Amoebic forms of Blastocystis spp. - evidence for a pathogenic role
© Rajamanikam and Govind; licensee BioMed Central Ltd. 2013
Received: 3 October 2013
Accepted: 3 October 2013
Published: 11 October 2013
Blastocystis spp. are one of the most prevalent parasites isolated from patients suffering from diarrhea, flatulence, constipation and vomiting. It’s pathogenicity and pathophysiology remains controversial to date. Protease activity and amoebic forms have been reported previously in symptomatic isolates but there has been no conclusive evidence provided to correlate the protease activity and any specific life cycle stage of the parasite thus far.
Symptomatic isolates with amoebic form were tested for protease activity and compared with symptomatic and asymptomatic isolates without amoebic form for 10 days culture period.
The present study demonstrates an elevated protease activity in cultures having a higher percentage of amoebic forms seen in symptomatic isolates. The growth curve demonstrated a significantly (p < 0.05) higher average number of parasite counts in asymptomatic compared to symptomatic isolates. Symptomatic isolates showed amoebic forms with percentages ranging from 5% to 17%. Elevated protease activity was demonstrated in isolates that had higher percentages of amoebic forms with intense bands at higher molecular weight proteases (60 – 100 kDa). As days of culture proceeded, the protease quantification also showed a steady increase.
This study elucidates a correlation between protease activity and percentage of amoebic forms. The finding implies that these forms could play a role in exacerbation of intestinal symptoms during Blastocystis spp. infection.
KeywordsBlastocystis spp Protease activity Amoebic form Gastrointestinal
Blastocystis spp., a controversial anaerobic parasite, has increasingly gained a reputation for being implicated in causing flatulence, diarrhea, constipation, vomiting and skin rash [1–3]. The mechanism of pathogenicity still remains unclear. The prevalence in humans is reported to be between 0.5%  to 60%  and 40-60% in animals [2, 6].
Genotypic variability has been reported to play an influential role in the pathogenicity of Blastocystis. This is evidenced by studies showing Blastocystis subtype 3 being the most common subtype isolated from patients with various gastrointestinal (GI) disorders. Despite a few prevalence studies that have been carried out in different countries, which showed subtype 3 followed by subtype 1 to be the highest [7–10], its pathogenic role has not been clearly defined. The question of whether there are other factors influencing the parasite in its ability to be opportunistic in immunocompetent and immunocompromised patients [2, 11] has not yet been clearly answered.
There are also reports that demonstrate the lack of correlation between the symptomatic and asymptomatic group in terms of genotype distribution [12–14] and whether this is related to factors such as age of host and genetics is still uncertain . Amoebic forms, one of the life cycle stages of the parasite, have been reported to be present in higher percentages in in vitro cultures of symptomatic isolates compared to asymptomatic isolates [15, 16]. Previously, amoebic forms were also observed in colonoscopic lavage and patients with acute diarrheal syndrome [17, 18].
Proteases from Blastocystis spp. have been proven to be one of the important candidates that contribute to the pathogenicity of this protozoan parasite [19, 20]. Proteases from Blastocystis spp. have been shown to be able to degrade immunoglobin A (Ig A) giving rise to the parasite’s ability to have greater virulence and colonization through the elicitation and alteration of immunological response as well as the disruption of barrier function [21–23].
To date there have been no studies to correlate the presence of amoebic forms in in vitro culture and protease activity of Blastocystis spp. The present study attempts to elucidate if there is a correlation between the percentage of amoebic forms in cultures isolated from asymptomatic and symptomatic patients to protease activity.
Source of Blastocystis spp
Parasites were obtained from random stool sample collection in a survey carried out in a particular rural area in Selangor, Malaysia. A total of 5 isolates from symptomatic (S1-S5) and asymptomatic (A1-A5) patients were continuously cultured in Jones’ medium. The symptomatic isolates originated from patients showing symptoms such as flatulence, abdominal pain, diarrhea and constipation. This information was obtained using a questionnaire. Samples with Blastocystis spp. were selected through direct fecal screening and cyst concentration technique to select the samples with only Blastocystis spp. as the sole symptom causative agent.
