determination of Microsatellite Markers and Primer Design

The complete genomic sequence of a human Blastocystis sp. ST3 isolate, an assemblage of 917 partial sequences, was acquired from the National Center for Biotechnology Information database (Genbank, Acc. no: JZRK00000000). Microsatellites mining was carried out using the software Msatcommander 0.8.1 and http://insilico.ehu.es/mini_tools/microsatellites/website with the motif criteria, di‐, tri‐, tetra‐, penta‐, and hexanucleotide repeats of microsatellites at least 8 within the complete genome of Blastocystis sp. ST3 (13). Primers for microsatellite loci (n=2) were designed with Primer3, the online version, with default settings (14, 15). For potential repetitive elements other than perfect repeats, the flanking regions were also analysed. In order to identify robust loci with an annealing temperature of at least 55 ºC, approximately 150-200 bp flanking each side of the repeat were included.

Blastocystis sp. ST3 Isolates

The ethical approval from a Local Ethical Committee in Aydın Adnan Menderes University Faculty of Medicine (no: 2015/10) was obtained. A total of 18 Blastocystis sp. ST3 isolates were used in the study. The isolates were previously acquired by culturing direct microscopy-positive faecal samples in the routine diagnostic laboratory at Aydın Adnan Menderes University Hospital. The cultures in 3 mL of Jones medium were subjected to genomic DNA isolation with DNAzol kit (Invitrogen, Life Technologies). Blastocystis sp. SSU-rDNA gene was partially amplified with the primers RD5 and BhRDr in a single round of polymerase chain reaction (PCR) (16). The amplicons were sequenced, and ST were detected using the Blastocystis sp. sequence typing database (pubmlst.org/Blastocystis) (17). 

PCR Amplification and Genotyping

The optimal annealing temperature for the PCR amplification for each microsatellite locus was determined with gradient PCR. The fluorophores FAM and HEX were used to label the 5′ ends of the forward primer. The amplifications were performed in 30 µL of volume: 0.5 mM each of the primers, 1.5 mM MgCl₂, 2.5 mM dNTP, 1.0 U Taq polymerase, and 1-2 µL of template DNA. The reaction for each locus was set as follows: 95 °C for 5 min, 35 cycles (at 95 °C for 30 s, 55-60 °C for 30 s and 72 °C for 45 s), and a final extension at 72 °C for 7 min. The length of alleles was detected with an automatic sequencer.

Statistical Analysis

The allele sizes of microsatellites were exactly determined with Genemarker 2.6.3 (Soft Genetics LLC, USA). The calculation of genetic variation in microsatellite loci was performed with GenAlEx 6.5 (18). GENEPOP 3.3 software was used to detect genotypic linkage disequilibrium between pairwise loci (19). GenAlEx 6.5 program was used to detect the allele numbers (N_A), effective allele numbers (N_E), the frequencies of alleles, intra-population diversity of alleles, and pairwise comparisons of the isolates (18). The expected heterozygosity (H_E) of loci was calculated using the Arlequin 3.11 (20). In multiple loci, haplotype overlaps were determined with GenAlEx 6.5 (18).

Population Structure and Microsatellite Typing

Population structure was analysed with a Bayesian clustering method in STRUCTURE ver. 2.3 program (21). The admixture model with correlated allele frequency parameters was used to to detect the estimated number of genetic clusters (K). Ten runs were performed for each K value (ranging from 1 to 10) with 100.000 MCMC repetitions and a burn-in periods of 10,000. The ad hoc estimated likelihood of K (ΔK) was included in the determination of the most likely number of populations (K) based on the rate of change in the log probability of the data [Ln Pr (X/K)] (22). Structure Harvester version 0.6.94 was used to infer the most likely number of genetic clusters (K) with both the Evanno and Delta K methods (23). The first isolate was defined as MT1 and the remaining different isolates (at least one different allele type) were annotated with a new MT number.

RESULTS

Microsatellite Variability and Population Structure

We detected 12 microsatellite loci with at least 8 repeats in Blastocystis sp. ST3 genome and of these 11 loci were polymorphic. All loci have three repeats, and most were characterized with (GAT)_n and (ATC)_n (Table 1).

