Chitinase-gene-based analysis of the genetic variability among the clinical isolates of Entamoeba dispar from Puducherry, India.

INTRODUCTION: Amebiasis is known to be caused by the protozoan parasite Entamoeba histolytica. Entamoeba dispar is considered to be a sibling species of E. histolytica, as the two are phylogenetically closest. There are reports that certain strains of E. dispar isolated were capable of causing hepatic lesions in the experimental animal models. The intra-/inter-species genetic variation has been found to have profound implication in the invasiveness of the disease. Thus, studying polymorphism in E. dispar aids to improve our perspective related to the variability in the genome of the parasite.
MATERIALS AND METHODS: The highly polymorphic region of the gene encoding the enzyme chitinase was targeted for the strain variation analysis in E. dispar. Isolates from the stool and liver abscess aspirate were subjected to the polymerase chain reaction (PCR) for the amplification of the targeted polymorphic loci. The PCR products were sequenced, and genetic variability analysis was carried out.
RESULTS: A total of 23 samples in the stool and 1 sample from liver abscess pus were positive for E. dispar by nested multiplex PCR which was confirmed by sequencing. Of these positive samples, 13 amplified for chitinase gene by PCR. We observed seven genotypes in our study isolates, of which four were found to be distinct.
CONCLUSION: This study shows that high degree of genetic variation exists among the clinical isolates of E. dispar in our location. The future studies including the analysis of other genetic makers such as serine-rich E. dispar protein or other loci have to be carried out to get an idea about the distribution of the different strains of E. dispar.

INTRODUCTION

The enteric protozoan parasite Entamoeba histolytica is said to cause amebiasis that results in about 100,000 deaths annually, placing it second only to malaria among the most common cause of morbidity and mortality due to parasitic infections.[12] The genus Entamoeba includes nearly seven species that can colonize the human intestine, of which E. histolytica is the only known human pathogen.[34] However, the pathogenic potential of the other morphologically indistinguishable species such as Entamoeba dispar, Entamoeba moshkovskii, and Entamoeba bangladeshi remains unclear.[567] The previous studies have shown that hepatic and intestinal lesions were found to be caused by E. dispar. Furthermore, E. dispar strains inoculated into hamster models were seen to produce liver abscess similar to E. histolytica.[891011]

The diagnosis, treatment, and epidemiology of amebiasis have undergone major revision after categorizing E. dispar as a distinct species and identification of other similar species such as E. moshkovskii.[12] To ascertain the disease outcome, distribution and transmission patterns of these parasites and to understand their pathogenic role polymorphism-based studies may be helpful. The intra-/inter-species genetic variation has been found to have profound implication in the invasiveness of the disease.[13] Thus, studying polymorphism in E. dispar aids to improve our perspective related to the variability in the genome of the parasite.

Several genes have been targeted for studying the intra-/inter-species genetic variability of the parasites.[12131415] The most extensively studied loci are the genes coding for serine-rich E. dispar protein (SREDP)[16] and amebic chitinase protein. Using these alleles, it is also possible to discriminate between E. histolytica and E. dispar. The parasite was found to be homozygous at the locus for chitinase, thus, making it an easier target for polymorphism-based studies.[15] Chitinase is expressed during encystment of the parasite. The protein consists of a ribosome-binding region in the 5'-UTR, a putative signal sequence, and a chitinase catalytic domain region in the carboxy terminal. Between the signal sequence and the carboxy terminal, there are seven amino acid long tandem repeats rich in hydrophilic amino acids, similar to the ones present in SREDP. These tandem repeats are absent in the chitinase isolated from the other organisms.[17] Our study aimed at identifying the distinct polymorphic patterns present in the genome of the E. dispar clinical isolates from our region with respect to the chitinase-coding gene.

MATERIALS AND METHODS

This work was approved by the Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER) Scientific Advisory Committee and Institute Ethics Committee.

Sample collection

Fresh stool (n = 1166) and liver abscess aspirate (n = 82) specimens were collected over a period of 4 years from May 2014 to April 2017 for analysis in the Department of Microbiology, JIPMER.

Differentiation of Entamoeba species

DNA was extracted from the stool and liver abscess aspirate specimens immediately on arrival at the parasitology laboratory or from samples stored at –20°C. DNA was extracted according to the manufacturer's instructions with the QIAamp Stool DNA Mini Kit and QIAGEN DNA extraction kit for sterile body fluids (QIAGEN, Hilden, Germany).

The nested multiplex polymerase chain reaction (PCR) with the primer targeting the 18S rRNA gene was carried out as previously described[18] in SureCycler 8800 (Agilent Technologies). The amplified product was visualized on 1.8% agarose in a gel documentation unit (GelDoc XR, BioRad). The results were further confirmed by sequencing.

Polymorphism analysis

The gene encoding the enzyme chitinase was the target for the analysis of the genetic variability among the clinical isolates of E. dispar. Primers that flank the tandem repeat units of the chitinase gene were used for the PCR analysis.[15] The cycling conditions are as follows: initial denaturation at 95°C for 2 min, followed by forty cycles of 1 min for denaturation at 94°C, 1.5 min for annealing at 55°C, and 2 min for elongation at 72°C. The PCR products were visualized on 1.8% agarose gels. Bands at the expected size (around 550 bp) were eluted and purified with Nucleospin Gel and PCR clean-up kit (Macherey-Nagel) and sequenced using ABI 3730 Genetic Analyzer (Applied Biosystems, USA). In case of the absence of a band, a second PCR round was performed on the amplified product with the same PCR condition.