Culture and purification of Blastocystis spp
All isolates were inoculated in Jones’ medium supplemented with 10% horse serum as described previously by Suresh et al.. Cultures were incubated at a constant temperature of 37°C before sub-culturing into fresh medium once every 3 days. Subsequently, the cells were purified from bacterial-contaminated culture using density gradient centrifugation. The cells were pooled into one tube and washed twice with phosphate buffered saline (PBS) for 5 minutes at 500 g. Five milliliters of the cell suspension was then layered carefully onto 6 ml of Ficoll-Paque without agitation. It was then spun for 20 mins at 700 g. Blastocystis spp. cells with minimal bacterial contaminants found above the thick layer of yellowish white clump was then gently isolated and washed with PBS. The pellet was stored at -20°C until further use.
Morphological study to elucidate morphological forms in culture
Isolates, maintained for a few weeks and showing consistent growth were used subsequently for the elucidation of morphological forms in culture. 105 cells in vacuolar form were counted and inoculated into culture tubes containing 1 ml of Jones’ medium with 10% horse serum and maintained to determine the growth profile at 37°C. 10 of culture suspension containing the parasites was mixed with an equal volume of Trypan blue to assess cell viability. The isolates were screened under a light microscope with 40X magnification everyday for 10 consecutive days and the percentages of amoeboid cells were observed by counting the number of amoeboid forms in a random count of 100 cells . Parasites from all culture tubes on day 5 were collected, purified and the respective pellets were then stored at -20°C. In a separate experiment, cell lysates of Blastocystis spp. isolates from all culture tubes were obtained at different periods of incubation (Day 1, 3, 5 and 7), which were then subjected to the Gelatin-SDS-PAGE and azocasein assays for the detection of protease activity using a standardized protein concentration of 0.1 mg/ml.
Gelatin-SDS-PAGE for the detection of protease activity
Purified isolates were washed twice with 0.85% NaCl. The respective pellets were then re-suspended with 200 of lysis buffer containing 4.3 g of sucrose and 100 mM of NaCl in 1% triton-X and kept overnight at 4°C. Protein concentration was then estimated using the Bio-Rad Microassay method (Bio-Rad). Twelve percent of resolving and 5% of stacking gel were prepared. Cell lysates with standardized protein concentration were then electrophoresed as described previously . The gel was then subjected to re-naturation and incubation to observe the protease activity. Subsequently, the gel was stained with 0.12% (w/v) Coomassie Brilliant Blue R-250 for 1 hour. The protease bands appear as colorless bands with a dark blue background.
Azocasein assay for colorimetric quantification of protease activity
Parasites were purified as described previously. The cells were lysed through a series of 15 cycles of freeze and thaw. The tubes containing the respective pellets were stored at 4°C overnight. The contents of each tube were centrifuged to sediment the debris and the protein content was estimated using the Bio-Rad Microassay method (Bio-Rad). The concentration of each sample was standardized before the assay. The cell lysates were initially incubated for 10 minutes with the supplementation of DTT (2 mM) (Sigma-Aldrich) at 37°C. This activated the parasitic proteases. 5 mg/ml of Azocasein was prepared in PBS at pH 7.4. One hundred microliters of lysate was mixed with 100 of pre-heated (37°C) azocasein solution and left to incubate at 37°C for 1 hour. The reaction was stopped by the addition of 300 of ice-cold Trichloroacetic acid and the mixture was then left on ice for 30 minutes. The tubes were then centrifuged at 8000 g to remove the undigested azocasein and the supernant was subsequently added to 500 of NaCl (500 mM). The clear orange solution that was produced was measured at 440 nm absorbance. Trypsin and inactivated lysate were used as positive and negative controls respectively. This experiment was carried out in triplicate.
All statistical analysis was carried out using IBM© SPSS© Statistics Version 21. Independent Students t-test was used to assess the difference in the number of cells and protease activity between symptomatic isolates with amoebic forms, symptomatic isolates without amoebic forms and asymptomatic isolates. Pearson Correlation Test was used to assess the correlation between protease activity and percentage of amoebic forms. A value of p < 0.05 is considered statistically significant.
