Can the pyruvate: ferredoxin oxidoreductase (PFOR) gene be used as an additional marker to discriminate among Blastocystis strains or subtypes?

Abstract

BackgroundBlastocystis spp. are the most prevalent intestinal eukaryotes identified in humans, with at least 17 genetic subtypes (ST) based on genes coding for the small-subunit ribosomal RNA (18S). It has been argued that the 18S gene should not be the marker of choice to discriminate between STs of these strains because this marker exhibits high intra-genomic polymorphism. By contrast, pyruvate:ferredoxin oxidoreductase (PFOR) is a relevant enzyme involved in the core energy metabolism of many anaerobic microorganisms such as Blastocystis, which, in other protozoa, shows more polymorphisms than the 18S gene and thus may offer finer discrimination when trying to identify Blastocystis ST. Therefore, the objective of the present study was to assess the suitability of the PFOR gene as an additional marker to discriminate among Blastocystis strains or subtypes from symptomatic carrier children.MethodsFaecal samples from 192 children with gastrointestinal symptoms from the State of Mexico were submitted for coprological study. Twenty-one of these samples were positive only for Blastocystis spp.; these samples were analysed by PCR sequencing of regions of the 18S and PFOR genes. The amplicons were purified and sequenced; afterwards, both markers were assessed for genetic diversity.ResultsThe 18S analysis showed the following frequencies of Blastocystis subtypes: ST3 = 43%; ST1 = 38%; ST2 = 14%; and ST7 = 5%. Additionally, using subtype-specific primer sets, two samples showed mixed Blastocystis ST1 and ST2 infection. For PFOR, Bayesian inference revealed the presence of three clades (I-III); two of them grouped different ST samples, and one grouped six samples of ST3 (III). Nucleotide diversity (π) and haplotype polymorphism (θ) for the 18S analysis were similar for ST1 and ST2 (π = ~0.025 and θ = ~0.036); remarkably, ST3 showed almost 10-fold lower values. For PFOR, a similar trend was found: clade I and II had π = ~0.05 and θ = ~0.05, whereas for clade III, the values were almost 6-fold lower.ConclusionsAlthough the fragment of the PFOR gene analysed in the present study did not allow discrimination between Blastocystis STs, this marker grouped the samples in three clades with strengthened support, suggesting that PFOR may be under different selective pressures and evolutionary histories than the 18S gene. Interestingly, the ST3 sequences showed lower variability with probable purifying selection in both markers, meaning that evolutionary forces drive differential processes among Blastocystis STs.

Succinate dehydrogenase gene as a marker for studying Blastocystis genetic diversity

“…not show high genetic diversity compared to the ssu rRNA locus (Figure 2, Supplementary material Table S1) and showed a grouping in the phylogenetic reconstruction that was relatively associated with the STs and the collection areas (Figures 3 and 5). This suggested that this gene was probably under different evolutionary forces, which were similar to the PFOR gene reported in the Mexican samples [26]. Also, it was important to consider that ribosomal genes were highly utilized in eukaryotes and required a large production of ribosomes in times of massive growth, that could encode hundreds of copies of their transcriptional units [55], leading to recombination events and potentially greater diversity compared to the constitutive metabolic genes, which did not require high variation in order to maintain their function.…”

“…But, due to the unknown role of this microorganism inside the gut, it has been proposed to explore additional markers with different evolutionary rate in order to elucidate what is happening in these populations. A few authors, have reported the need for use of other genes or non-coding regions of the genome, such as the internal transcribed spacers (ITS) [27], the use of single copy markers from MROs, which allowed for the detection of coinfections even in the same ST [25] or the use of the PFOR gene that generated clades that were different from the STs, because they were subject to different selective pressures that showed an evolutionary history different from that of the ssu rRNA gene [26].…”

“…However, due to the increasing number of STs found, it would be important to add other markers with different resolution power to explore the diversity in other genetic targets and conjunction with ssu rRNA, solving the variation both inter and intraspecific, even more considering the heterogeneity of ssu rRNA which was reported in the ST7 strain B isolate [25], with 17 non-identical copies [20,25]. A few additional markers have also been evaluated, such as the pyruvate ferredoxin oxidoreductase (PFOR) gene, which allowed finding three clades with different ST samples, a lower nucleotide diversity and haplotype polymorphism for clade III [26]. Other study used the internal transcribed spacer ITS which revealed novel variants intra ST1 and a high gene flow among different countries of Europe and the Americas [27].…”

A summary of Blastocystis subtypes in North and South America

“…Our review of Blastocystis in the different countries of North and South America identified 39 articles that met the selection criteria, for which the distribution of Blastocystis and its subtypes was analyzable. However, only nine countries in North and South America (Argentina [9, 13], Brazil [9, 11, 21–31], Bolivia [9, 32, 33], Colombia [2, 9, 10, 34–37], Chile [38], Ecuador [9, 39], USA [17, 36, 40–47], Peru [9] and Mexico [48–50] were found to have carried out this type of study. From these countries, Blastocystis was identified in samples from both human and other hosts.…”

“…Among the studies conducted in North and South America, the most widespread subtypes we collated were ST1 and ST2, which were present in the samples from eight of the nine countries that were studied [2, 9–11, 17, 21–27, 29–39, 42, 43, 45–50]. Subsequently, ST3 was found in seven of the nine countries [2, 9–11, 13, 17, 21, 23–25, 27, 30, 31, 33–37, 39, 42, 43, 46–50].…”