RESULTS

Of the 1166 intestinal specimens screened, 24 samples were positive for E. dispar and among the 82 liver abscess aspirates tested 1 sample showed the presence of E. dispar along with E. histolytica. The results were confirmed by sequencing the PCR products. Following which, the 24 samples were subjected to polymorphic analysis by targeting the chitinase gene of E. dispar. Only 13 samples showed amplification of the chitinase gene in a single or two PCR rounds and visualization on 1.8% agarose gel [Figure 1]. The amplified region was sequenced. The obtained sequence reads were translated and aligned for analysis of the repeat patterns. Nearly nine samples were negative in spite of subjecting the samples for a second PCR round. Lesser parasite load, low DNA quantity, or poor quality of the DNA due to time and freeze-thawing can be the possible explanations for the negative results in few isolates. Furthermore, chitinase is expressed only during encystation, that could have also led to negativity in the isolates tested for this gene.

Figure 1

1.8% agarose gel image of the chitinase gene amplified by Polymerase Chain Reaction (Lane 1–5: Clinical samples, Lane 6: Positive control SAW760, Lane 7: Negative control, and Lane 8: 100bp DNA Ladder)

Chitinase gene has 21 nucleotide long repeats that codes for heptapeptides.[17] The variation in the number and location of these nucleotide units may contribute to the variation of strains that in turn may lead to the differential disease outcome. Based on the difference in the number and location of the repeat units, the strains were coded and analyzed.

In this study, we observed, 7 different gene repeat patterns among the 13 samples positive for chitinase gene in our study isolates. Wherein, we found that two isolates were similar to the standard strain SAW760, two isolates were similar to the isolate from Kolkata Ed K1, and three isolates were like the strain Ed K2 from Kolkata. PDY I-IV was the distinct isolate observed in our study.

The analysis showed that patterns similar to the standard axenic strains also exist in our study population. Three isolates were similar to SAW760.

DISCUSSION

A single PCR product was obtained for each isolate by targeting the polymorphic E. dispar chitinase gene. Thirteen isolates showed the presence of polymorphic patterns from the total 24 E. dispar clinical isolates in our study population. The distinct polymorphic patterns were coded with Roman numerals (PDY I-IV) based on the difference observed. Nearly seven polymorphic patterns that differed in the number and location of the EVKPDSS heptapeptides were identified. The previous studies show the similar findings in the isolates from Asia.[15] The PCR products of the positive samples showed the presence of about 14–21 heptapeptides. However, a majority of the sequences from the study isolates had 18 repeat units.

We observed one polymorphic pattern of the chitinase gene similar to the standard strains SAW760 in our study isolates. This suggests that the previously axenized classical strains are still in circulation around the globe. The polymorphic patterns in the repeat units of two E. dispar strains were similar to that of Ed H15 from Mexico or Ed K1 from Kolkata or TNX14a from Tunisia. Furthermore, one isolate showed a similar pattern as the Ed K2 Kolkata isolate from the previous studies.[1415] The existence of similar sequence patterns in the isolates from the different geographic areas could suggest the worldwide spread of certain strains of these parasites.

Six isolates showed distinct polymorphic patterns (PDY I-IV) that had not been observed from the previous studies. Two isolates each were designated as PDY I and II, and one isolate each was PDY III and IV seen in our study isolates. Sequence analysis distinguished these based on the number and location of the heptapeptide unit EIKPDSS [Figure 2]. The difference in the numbers was too small to be identified based on the amplicon size in gel analysis. Thus, it has to be noted that gel electrophoresis based on the size of the PCR products may alone not be helpful in detecting the intra-/interspecies genetic variation.[19]

Figure 2

Schematic representation of the polymorphism in the chitinase gene of the Entamoeba dispar clinical isolates

One of the isolates showing distinct pattern was extracted from the liver abscess aspirate specimen, wherein it was found to be present along with E. histolytica. The pattern EIKPDSS predominantly occurring in the American isolates was also found to be present in our isolates in the varying numbers. The repeat unit DTKPDSS specifically found in North America was not present in the isolates from our region that correlated with the previous studies.[15] A correlation could not be obtained between the genetic variability observed, and how one E. dispar isolate had migrated to the liver with the pathogenic E. histolytica. However, the further studies are required to determine the pathogenic ability of E. dispar. This observation suggests that the parasite has a widespread geographic distribution. These findings could not efficiently trace back to the travel of E. dispar; however, the transcontinental spread of the parasite was confirmed.

CONCLUSION

A definitive conclusion could not be drawn with the preliminary results obtained from our study. However, we have shown that high degree of genetic variation exists among the clinical isolates of E. dispar in our study location. The further studies including the analysis of other genetic makers such as SREDP or other loci have to be carried out to understand the spread and geographic distribution of the clinically important strains.

Financial support and sponsorship

This work was supported by the JIPMER Intramural Research Grant.

Conflicts of interest

There are no conflicts of interest.