Protease activity using the azocasein assay and gelatin-SDS-PAGE demonstrated higher activity in symptomatic isolates with the highest observed in S2 followed by S1, S5, S4 and S3. Asymptomatic isolates demonstrated a consistently lower activity of protease in all five isolates.
Pearson’s correlation of average protease activity and average amoebic form count
Protease activity and amoebic cell count
Discussion and conclusions
This study is the first to report a correlation between the presence of amoebic forms and protease activity of Blastocystis spp. Proteases have previously been shown to be involved as one of the pathogenic factors of Entamoeba histolytica, Trichomonas vaginalis and Acanthamoeba sp. [25–27]. In a previous study on Blastocystis spp., subtype 3 of symptomatic isolates were shown to frequently posses protease activity especially at 32 kDa, which was postulated to be the virulent factor .
Cysteine-type activity of protease was observed in Blastocystis spp. isolated from patients with gastrointestinal symptoms  with its effect on intestinal cells and gut functions . Proteases from Blastocystis spp. have also been shown to cleave intestinal IgA and elicit inflammatory cytokines [21, 22]. However, these reports did not attempt to provide credible evidence on the life cycle stage of Blastocystis spp. responsible for protease activity.
The relationship of proteases to pathogenicity can be better illustrated with the example of Entamoeba histolytica where its membrane-associated serine proteases, with a molecular weight 60 kDa, were shown to be responsible for the degradation lysosamal proteins and tight junctions in target cells. These proteases were found in low levels in the non-pathogenic strain, Entamoeba dispar. Proteases from Trypanosoma cruzi and Acanthamoeba have been shown to play a role in immune evasion during Chagas disease and be responsible for extracellular membrane degradation during granulomatous encephalitis respectively.
Amoebic forms were shown to be a life cycle stage of Blastocystis spp.  as well as being seen in cultures isolated from symptomatic patients [16, 32, 33]. Amoebic forms have also been reported in stool specimens of patients with diarrhea . In the present study we also saw amoebic forms only in symptomatic isolates. Amoebic forms have been shown to be responsible for the engulfment of bacteria  which could then survive and replicate within lysosomes or phagosomes . Whether bacteria contribute to the pathogenic role of the parasite still remains in question.
The present study demonstrated intense protease activity seen on day 5 in all symptomatic isolates, which correspondingly co-related to the intense bands of 60–100 kDa seen in the same day having the higher percentage of amoebic forms observed in all isolates. The presence of lower molecular weight (17 – 28 kDa) protease in isolates S2 and S4 as well as in asymptomatic isolates (A1 and A3) elicits a question on the significance of this band, which remains unclear although lower molecular weight bands in other studies have been suggested to be a virulence factor [19, 20].
Moreover, our study suggests a steady increase in protease activity during the incubation corresponding to an increase in amoebic forms and a slight decrease on day 7, which relates to the drop in amoebic forms. A previous study reported that the fluctuation of cell size and protease activity appears to have a strong correlation when examined over a 96 hour incubation period . This study contradicts our observation in that other than the percentage of amoebic forms, size of cell showed no influence on the protease activity. The present study involved a painstaking and tedious process to screen large samples of stools to obtain Blastocystis infected stool samples. We limited the study to a sample size of 5 as we had to obtain positive stool samples from symptomatic and asymptomatic patients, given that other published papers relating protease activity and Blastocystis spp. as well as other parasites have been carried out on a smaller sample size yet demonstrated, as in our study, significant and consistent results [19, 21, 27, 36]. Similar studies should be carried out with larger sample sizes with isolates that have undergone genotypic characterization to statistically concrete our postulation and to completely understand the variation of protease activity in different life cycle stages of Blastocystis spp.
The peak parasite count (parasite count between day 5 – 7) was used as one of the parameters in a previous study to differentiate the phenotypic characteristics of Blastocystis spp. It has been reported that asymptomatic isolates grew faster and the average total number of cells reached up to 7 times more than symptomatic isolate. The generation time was twice as high in asymptomatic isolates . The present study concurred with these findings in that the asymptomatic isolates showed higher parasite peak counts than symptomatic ones (Figure 2). It is therefore evident that protease activity did not arise despite the higher parasite count consisting of mostly vacuolar and granular forms seen in asymptomatic isolates but from the larger percentage of amoebic forms seen in symptomatic isolates.