There was no significant linkage disequilibrium when the loci were compared dually (p>0.05). The total number of alleles (N_A) for each locus changed from three to 11, the average was 6.55 alleles per locus (Table 1). The mean effective number of alleles (N_E) was 4.06 (range from 1.841 to 8.526). BHST3-142 loci (h=0.457) provided the minimum intra-population diversity, while BHST3-838 locus (h=0.883) provided the highest. The average expected heterozygosity value was determined as 0.740±0.146 (min: 0.399 and max: 0.935). Two distinct genetic groups were suggested with the analyses of microsatellite data by a structure clustering algorithm (K=2.297) (Figure 1).

Microsatellite Typing

We selected eight loci: BH142, BH217, BH278, BH302, BH399, BH476, BH806 and BH808 for MT typing of Blastocystis sp. ST3 isolates. These loci were selected relying on the reliability of microsatellite length, clarity in representations, and amplification/reproducibility. Microsatellite based typing of isolates revealed that each isolate had a unique MT type (Table 2).

DISCUSSION

Blastocystis sp. is a common enteric stramenophile with a high genetic diversity between isolates and an unclear role in the development of intestinal diseases and pathogenicity. The molecular typing of Blastocystis sp. isolates is valuable to study its population structure of parasites on a global scale. Genetically diverse isolates may influence the frequency of Blastocystis sp., for example, host specificity and adaptation to novel hosts (24). In addition, several studies reported a decreased bacterial diversity and changed intestinal microbiome in certain Blastocystis sp. ST (6). The development of novel genotyping methods is important for an improved understanding of the genotype distribution of Blastocystis sp. isolates and phenotypic characteristics. In the present study, we characterised the microsatellite markers for the most common Blastocystis sp. subtype in human faecal samples, namely ST3. The most significant finding from this study was that the microsatellite loci in Blastocystis sp. ST3 isolates were highly variable, with each of the 18 isolates exhibiting a distinct or unique microsatellite type. A great genetic difference between Blastocystis sp. ST was noted in the literature (25). The comparison of Blastocystis sp. ST1, ST4 and ST7 genomes found great divergence in terms of genome assembly size, number of protein-coding genes, guanine-cytosine content, average gene size (bp) and also number of introns (25, 26). In addition to subtype level genetic difference in Blastocystis sp., the isolates were highly separated in terms of intra-subtype variation. A previous study investigated the intra-subtype genetic diversity and host specificity of Blastocystis sp. ST3 with multilocus sequence typing including SSU-rDNA and mitochondrion-like organelle sequences (27). The authors reported that human isolates were restricted to a single clade suggesting relatively high host specificity of ST3 human-to-human transmission. Another study reported that 11 human Blastocystis sp. ST3 isolates were distributed in nine sequence types with the same method. There were only three isolates that shared the same sequence type (28). However, the use of a single gene, most commonly SSU rRNA, detected a limited intra-subtype genetic diversity in Blastocystis sp. ST3 STs. For example, intra-subtype genetic polymorphisms were identified five in ST3 isolates in China and the similarity of SSU rRNA sequences was 99.8% in Iran (29, 30).

In the present study, the population structure analysis of Blastocystis sp. ST3 isolates revealed two distinct groups. The genomic DNA samples in our study were limited to a specific location, the same hospital laboratory. Collecting samples from various localities, such as different cities or countries, will enable a detailed phylogenetic analysis of microsatellites in Blastocystis sp. ST3. The major contribution of the present study is the characterization of microsatellites in Blastocystis sp. ST3 and the presentation of the associated methodology. This characterization has important implications for future research and practices, particularly in areas such as zoonotic transmission, microbiota interactions, and pathogenesis. These are the widely discussed topics in the literature on Blastocystis (5). Microsatellite polymorphisms have previously been described as valuable genetic markers for investigating zoonotic transmission in both parasitic microorganisms. For instance, certain microsatellite alleles in Cryptosporidium parvum have been associated with human-adapted or zoonotic strains (31). Similarly, microsatellite markers have been used to compare Plasmodium falciparum isolates from patients with cerebral malaria and those with uncomplicated malaria, to determine if there is a possible link between genetic variation and pathogenicity (32).

CONCLUSION

In conclusion, the characterisation of microsatellite loci in Blastocystis sp. ST3 revealed different profiles or types showing high intra-subtype diversity in terms of microsatellite repeats. This novel genotyping approach may be used in molecular epidemiology and population structure screening studies.