The amoebic form of Blastocystis spp. has been postulated previously to posses a sticky surface, which enhances the adherence to the intestinal epithelial cell lining . This adherence could facilitate the release of proteases from amoebic forms to degrade extracellular membrane similar to that as seen in Entamoeba histolytica. Higher occurrence of Blastocystis spp. has been seen in cancer patients undergoing chemotherapy . Exacerbation of symptoms in such situations could be attributed to vacuolar stages reverting to amoebic forms, which in turn could be causing the related symptoms due to the release of proteases.
In conclusion, this is the first study to provide evidence co-relating amoebic forms of Blastocystis spp. and protease activity, which suggests a strong pathophysiological role that amoebic forms can play in exacerbating symptoms in patients infected with Blastocystis spp.
This study was approved by the Medical Ethics Committee of the University Malaya Medical Centre (UMMC) (Kuala Lumpur, Malaysia) according to the Declaration of Helsinki approved this study.
We would like to extend our gratitude and heartfelt thanks to our lab collogues and all the staff in the Department of Parasitology, University of Malaya.
This study was entirely supported by Postgraduate Research Grant from University of Malaya (PG108-2012B) and High Impact Research Grant (HIR) (UM.C/625/1/HIR/031).
- Hussainqadri SM, Al-Okaili GA, Al-Dayel F: Clinical significance of Blastocystis hominis. J Clin Microbiol. 1989, 27: 2407-2409.Google Scholar
- Tan KSW: New insights on classification, identification, and clinical relevance of Blastocystis spp. Clin Microbiol Rev. 2008, 21: 639-665. 10.1128/CMR.00022-08.PubMed CentralView ArticlePubMedGoogle Scholar
- Stenzel DJ, Boreham PFL: Blastocystis hominis revisited. Clin Microbiol Rev. 1996, 9: 563-584.PubMed CentralPubMedGoogle Scholar
- Horiki N, Maruyama M, Fujita Y, Yonekura T, Minato S, Kaneda Y: Epidemiologic survey of Blastocystis hominis infection in Japan. Am J Trop Med Hyg. 1997, 56: 370-374.PubMedGoogle Scholar
- Pegelow K, Gross R, Pietrzik K, Lukito W, Richards A, Fryauff DJ: Parasitological and nutritional situation of school children in the Sukaraja district, West Java. Indonesia. Southeast Asian J Trop Med Public Health. 1997, 28: 173-190.PubMedGoogle Scholar
- Tan TC, Tan PC, Sharma R, Sugnaseelan S, Suresh KG: Genetic diversity of caprine Blastocystis from Peninsular Malaysia. Parasitol Res. 2012, 112: 85-89.View ArticlePubMedGoogle Scholar
- Souppart L, Sanciu G, Cian A, Wawrzyniak I, Delbac F, Capron M, Dei-Cas E, Boorom K, Delhaes L, Viscogliosi E: Molecular epidemiology of human Blastocystis isolates in France. Parasitol Res. 2009, 105: 413-421. 10.1007/s00436-009-1398-9.View ArticlePubMedGoogle Scholar
- Dogruman-Al F, Dagci H, Yoshikawa H, Kurt Ö, Demirel M: A possible link between subtype 2 and asymptomatic infections of Blastocystis hominis. Parasitol Res. 2008, 103: 685-689. 10.1007/s00436-008-1031-3.View ArticlePubMedGoogle Scholar
- Li LH, Zhang XP, Lv S, Zhang L, Yoshikawa H, Wu Z, Steinmann P, Utzinger J, Tong XM, Chen SH, Zhou XN: Cross-sectional surveys and subtype classification of human Blastocystis isolates from four epidemiological settings in China. Parasitol Res. 2007, 102: 83-90. 10.1007/s00436-007-0727-0.View ArticlePubMedGoogle Scholar
- Kaneda Y, Horiki N, Cheng X-J, Fujita Y, Maruyama M, Tachibana H: Ribodemes of Blastocystis hominis isolated in japan. Am J Trop Med Hyg. 2001, 65: 393-396.PubMedGoogle Scholar
- Moosavi A, Haghighi A, Mojarad EN, Zayeri F, Alebouyeh M, Khazan H, Kazemi B, Zali MR: Genetic variability of Blastocystis sp. isolated from symptomatic and asymptomatic individuals in Iran. Parasitol Res. 2012, 111: 2311-2315. 10.1007/s00436-012-3085-5.View ArticlePubMedGoogle Scholar
- Böhm-Gloning B, Knobloch J, Walderich B: Five subgroups of Blastocystis hominis from symptomatic and asymptomatic patients revealed by restriction site analysis of PCR-amplified 16S-like rDNA. Trop Med Int Health. 1997, 2: 771-778. 10.1046/j.1365-3156.1997.d01-383.x.View ArticlePubMedGoogle Scholar
- Yoshikawa H, Abe N, Wu Z: PCR-based identification of zoonotic isolates of Blastocystis from mammals and birds. Microbiology. 2004, 150: 1147-1151. 10.1099/mic.0.26899-0.View ArticlePubMedGoogle Scholar
- Özyurta M, Kurtb Ö, Mølbakc K, Nielsend HV, Haznedaroglua T, Stensvold CR: Molecular epidemiology of Blastocystis infections in Turkey. Parasitol Int. 2008, 57: 300-306. 10.1016/j.parint.2008.01.004.View ArticleGoogle Scholar
- Tan TC, Suresh KG: Predominance of amoeboid forms of Blastocystis hominis in isolates from symptomatic patients. Parasitol Res. 2006, 98: 189-193. 10.1007/s00436-005-0033-7.View ArticlePubMedGoogle Scholar
- Tan TC, Suresh KG: Amoeboid form of Blastocystis hominis—a detailed ultrastructural insight. Parasitol Res. 2006, 99: 737-742. 10.1007/s00436-006-0214-z.View ArticlePubMedGoogle Scholar
- Stenzel DJ, Boreham PFL, McDougall R: Ultrastructure of Blastocystis hominis in human stool samples. Int J Parasitol. 1991, 21: 807-812. 10.1016/0020-7519(91)90149-2.View ArticlePubMedGoogle Scholar
- Lanuza M, Carbajal J, Villar J, Borrás R: Description of an improved method for Blastocystis hominis culture and axenization. Parasitol Res. 1997, 83: 60-63.View ArticlePubMedGoogle Scholar
- Sio SWS, Puthia MK, Lee ASY, Lu J, Tan KSW: Protease activity of Blastocystis hominis. Parasitol Res. 2006, 99: 126-130. 10.1007/s00436-006-0131-1.View ArticlePubMedGoogle Scholar
- Abdel-Hameed DM, Hassanin OM: Proteaese activity of Blastocystis hominis subtype3 in symptomatic and asymptomatic patients. Parasitol Res. 2011, 109: 321-327. 10.1007/s00436-011-2259-x.View ArticlePubMedGoogle Scholar
- Puthia MK, Lu J, Tan KSW: Blastocystis ratti contains cysteine proteases that mediate interleukin-8 response from human intestinal epithelial cells in an NF-kB-dependent manner. Eukaryot Cell. 2008, 7: 435-443. 10.1128/EC.00371-07.PubMed CentralView ArticlePubMedGoogle Scholar
- Puthia MK, Vaithilingam A, Lu J, Tan KSW: Degradation of human secretory immunoglobulin a by Blastocystis. Parasitol Res. 2005, 97: 386-389. 10.1007/s00436-005-1461-0.View ArticlePubMedGoogle Scholar
- Mirza H, Tan KSW: Blastocystis exhibits inter- and intra-subtype variation in cystiene protease. Parasitol Res. 2009, 104: 355-361. 10.1007/s00436-008-1203-1.View ArticlePubMedGoogle Scholar
- Suresh K, Ng GC, Ho LC, Yap EH, Singh M: Differentiation of the various stages of Blastocystis hominis by acridine orange staining. Int J Parasitol. 1994, 24: 605-606. 10.1016/0020-7519(94)90152-X.View ArticlePubMedGoogle Scholar
- Que X, Reed SL: Cysteine proteinases and the pathogenesis of amebiasis. Clin Microbiol Rev. 2000, 13: 196-206. 10.1128/CMR.13.2.196-206.2000.PubMed CentralView ArticlePubMedGoogle Scholar
- Yadav M, Dubey ML, Gupta I, Bhatti G, Malla N: Cysteine proteinase 30 in clinical isolates of T. vaginalis from symptomatic and asymptomatic infected women. Exp Parasitol. 2007, 116: 399-406. 10.1016/j.exppara.2007.02.007.View ArticlePubMedGoogle Scholar
- Khan NA, Jarroll EL, Panjwani N, Cao Z, Paget TA: Proteases as markers for differentiation of pathogenic and nonpathogenic species of acanthamoeba. J Clin Microbiol. 2000, 38: 2858-2861.PubMed CentralPubMedGoogle Scholar
- Wawrzyniak I, Texier C, Poirier P, Viscogliosi E, Tan KSW, Delbac F, Alaoui HE: Characterization of two cysteine proteases secreted by Blastocystis ST7, a human intestinal parasite. Parasitol Int. 2012, 61: 437-442. 10.1016/j.parint.2012.02.007.View ArticlePubMedGoogle Scholar
- Barrios-Ceballos MP, Martínez-Gallardo NA, Anaya-Velázquez F, Mirelman D, Padilla-Vaca F: A novel protease from Entamoeba histolytica homologous to members of the family S28 of serine proteases. Exp Parasitol. 2005, 110: 270-275. 10.1016/j.exppara.2005.02.022.View ArticlePubMedGoogle Scholar
- Doyle PS, Zhou YM, Hsieh I, Greenbaum CD, McKerrow JH, Engel JC: The trypanosoma cruzi protease cruzain mediates immune evasion. PLoS Pathog. 2011, 7: 1-11.Google Scholar
- Sissons J, Alsam S, Goldsworthy G, Lightfoot M, Jarroll EL, Khan NA: Identification and properties of proteases from an Acanthamoeba isolate capable of producing granulomatous encephalitis. BMC Microbiol. 2006, 6: 42-50. 10.1186/1471-2180-6-42.PubMed CentralView ArticlePubMedGoogle Scholar
- Singh M, Suresh K, Ho LC, Ng GC, Yap EH: Elucidation of the life cycle of the intestinal protozoan Blastocystis hominis. Parasitol Res. 1995, 81: 446-450.View ArticlePubMedGoogle Scholar
- He N, Zhang Y, Hong M, Cong M: Morphological and ultrastructural observation of Blastocystis hominis. Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2001, 19: 169-172.PubMedGoogle Scholar
- Zhang X, Zhang S, Qiao J, Wu X, Zhao L, Liu Y, Fan X: Ultrastructural insights into morphology and reproductive mode of Blastocystis hominis. Parasitol Res. 2012, 110: 1165-1172. 10.1007/s00436-011-2607-x.View ArticlePubMedGoogle Scholar
- Scanlan PD: Blastocystis: past pitfalls and future perspectives. Trends Parasitol. 2012, 28: 327-334. 10.1016/j.pt.2012.05.001.View ArticlePubMedGoogle Scholar
- Carvalho TB, David EB, Coradi ST, Guimarães S: Protease activity in extracellular products secreted in vitro by trophozoites of Giardia duodenalis. Parasitol Res. 2008, 104: 185-190. 10.1007/s00436-008-1185-z.View ArticlePubMedGoogle Scholar
- Tan TC, Suresh KG, Smith HV: Phenotypic and genotypic characterisation of Blastocystis hominis isolates implicates subtype 3 as a subtype with pathogenic potential. Parasitol Res. 2008, 104: 85-93. 10.1007/s00436-008-1163-5.View ArticlePubMedGoogle Scholar
- Schulte W, Scholze H: Action of the major protease from Entamoeba histolytica on proteins of the extracellular matrix. J Protozool. 1989, 36: 538-543. 10.1111/j.1550-7408.1989.tb01092.x.View ArticlePubMedGoogle Scholar
- Samudi C, Kumar S, Anitab ZB, Kuppusamy UR: Infections of Blastocystis hominis and microsporidia in cancer patients: are they opportunistic?. Trans R Soc Trop Med Hyg. 2012, 106: 267-269. 10.1016/j.trstmh.2011.12.008.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.